Angioleiomyoma of the pulmonary artery is rare in the literature and few studies have been reported. Here we present a rare case of angioleiomyoma arising from the pulmonary artery in a young patient.A 27-year-old male patient presented to our clinic due to the incidental finding of a nodule in the right lower lobe of the lung, which was unchanged from the prior year. Preoperative CT scans showed a well-demarcated nodule of soft tissue density penetrated by the basal branch of the right anterior basilar artery (RA8b). Single-port video-assisted RS8 segmentectomy was performed under the guidance of preoperative 3-dimensional reconstruction for histologic confirmation of the tumour. The tumour appeared as a solid tumour of a tube-like structure with vascular endothelium, composed of spindle-shaped smooth muscle cells lacking nuclear atypia and homogenous red-dye substances. The spindle cells were positive for immunostaining for smooth muscle actin (SMA), desmin and Ki-67 and were negative for immunostaining for Dog-1, HMB45, and Melan-A. A pathological diagnosis of primary angioleiomyoma of the pulmonary artery was finally made.This report is a reminder for thoracic surgeons that angioleiomyoma should be included in the differential diagnosis of lung neoplasms, especially for the mass of soft tissue density penetrated by pulmonary blood vessels shown by CT. Awareness of this rare entity should potentially prevent underdiagnosis and improper surgical treatment. Angioleiomyoma is a benign soft tissue tumour comprising mature smooth muscle cells with a prominent vascular component . It typiA 27-year-old male patient complained of the incidental finding of a nodule in the right lower lobe of the lung on a routine health examination in October 2019, which was unchanged on repeat examinations from the prior year. No related subjective symptoms were noted and the physical examination showed no abnormalities. The results of routine blood biochemistry, oncological biomarkers and flexible bronchoscopy were normal. Contrast-enhanced computerized tomography of the chest showed a well-demarcated mass with a maximum diameter of 1.7 cm in the right anterior basilar segment of the lung (RS8). The lesion, which displayed a soft tissue density in the periphery, was penetrated by the basal branch of the right anterior basilar artery (RA8b)  in 2002, but reclassified under ‘pericytic tumours’ in the updated classification because of morphological features shared with myopericytoma, that is, showing a perivascular concentric arrangement of smooth muscle cells . AdditioClinically, angioleiomyoma usually occurs in the lower extremities and appears as a solitary, slow-growing, mobile, firm and occasionally painful cutaneous mass . The paiPreoperative diagnosis of angioleiomyoma is difficult before histopathology . CompletTo the best of our knowledge, only two cases of angioleiomyoma of the pulmonary artery have been reported so far , 7. The We believe this report emphasizes the necessary awareness of thoracic surgeons that angioleiomyoma should be included in the differential diagnosis of lung neoplasms, Although an accurate preoperative diagnosis can be challenging, in our opinion, CT imaging findings presenting as a soft-tissue density nodule penetrated by the pulmonary blood vessel may indicate the possibility of angioleiomyoma. Due to the benign nature of this tumour, sublobar resection is appropriate for the treatment of angioleiomyoma of the pulmonary artery.Additional file 1. Video 1 Legend. Single-port RS8 segmentectomy was performed through the fifth intercostal space between anterior axillary line and midaxillary line. Insufflation technique was utilized to establish intersegmental border (Asian Cardiovasc Thorac Ann 2019 Nov; 27 [


This is in stark contrast to the otherwise similar metabolism of cancer cells, and previous results obtained in activated macrophages and dendritic cells. Our results establish a novel metabolic pathway whereby glucose provides glycerol to the headgroup of TAG during classical macrophage activation.Altered lipid metabolism in macrophages is associated with various important inflammatory conditions. Although lipid metabolism is an important target for therapeutic intervention, the metabolic requirement involved in lipid accumulation during pro-inflammatory activation of macrophages remains incompletely characterized. We show here that macrophage activation with IFNγ results in increased aerobic glycolysis, iNOS-dependent inhibition of respiration, and accumulation of triacylglycerol. Surprisingly, metabolite tracing with  Activation of macrophages with pro-inflammatory stimuli, also known as classical M1 activation, induces a profound shift in energetic metabolism characterized by aerobic glycolysis and decreased mitochondrial substrate oxidation . Lipid ade novo fatty acid synthesis from glucose contributes to lipid accumulation in macrophages in murine models of sterile inflammation . Finally, we show that nitric oxide produced by inducible nitric oxide synthase (iNOS) inhibits mitochondrial respiration and therefore oxidation of FA, which instead accumulates in lipid droplets.We show here that activation of macrophages with interferon gamma (IFNγ), a major mediator of sterile and bacterial-induced inflammation, increases glucose uptake and lactate release. Further, IFNγ increases total TAG levels, and induces lipid droplet accumulation that depends on exogenous lipids. Metabolite tracing with In order to study the metabolic basis of lipid droplet accumulation, we used IFNγ to activate MafB/c-Maf double deficient Maf-DKO) primary mouse macrophages. These cells are a bona fide alternative to other macrophage sources such as RAW cells as they are not transformed cells with distorted metabolism typical of cancer cells; maintain a differentiated macrophage phenotype when expanded in culture; and functionally integrate into tissues without causing tumors when transplanted into mice , 21. ActKO primarIFNγ induced a 2-fold increase in glucose uptake rate and a 2-fold increase in lactate release rate . Moreove13C glucose. We then monitored 13C incorporation into TAG using liquid chromatography-mass spectrometry (LC-MS). We consistently observed a mass increase of 3 Da in major TAG species extracted from cells activated in medium containing U-13C glucose compared to TAG from cells activated in medium containing unlabeled glucose  glucose . Using c carbons . Importa labeled . Similar glucose , indicat glucose , providied cells . Our resde novo fatty acid synthesis, we activated macrophages in the presence of the fatty acid synthase  glucose concentration, there were mass shifts of 3 Da in major TAG species extracted from cells activated in medium containing 24 mM U-13C glucose . BODIPY-fatty acid accumulated within lipid droplets, suggesting that exogenous fatty acid incorporated into TAG contained in these organelles . We confinto TAG .de novo fatty acid synthesis.Externally derived fatty acids must be activated before incorporation into TAG by esterification with coenzyme A through a reaction catalyzed by fatty acyl-CoA synthetase. Indeed, the fatty acid CoA synthetase inhibitor triacsin prevented BODIPY-fatty acid accumulation in lipid droplets , reducedLipid droplets might have accumulated because of increased fatty acid uptake. We observed increased expression of the scavenger receptor CD36 upon activation with IFNγ, but addition of a blocking anti-CD36 antibody previously shown to inhibit fatty acid uptake  did not q < 0.1, fold change > 2; q < 0.05)  . The pat < 0.05) ,B, consi < 0.05) –35, hintTo test this possibility, we first determined macrophage β-oxidation activities under basal conditions. Incubation of macrophages for 24 h with the carnitine palmitoyltransferase I inhibitor etomoxir reduced OCR by 23.5 ± 0.4% , which iNext, we tested if inhibition of mitochondrial respiration by nitric oxide contributed to accumulation of neutral lipid upon activation with IFNγ , 37. We de novo fatty acid synthesis. In order to study the contribution of metabolites to lipid synthesis, previous studies have relied on the detection of radioactivity in total lipids extracted from cells incubated in the presence of 14C-labeled glucose  which contained DMEM, 44 mM sodium bicarbonate, 10% FCS, glucose (4.8 mM), and glutamine (0.8 mM) with or without IFNγ 10 ng/mL or IL-4 10 ng/mL (both from Preprotec), and C75, etomoxir, triacsin, oligomycin, DETA/NO, or SEITU  at the indicated concentrations. In a set of experiments, FCS was replaced with delipidated serum  plus a 1:100 dilution of lipid mixture  containing cholesterol, and arachidonic, linoleic, linolenic, myristic, oleic, palmitic, and stearic acids.MafB/c-Maf double deficient (Maf-DKO) macrophages were a kind gift from Dr. Michael H. Sieweke (Center d'Immunologie de Marseille-Luminy). Maf-DKO cells were grown in DMEM containing 20% L929-conditioned medium and 10% FCS. BMDM were differentiated from bone marrow cells obtained from C57BL6 mice in the presence of DMEM containing 20% L929-conditioned medium and 10% FCS. Maf-DKO cells were predominantly octaploid (8c) whereas BMDM were diploid (2c) and octaploid (8c) as determined by DNA quantification with DAPI staining (data not shown). For experiments, cells were incubated for 24 h at 37°C and 5% COCells were incubated in 24-well plates for 24 h after which they were washed once with cold PBS and detached with cold PBS/EDTA 1 mM. Cells were stained with anti-CD86 , I-A/I-E  or anti-CD36  antibodies in FACS buffer  for 30 min on ice, washed once, and resuspended for cytometry analysis. Cells were acquired with an LSRFortessa II flow cytometer (BD Biosciences) and analyzed with FlowJo .Cells were incubated in 24-well plates for 24 h after which they were washed once with cold PBS, fixed with PBS containing formaldehyde 4% for 10 min, and washed three times with PBS. Cells were incubated at room temperature (RT) for 30 min with HCS LipidTOX Green Neutral Lipid (ThermoFisher Scientific) diluted 1:1,000 in PBS, washed three times with PBS, scraped off, and resuspended in PBS for flow cytometry analysis. For microscopy analysis, cells were grown on microscope slides inside wells of 24-well plates followed by the same procedure as for flow cytometry with the addition of DAPI. For ADRP staining, cells were incubated at RT for 1 h in blocking buffer . Then, rabbit polyclonal anti-ADRP antibody (Abcam) was added at a final 1:200 dilution, and cells were incubated for 2 h at RT. After washing with blocking medium, cells were incubated at RT for 2 h in blocking medium containing Alexa Fluor 647-conjugated goat anti-rabbit antibody (ThermoFisher Scientific) at a 1:1,000 dilution. After washing with blocking medium, cells were mounted with Fluoromount-G (Southern Biotech). Images were taken with a Leica SP8 confocal microscope, and analyzed with Fiji.Cells were washed once with cold PBS. PBS was then removed and cold PBS/EDTA 5 μM solution was added. Cells were incubated for 5 min at RT, gently detached by pipetting, and transferred to FACS tubes and kept on ice. Tubes were spun down at 200 g for 5 min at 4°C, after which PBS/EDTA was removed. Cells were resuspended in 200 μL of LGLG medium containing 2 μL of a 1:10 dilution of Mitosox (ThermoFisher Scientific), and incubated at 37°C in a water bath for exactly 25 min. DAPI was then added to exclude dead cells and cells were analyzed by flow cytometry.Glucose, lactate, and triacylglycerol were determined using commercially available enzymatic assay kits (BioAssay Systems or BioVision) following the manufacturers' instructions. Glycerol 3-phosphate and activity of cG3PDH were quantified as previously described , 59. CytTotal cell protein was extracted from cells using CelLytic M (Sigma) and kept at −20°C until further analysis. Protein was heated for 5 min at 50°C  or 95°C (for iNOS and arginase-1) in 5x Laemmli buffer containing 2-mercaptoethanol. Samples were loaded into acrylamide gels and protein was transferred to PVDF membranes followed by detection with chemiluminiscence using the total OXPHOS rodent WB antibody cocktail (Abcam), or anti-INOS , anti-arginase-1  or anti-β-actin antibodies  plus appropriate HRP-conjugated secondary antibodies.e Extracellular Flux Analyzer (Seahorse Bioscience). Cells were incubated for 24 h in XF-96 cell culture plates at a density of 105 cells/200 μL per well in LGLG medium in the presence or absence of IFNγ 10 ng/mL, and SEITU, etomoxir, DETA-NO and oligomycin at the indicated concentrations. One hour prior to the experiment, LGLG medium was removed and 175 μL of assay medium (LGLG medium containing 44 mM sodium chloride instead of 44 mM sodium bicarbonate in order to prevent pH buffering and to maintain medium osmolarity) were added. Drugs (25 μL) were injected during the assay at the following final concentrations: oligomycin (1 μM), CCCP (carbonyl cyanide m-chloro phenyl hydrazone) as uncoupler (2 μM), and rotenone (100 nM) plus antimycin (1 μM). The experiment was performed at 37°C and 20% oxygen using a mix-wait-measure protocol of 3-0-3 min with three initial basal rate measurements.Oxygen consumption rate was measured with an XF1-BODIPY 500/510-C12 (Life Technologies) into cells using a fluorescent plate reader, as previously described (5 cells per well in the presence or absence of IFNγ 10 ng/mL. Cells were washed once with warm PBS and incubated at 37°C for 30 min with 100 μL of LGLG without FCS (serum free LGLG medium). Staining solution consisted of one part 2x serum free LGLG medium plus one part 8% trypan blue that served as fluorescence quencher. To this staining solution C1-BODIPY 500/510-C12 diluted 1:4,000 from a 1 mg/mL stock was added. Fatty acid incorporation was started by adding 100 μL of pre-warmed (37°C) staining solution  to each well already containing 100 μL of serum free LGLG. Fluorescence (Ex485/Em528) was measured from the bottom of the plate on a Synergy H4 (BioTek) plate reader set at 37°C. Data were acquired at intervals of 80 s for up to 30 min, time at which fluorescence plateaued. In a different experiment, cells were incubated for 24 h in LGLG medium containing delipidated serum, IFNγ 10 ng/mL and C1-BODIPY 500/510-C12 at a final dilution of 1:4,000. In another set of experiments, cells were incubated for 24 h in LGLG medium containing FCS and IFNγ 10 ng/mL. Cells were washed once with warm PBS, and incubated at 37°C for 30 min in LGLG medium containing delipidated serum plus triacsin (1 μM), after which C1-BODIPY 500/510-C12 was added. After 10 min of incubation at 37°C cells were washed swith PBS, fixed, and stained with DAPI and anti-ADRP antibody as mentioned above.Fatty acid uptake was determined by measuring incorporation of the fluorescent fatty acid analog Cescribed . Briefly13C) glucose (Cambridge Isotope Laboratories) instead of glucose. In another set of experiments, cells were incubated in the presence of IFNγ 10 ng/mL in medium containing 24 mM uniformly-labeled (U-13C) glucose plus L-glutamine 4, or 24 mM of glucose plus 4 mM U-13C, U-15N L-glutamine (Cambridge Isotope Laboratories). In another set of experiments, cells were incubated in the presence of LGLG containing delipidated serum instead of FCS plus 50 μM oleic acid or U-13C oleic acid (Sigma) conjugated to BSA. After 24 h, cells were washed once with cold PBS, scraped off, counted, resuspended in 100 μL of PBS, and kept frozen at −80°C until further analysis. For lipid extraction, cells were thawed and extracted using modified Bligh and Dyer method (Cells were incubated in 6-well plates in the presence of IFNγ 10 ng/mL in LGLG medium. Some wells contained 4.8 mM uniformly-labeled (U-r method . Samplesr method . For fatMus musculus downloaded from SwissProt (2016/10/05). Proteins were quantified by MS1-based label-free quantification and statistical analysis was performed using SafeQuant .Comparisons between two groups were made using a two-tailed unpaired The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD017148 and 10.6019/PXD017148. Other raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.MR-B conceived the study. MR-B, DB, and XG designed experiments. XG, AS, and MR-B performed experiments and analyzed data. XG, AS, and DB provided resources. MR-B and XG wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Prostate cancer (PCa) is the most common malignant tumor affecting males worldwide. The substantial heterogeneity in PCa presents a major challenge with respect to molecular analyses, patient stratification, and treatment. Least absolute shrinkage and selection operator was used to select eight risk-CpG sites. Using an unsupervised clustering analysis, called consensus clustering, we found that patients with PCa could be divided into two subtypes (Methylation_H and Methylation_L) based on the DNA methylation status at these CpG sites. Differences in the epigenome, genome, transcriptome, disease status, immune cell composition, and function between the identified subtypes were explored using The Cancer Genome Atlas database. This analysis clearly revealed the risk characteristics of the Methylation_H subtype. Using a weighted correlation network analysis to select risk-related genes and least absolute shrinkage and selection operator, we constructed a prediction signature for prognosis based on the subtype classification. We further validated its effectiveness using four public datasets. The two novel PCa subtypes and risk predictive signature developed in this study may be effective indicators of prognosis. Despite the ongoing development of therapeutic strategies, the heterogeneity of PCa contributes to treatment failure  is a major public health threat . Based o failure . TherefoDuring the process of DNA methylation, methyl groups are added to CpG islands on the DNA molecule. Hypermethylation acts on promoters and could lead to gene silencing, whereas hypomethylation is associated with chromosomal instability and a loss of imprinting . In manyNovel methylation-based subtypes have been reported in PCa. For example, the Cancer Genome Atlas Research Network  conducte2 (3 (4 (5 (RNA-seq data (in the form of HTSeq-Counts and HTSeq-FPKM), DNA methylation (450 K), somatic variation, copy number alterations (CNA), and clinical information for patients with PCa were downloaded from The Cancer Genome Atlas (TCGA) database2 . The gen2  . Least aSPOP) was evaluated. Then, the mRNA levels of SPOP in different subtypes were compared by Wilcoxon’s test.Simple nucleotide variants were compared between subtypes using the GenVisR R package . Genes wRND3 was differentially expressed between the subtypes, as determined by a Wilcoxon test. The type and frequency of CNAs in RND3 were explored. Furthermore, the relationship between CNA types and mRNA expression levels of RND3 were evaluated by the Wilcoxon test.To explore the difference in CNAs between subtypes, genes with significant differences in copy number between subtypes were identified by chi-squared tests. Among these genes, https://www.tumorfusions.org/) were used to analyze the difference in TMPRSS2–ERG fusion gene expression between the subtypes . To obtain fold changes, HTSeq-Counts were analyzed using the DESeq2 R package . The halthe GSEA . Finallythe GSEA .Furthermore, expression levels of genes that were crucial for PCa were compared between the subtypes by Wilcoxon tests.p < 0.05. Then, survival-associated genes were screened from the DEGs by Cox regression and log-rank tests. Finally, genes for LASSO were filtered out.A weighted correlation network analysis (WGCNA) could be used find phenotype-associated gene modules . TherefoBefore training, 477 patients were randomly divided into a training set and internal validation set using the caret R package . InformaFirst, time-dependent receiver operating characteristic (tdROC) curves were used to evaluate the predictive accuracy of the signature in the training set, internal validation set, and external validation sets  using the timeROC R package . Then, aUnivariate and multivariate Cox regression analyses were used to explore whether the risk score is an independent predictor of prognosis. Finally, the clinical diagnostic value of the signature was compared with that of clinical features (Gleason score and PSA) by a decision curve analysis (DCA) . DCA is p < 0.05 were defined as statistically significant. In the survival analysis, the survival outcome was defined as DFS or biochemical recurrence-free survival (BCR) based on clinical records.R 3.6.3 was used for all statistical analyses. Values of k = 5, indicating that the cohort could be divided into up to five subtypes. However, one cluster consisted of only a single patient when k = 4 or 5. Additionally, the cluster-consensus value for each cluster was not large enough under k = 4 or 5  and relative change in the area under the CDF curve are shown in = 4 or 5 . Thereforognosis . For k =lation_L . Furtherlation_L . As showSPOP was higher in the Methylation_H subtype than in the Methylation_L subtype. SPOP is one of the most frequently mutated genes in primary PCa. Based on the tumor-suppressive role of SPOP in PCa and the results of loss-of-function assays, SPOP mutations are expected to include the invasion and proliferation of PCa cells  for the training set, internal validation set, and external validation sets  were 0.72, 0.66, 0.76, 0.76, 0.84, and 0.74, respectively . FurtherIn univariate Cox regression analyses, six variables  were associated with a worse prognosis . In a muWe have recently described the advantages and necessity of multi-omics approaches for studies of PCa . In thisSPOP, which is the most frequently mutated tumor-suppressor gene in primary PCa, were lower in the Methylation_H subtype than in the Methylation_L subtype . With respect to the clinical applications of these findings, we have the following suggestions. Because RNA-seq data in TPM format were used to train the signature, we suggest employing the same data format of data in clinical applications. Considering batch effects of measurement techniques, gene expression levels should be measured by similar techniques, even though the signature performed well in the validation data sets, in which genes were profiled by array-based methods. Furthermore, the risk levels were determined by the median risk score in the patient cohorts. In the future, the study cohort should be further expanded to obtain a more objective and stable threshold range.Collectively, we identified two subtypes with different methylation statuses at eight CpG sites and evaluated the high-risk characteristics of the Methylation_H subtype based on epigenomics, genomics, transcriptomics, disease status, immune cell infiltration, and functional analyses. Finally, based on these two novel subtypes, an eight-gene predictive signature was constructed and validated using various public datasets.The original contributions presented in the study are included in the article/EZ, MZ, and YS were responsible for the design and conception of the research project. EZ, YS, MZ, FS, OM, JH, YG, and HW contributed to the data acquisition or data analysis and data cleaning. EZ and YS participated in the drafting of the manuscript and the rigorous modification of the manuscript to clearly convey the research contents. All authors are responsible for the authenticity and reliability of this study and have no objection to the final submitted manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Periodontal diseases can lead to chronic inflammation affecting the integrity of the tooth supporting tissues. Recently, a striking association has been made between periodontal diseases and primary cancers in the absence of a mechanistic understanding. Here we address the effect of periodontal inflammation (PI) on tumor progression, metastasis, and possible underlining mechanisms. We show that an experimental model of PI in mice can promote lymph node (LN) micrometastasis, as well as head and neck metastasis of 4T1 breast cancer cells, both in early and late stages of cancer progression. The cervical LNs had a greater tumor burden and infiltration of MDSC and M2 macrophages compared with LNs at other sites. Pyroptosis and the resultant IL-1β production were detected in patients with PI, mirrored in mouse models. Anakinra, IL-1 receptor antagonist, limited metastasis, and MDSC recruitment at early stages of tumor progression, but failed to reverse established metastatic tumors. PI and the resulting production of IL-1β was found to promote CCL5, CXCL12, CCL2, and CXCL5 expression. These chemokines recruit MDSC and macrophages, finally enabling the generation of a premetastatic niche in the inflammatory site. These findings support the idea that periodontal inflammation promotes metastasis of breast cancer by recruiting MDSC in part by pyroptosis-induced IL-1β generation and downstream CCL2, CCL5, and CXCL5 signaling in the early steps of metastasis. These studies define the role for IL-1β in the metastatic progression of breast cancer and highlight the need to control PI, a pervasive inflammatory condition in older patients. Porphyromonas gingivalis , Fusobacterium nucleatum (F. nucleatum), and Prevotella intermedia can potentially serve to initiate or promote tumor development, as seen for gastric cancer with Helicobacter pylori infection [P. gingivalis and F. nucleatum have been attributed to pancreatic and colorectal cancer in lymph node (LN) metastases [Periodontal diseases are characterized by inflammation affecting the integrity of the tooth supporting tissues, among them gingivitis and periodontitis are the most common. Periodontitis affects nearly half of the US adult population . Strikinnfection . Interesnfection –11. Hightastases , 13. Thetastases . PeriodoSeveral animal models have been used to mimic cellular complexities that occur in human periodontal diseases. Gram-negative bacteria are thought to be important periodontal pathogens. The lipopolysaccharide (LPS) component of the cell wall of these microorganisms, is a significant inflammatory stimulus that triggers an innate immune response. Thus, injections of LPS into the mouse gingival tissues is capable of recreating a sterile model of periodontal inflammation (PI), not involving the pervasive nature of untreated bacterial infections , 16. TolInflammatory pathways are key mediators for cancer development and metastatic progression. Using the PI model of chronic LPS injection in the gingiva we observed an immune response after 3 weeks Fig. . As expeP < 0.0001). Previous studies have highlighted the recruitment and expansion of MDSC in the periodontal inflammatory process induced by P. gingivalis infection [+ and CD206+ expression in the tumor bearing mice with PI, compared with sham injected mice allografted with tumor alone (P < 0.05). In addition, M1 macrophages, characterized by F4/80+ and MHCII+ staining, were similarly increased in mice with PI. The presence of MDSC and M2 macrophage in the cervical LNs was suggestive of a tumorigenic niche. Not entirely surprising, the M1 and M2 macrophages in the spleen seemed to be depleted in tumor bearing mice with PI, compared with mice with either tumors or PI alone, in support of an active recruitment process to the site of inflammation. We observed recruitment of macrophage and MDSC to the cervical LNs of mice with PI, concomitant with tumor cells.To better characterize the tumor tropism mediated by PI, a syngeneic metastatic model was tested using orthotopically grafted 4T1 cells in the fourth mammary gland Fig. . LN micrnfection . LikewisNumerous factors can induce the recruitment and differentiation of MDSCs. The chronic injection of LPS into the gingival tissue, used to initiate the sterile PI model, could mediate a host response by the production of proinflammatory factors capable of recruiting macrophages and MDSC . In tryiWe next sought to determine the relevance of IL-1β expression by gingival fibroblasts on breast cancer metastatic progression. We found that PI tissues had expression of NF-κB phosphorylated-p65 and NLRP3 in the fibroblastic cells, indicating active inflammasome signal I and signal II signaling Fig. . HoweverTo evaluate the role of IL-1β in immune cell recruitment, a panel of chemokines and cytokines were measured from gingival fibroblasts treated with LPS with or without anakinra. LPS was found to elevate CCL2 expression by almost sixfold and CXCL12 expression by threefold Fig. . Anakinr+ and CD33+, compared with the healthy gingiva  recruitment to the LNs in the neck and associated TGF-ß expression seemed to be the greatest in the PI mouse model, compared with either control or the PI mice treated with anakinra , and to a lesser extent macrophage  and functional  markers. Granulocytic MDSC  in the premetastatic niche, as well as in the tumor of the head and neck tissues. This data also confirmed a colocalization of IL-10, IL-2, and TGF-β showing the immunosuppressive expression pattern initiated by these MDSC. While TGF-ß expression is also a feature of M2 macrophage, no such co-expression was observed in our tissues. Activated M2 macrophage are reported to have low IL-2 expression in response to IL-6 [P. gingivalis is reported to promote the expansion of MDSC and consequently suppressed the host response [+/CD33+ MDSC, suggested that its infiltration can be an inherent feature of periodontitis, rather than tumor progression  in PBS or 10 μl endotoxin-free PBS vehicle into mandibular buccal mucosa 2 times a week for 3 weeks [4 cells) intracardially (early metastasis model 1) or received 4T1 cells (105 cells) in the mammary fat pad (early metastasis model 2). Sample size of at least five mice per group were selected to achieve 80% power. Both experiments were terminated 2 weeks following cancer cell injections. Tumor size was measured by Vernier caliper and tumor volumes calculated according to the formula V (mm3) = L (major axis) × W (minor axis) × H × 6/π. The axillary, cervical LNs, and gingival tissues were collected for further experiment. The late metastasis model was established after 3 weeks of PI model establishment, where the 4th mammary fat pad was injected with 105 4T1 cells. The anakinra  was administered daily, starting on the day of tumor introduction. Following the injection/grafting of tumors the mice were randomized prior to allocating to the control and treatment groups. Tumors were resected when they reached 100 mm2 and metastatic progression allowed to progress for up to 10.5 weeks. The axillary, cervical LNs, the neck tissue, and gingival tissue were collected for further evaluation. The investigators were blinded to the group at the time of allocation to the tumor or treatment groups. However, the groups were known to the investigators during data collection and analysis.Procedures and animal experiments were approved by Institutional Animal Care & Use Committee (IACUC003638) at Sinai Medical Center. Four-week-old, female BALB/c mice  were used in the syngeneic xenografting and PI models described. The PI model was generated as previously reported by injecting 10 μg LPS  for 10 min. Luciferase activity was assayed using luciferase substrate buffer . Protein concentrations were evaluated using BCA protein assay kit  on a microplate reader . Relative luciferase Unit was calculated on a Luminometer  according to the formula, Relative luciferase Unit = (reporter activity − background)/protein concentration.LNs and spleen tissues were prepared using mechanical dissociation of minced tissue to obtain single cell suspensions. The following antibodies were used for flow cytometry: anti-Ly6G/Ly6C (Cat #58-5931-82), anti-Cd11b (Cat #12-0112-82), anti-CD86 (Cat #11-0862-82), anti-F4/80 (Cat #17-4801-82), anti-MHCII (Cat #13-5321-82), and anti-CD11c (Cat #11-0116-42) all from eBioscience, San Diego, CA) and anti-CD206 . After being stained with a standard protocol, all events were analyzed with FlowJo software  and frequencies among live cells were obtained.3 pieces and maintained in Alpha Modifications Minimum Essential Medium (α-MEM) with 10% fetal bovine serum supplemented with 100 U/ml penicillin, and 100 μg/ml streptomycin  at 37 °C with 5% CO2. Cells between passage 3 and 7 were used.Mouse gingival fibroblasts were cultured from mandibular buccal gingival tissues obtained from 6-week-old female Balb/c mice. The gingival tissues were cut into 1 mmThe 4T1 mouse mammary carcinoma cells were obtained from the American Type Culture Collection  and were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Human gingival fibroblasts were cultured following a previously published report .4T1-Luc cells were established as follows: the pGL4 luciferase reporter vector was obtained from Promega. This vector containing a hygromycin B selectable marker and was transfected into 4T1 cells by using Nucleofector™ Kit V  with the Amaxa Nucleofector (Lonza Group Ltd.), followed by a 30 μg/ml hygromycin B  selection for 7 days. The expression level of luciferase was then evaluated by luciferase assay.Mouse gingival fibroblasts were serum-starved for 24 h, then treated with 1 μg/ml LPS with or without 500 ng/ml anakinra for 24 h. Total mRNA was isolated from gingival fibroblasts or gingival tissue using RNeasy mini kit . cDNA was synthesized from 1 μg of total RNA with the iScript cDNA kit (Bio-Rad Laboratories). Receptors mRNA expression was determined by quantitative RT-PCR using primers in Supplementary Table Gingival fibroblasts were serum-starved for 24 h, then treated with 1 μg/ml LPS for 24 h. Cells were further cultured in the presence of 20 μg MDCF-DA (Sigma-Aldrich) for 20 min and imaged with a fluorescence microscope .Gingival fibroblasts were serum-starved for 24 h, then treated with 1 μg/ml LPS with or without 500 ng/mg anakinra for 24 h. Cytokine concentrations in cell lysates were then determined using the Mouse Cytokine Array C3  following manufacturer’s instructions. Data were quantitated with Quantity One (Bio-Rad Laboratories)For short-term cultures, gingival fibroblasts were serum-starved for 24 h, then treated with 1 μg/ml LPS for 24 h. For long-term cultures, gingival fibroblasts were treated with 1 μg/ml LPS in α-MEM containing 2% FBS for 14 days. Proteins were detected following a previously published western blot procedure . The fol2 laser ablation spot size at a frequency of 200 Hz and ablation energy of 3 dB. Area of analyzed tissue varied from 400 um2 up to 1000 um2 with multiple areas imaged per specimen. Image analysis was performed with MCD viewer (Fluidigm) and HistoCAT [Literature was reviewed to identify markers relevant to the tumor and premetastatic niche. Metal conjugated antibodies were obtained with custom conjugations with Fluidim Maxpar Antibody Labeling Kit . Human gingival specimens were collected from crown lengthening surgery  and gingivectomy (periodontitis tissues) by informed consent.Immunohistochemical localization of select proteins were performed on standard protocol on 4% paraformaldehyde fixed, paraffin embedded tissues using anti Luciferase , NLRP3 (LifeSpan Biosciences), 8-OHdG (Abcam), TUNEL , IL-1β (R&D Systems), Gr1.1 , CD33 (Abcam), CD11b (Abcam), and Pan cytokeratin antibodies (Santa Cruz Biotechnology). Quantitation of positive staining was normalized to total number of cells per field by NIH Image software.ELISA was performed for the expression of IL-1β , CCL2, CCL5, CXCL5 and CXCL12  on conditioned media from human gingival fibroblasts expanded for 24 h in the presence or absence of LPS. IL-1β ELISA was also performed on conditioned media from mouse gingival fibroblasts and blood plasma from mice of early metastasis models.t test. Difference was considered significant when P < 0.05.Data were expressed as mean ± SEM. The statistical significance of differences was assessed by using ANOVA or student’s Supplement table 1Supplement table 2Supplemental Figure 1Supplemental Figure 2Supplemental Figure 3


While being a monomer in physiological conditions, under the necessary stimulus—usually a mutation, it tends to form fibrils, which later participate in the disease development. This process can potentially be regulated  by many cellular components and it is being hypothesized that the cell membrane might play a key role in the oligomerization pathway. Studies involving cell membranes pose several difficulties; therefore, an alternative in the form of membrane mimetics is a very attractive solution. Here, we would like to present the first study on hCC oligomerization under the influence of phospholipid liposomes, acting as a membrane mimetic. The protein–mimetic interactions are studied utilizing circular dichroism, nuclear magnetic resonance, and size exclusion chromatography.Studies revolving around mechanisms responsible for the development of amyloid-based diseases lay the foundations for the recognition of molecular targets of future to-be-developed treatments. However, the vast number of peptides and proteins known to be responsible for fibril formation, combined with their complexity and complexity of their interactions with various cellular components, renders this task extremely difficult and time-consuming. One of these proteins, human cystatin C ( The process of hCC interaction with the membranes constitutes an interesting and important target of studies. It may explain the process of hCC toxicity against cells and its consequences, e.g., the processes leading to the occurrence of hereditary cystatin C amyloid angiopathy [Amyloid is a name used for the description of a specific protein (or peptide) that aggregated into its insoluble form featuring characteristic fiber-like shape . The pheide (Aβ) , immunogide (Aβ) , and humβ) [hCC) . The lasgiopathy  or Alzhegiopathy .The studies of protein–membrane interactions are, however, complicated, time-, and cost-consuming experiments when performed on biological membranes isolated from natural sources. Previously, we performed a structural and dynamic analysis of the membrane mimetic—mixed micelle combining zwitterionic dodecylphosphocholine (DPC) and anionic sodium dodecyl sulfate (SDS) components . In factThe number of techniques allowing to study interactions between proteins and membrane mimetics is rather scarce. Current techniques involve electrochemical techniques ,16, micrhCC protein  reaches an equilibrium state between the monomer, being the major component, and the dimer. This results in two sets of overlapping signals observed in the NMR spectra, making them difficult for interpretation [hCC V57P variant (stable dimer) is a demanding task as the subunits of the dimeric variant are virtually identical, once again causing an overlap of NMR signals. As a result, the assignment of the backbone resonances was not yet performed for hCC V57P dimer. The oligomeric state of hCC is important in context of its structural studies since any interaction with ligands may cause changes in the oligomeric equilibrium of the amyloidogenic protein. However, due to technical difficulties  at present, NMR measurements can be performed on stable monomeric hCC V57G variant only.The studies performed so far revealed that in solution the wild type retation . SimilarhCC V57G mutant (stable monomer) to explore the structural and dynamic aspects of the interactions between the hCC V57G protein and the DMPC phospholipid bilayer. For the purpose of this study, DMPC was chosen as a membrane mimetic due to its high abundance in mammalian membranes and relatively easy procedure of liposome preparation [hCC V57G sequence involved in interaction with DMPC bilayer.In this study, we used the paration . DMPC isparation ,27,28,29hCC protein resulting from the interactions of the protein with the dimyristoylphosphocholine (DMPC) phospholipid. Recording of the CD spectra turned out to be difficult, due to high light scattering propensities of the DMPC liposome solution. However, after the prolonged process of liposome formation , no increase of Membranes). It is possible that the interactions between hCC V57G and the liposomes are rather weak and require higher liposome concentrations for the changes of protein structure to be observed. On the other hand, performing the experiments at higher DMPC concentrations proved not to be possible due to the high light-scattering properties of the solution.Circular dichroism (CD) spectroscopy experiments were performed to visualize the changes in the secondary structure of the trations . Collect protein . ContrarMost of the phospholipid-based liposomal solutions  have a tendency for strong light scattering. The only way to make the solutions more transparent is to decrease the liposome size, as the intensity of light scattered by a sphere is directly related to its radius . In expetion see . HoweverhCC protein caused by the presence of different concentrations of DMPC liposomes, indicating interactions between the molecules and possible impact on further oligomerization of protein. The incubation of the hCC V57G with DMPC solutions did not cause any particular changes in the oligomeric state of the analyzed protein. No changes in the intensity of signals or retention times were observed in the chromatograms indicating that, regardless of the incubation temperature, the hCC V57G monomer is stable in the presence of DMPC liposomes  experiments were performed to verify if there are any changes in the oligomeric state of iposomes A,B. This factors ,31. The hCC V57G and cell membrane were monitored by NMR spectroscopy using the DMPC phospholipid as a membrane model. The hCC V57G exhibit good dispersion, confirming that in this experimental condition the hCC V57G is properly folded  . For sevhCC V57G mutant by PGSE-NMR deliver an additional confirmation of the stability of the 3D structure at different experimental conditions. The translation diffusion coefficient  . The obtsolution .S, which is a measure of the degree of spatial restriction of the motion, an effective correlation time The analysis of tumbling ,34. Fourme scale .The experimental sonances . The relhCC V57G in solution reveals that rotational diffusion tensor can be presented as prolate axially symmetric [hCC V57G in solution [The structural analysis performed for ymmetric . The D∥ on rates ,37. The solution .hCC V57G comprises two The secondary structure of than 0.8 . At the The values of local parameters—residues .hCC V57G sequence, we can conclude the low intensity of chemical exchange . As a result, only a small amount of molecules in the bound state are present in the solution. Therefore, the rotational and translational motions observed during the measurement are very similar to those observed for the V57G variant in solution. The relaxation parameters, on the other hand, are quite sensitive to the existence of a transition state, even a couple of percent of a different conformation can be detected by the analysis of relaxation data.An additional possibility of fast and simple identification of the residues undergoing conformational exchange slower than the overall molecular tumbling can be derived from analysis of  product . The R1Rme frame . InspecthCC V57G in the environment of DMPC-The experimental data acquired for db 6RPV) , suggestPV) [csp A,B, whicPV) [csp C. The mahCC V57G interaction with the DMPC liposome. The presence of the DPMC-hCC interactions with the DMPC phospholipid.The backbone mobility in a low-frequency time frame is another important aspect of  product A. Inspec-strands . They prhCC V57G and DMPC membrane mimetic. The acquired experimental data demonstrated that the hCC V57G variant does not migrate into the DMPC bilayer but locates itself on the lipid surface. The bilayer exhibits a rather weak attraction to the N-terminal part of the protein and hCC oligomerization seems to be rational. However, to fully verify it further, detailed studies involving advanced membrane constructs, natural membranes, and hCC variants prone to oligomerization processes are required.The presented study focused on the description of the interaction sites between hCC V57G variant was obtained using site-directed mutagenesis as previously described [E. coli OmpA protein (causes the secretion of a protein into periplasmic space), temperature-sensitive E. coli BL21(DE3)-competent cells (Novagen) using the standard LB medium and temperature-induced expression, according to the protocol described earlier [The DNA of escribed . Plasmidescribed ) contain earlier .The lyophilized DMPC powder was suspended in PBS buffer . If the solution was used in NMR experiments, the buffer contained 10% Dof times . After thCC V57G protein was dissolved at the concentration of 10 v/v five step dilution series with the highest concentration of g for 5 min to remove any insoluble particles from the solution. The CD spectra were registered with JASCO J-815 spectropolarimeter for supernatants at 295 K in the UV range of 195 to 260 nm and analyzed with Jasco Spectra Manager software.The hCC V57G protein was dissolved at the concentration of 30 The hCC monomer–dimer equilibrium has changed.The results were analyzed with Chromax 2007  and OriginPro 2018  software to verify if the z-gradient unit and NMR measurements were performed on Agilent DDR2 800 NMR spectrometer operated at magnetic field strength of 18.8 T =25S form of J(ω)=S2AG is the gradient strength. The Diffusion experiments were conducted on an Agilent DDR2 800 NMR spectrometer utilizing a standard PGSE (Pulsed Gradient Spin Echo) pulse sequence  containined from .


All molecular systems, from small molecules to macromolecules, exhibit specific characteristics for a specific environment and time. In order to gain an accurate understanding of the functions of all types of molecules, studies of their structure and dynamics are essential. Through dynamic studies, using techniques such as spectroscopy, structure determination, and computer analysis, it is possible to collect functional information on molecules at specific times and in specific environments. Such information not only reveals the properties and mechanisms of action of molecules but also provides insights that can be applied to various industries, such as the development of new materials and drugs. Herein, I discuss the importance of molecular dynamics studies, present the time scale of molecular motion, and review techniques for analyzing molecular dynamics. Imagine this example of why molecular dynamics research is important. There is an advertisement for a new vehicle in the newspaper. It is built primarily for driving on the road like a car, but it can fly like a light aircraft and can be sailed like a ship on rivers or seas. Of course, it is equipped with fully autonomous driving capability, and powered by sustainable energy. Below the introduction to each of these versatile features is a picture of the vehicle engaged in each mode of transport . We can extrapolate a lot from these three pictures by imagination, but some key questions remain unanswered. How will this vehicle transform from one mode of transport to another ? How long will each transformation take? How long can this transport run without replacing its energy source? How perfect/safe is its autonomous driving? How does it handle traffic conditions on the road, sky, and sea? We cannot say anything with confidence with just three pictures. Each mode of transport will have its own operating characteristics, and these characteristics will also change in adaptation to changes in operating environment. We will need tons of pictures and/or intelligently produced videos to accurately understand the vehicle’s characteristics under all conditions we may encounter in each mode of transportation. With continuous information (as is provided by videos), we can accurately understand the vehicle’s performance, whereas fragmented information (such as that from pictures) is insufficient to understand the new vehicle.This transportation-based analogy illustrates the importance of continuous observation, like movies, for the understanding of complex processes such as those studied in the sciences. In nature, there are many molecules  with interesting and important molecular properties that can be elucidated using various scientific techniques.Among these, techniques for obtaining high-resolution structural information are essential as they provide intuitive insights into important structural and functional characteristics of molecules that play important roles in life. Currently, the three dimensional structures of small molecules can be obtained from repositories such as the Cambridge Structural Database (CSD) , which cVarious techniques such as spectroscopy, structure determination and computer analysis are applied to the study of molecular dynamics . In ordeMeanwhile, techniques such as X-ray diffraction/scattering, NMR, Cryo-EM, MicroED, and neutron scattering/diffraction are widely applied to study the mechanism of action by revealing the structure of a molecule ,15,20. TAdditionally, computational molecular dynamics analysis by density-functional theory (DFT) or MD simulation provides an understanding of the functions of molecules and insights into their industrial or medical applications, such as in drug design ,35,36,37Comprehensive molecular dynamics information for pinpointing the functions and properties of molecules can be inferred from information gleaned from multiple snapshots obtained using traditional techniques and the most advanced techniques. Examples of these include temporal characterization based on autocorrelation and pump probe technology combined with microscopy, spatial evolution analysis using X-ray and electron diffraction techniques, and monitoring of the temporal and spatial evolution of materials using nonlinear time-resolved spectroscopy .In this special issue, we deal with the topic of molecular dynamics—those of small molecules to macromolecules. The issue includes a collection of comprehensive articles and reviews of research findings on a wide range of subjects related to molecular dynamics and the development of the technologies that makes its study possible. The results from this broad range of studies not only provide important insights about the technology but also facilitate a broader understanding of molecular dynamics studies across the various sciences.


Oesophageal squamous cell carcinoma (OSCC) is a prevalent malignancy with high morbidity and mortality as a result of early metastasis and poor prognosis. Metastasis is a multistep process, involving various signalling pathways. Circular RNAs (circRNAs) are a class of covalently closed noncoding RNAs, the aberrant expression of which is reported to be involved in several biological events, including cell transformation, proliferation, migration, invasion, apoptosis and metastasis. Several studies have reported interactions between circRNAs and metastasis‐associated signalling pathways. The abundance, stability and highly specific expression of candidate circRNAs make them potential biomarkers and therapeutic targets in OSCC. In this review article, we comprehensively describe metastasis‐related circRNAs and their interactions with epithelial–mesenchymal transition‐associated molecules. We also describe the molecular mechanisms and clinical relevance of circRNAs in OSCC progression and metastasis. The aberrant expression of circular RNAs (circRNAs) is reported to be involved in several biological events, including proliferation, cell cycle, apoptosis and metastasis. In this review article, we comprehensively describe metastasis‐related circRNAs and their interactions with epithelial–mesenchymal transition‐associated molecules, as well as the molecular mechanisms and clinical relevance of circRNAs in oesophageal squamous cell carcinoma progression and metastasis. CDCcell division cyclecircRNAcircular RNAEMTepithelial–mesenchymal transitionECesophageal carcinomaFERMT1fermitin family member 1Foxoforkhead box O classGLUT1glucose transporter 1GSK3βglycogen synthase kinase‐3βHMGA2high mobility group AT‐hook 2ITCHItchy E3 Ubiquitin Protein LigaseLNMlymph node metastasisLPAR3lysophosphatidic acid receptor 3miRNAmicroRNAMMPmatrix metalloproteinaseNF‐κBnuclear factor‐κBNRIP1nuclear receptor‐interacting protein 1NTRK2neurotrophic receptor tyrosine kinase 2OCoesophageal cancerOSoverall survivalOSCCoesophageal squamous cell carcinomaPI3Kphosphatidylinositol‐4,5‐bisphosphate 3‐kinasemTORmammalian target of rapamycinPPARperoxisome proliferator‐activated receptorPTENphosphatase and tensin homologRBBP7RB‐binding protein 7RBPRNA‐binding proteinTNMtumour node metastasisTTC17tetratricopeptide repeat domain 17XRCC1X‐ray repair cross‐complementing 1ZEB1zinc‐finger E‐box‐binding homeobox 1Oesophageal cancer (OC) is one of the major challenges to human health, representing the eighth most common malignancy worldwide . AlthougTP53, a pivotal gene that affects tumour progression, chromosomal instability and the regulation of metastasis, are corroborated in more than 90% of cases [CDKN2A, which controls cell‐cycle arrest, were reported in more than 60% of cases and were found to be associated with stepwise progression from inflammation to cancerous lesions [Oesophageal squamous cell carcinoma (OSCC) is the major histological type of OC, constituting approximately 90% of the annual OC incidence . The devof cases , 11. Mor lesions , 12. Oth lesions . Further lesions . Meanwhi lesions . For ins lesions . ConsideIn the years after the initial discovery of circRNA via electron microscopy in 1976 , only spIn vitro, cell proliferation and migration increase with circ‐TTC17 expression. Statistical analysis demonstrated that a relatively high circ‐TTC17 expression level is correlated with tumour node metastasis (TNM) stage, lymph node metastasis (LNM) and inferior overall survival (OS) [SMAD7 intron 1 on chr18:46470601–46470865. The down‐regulation of circ‐SMAD7 was verified in OSCC plasma samples and specimens and reported as negatively correlated with TNM stage and LNM. Furthermore, the high expression level of circ‐SMAD7 positively affected the proliferation and migration of OSCC cell lines. Although further investigations are required to determine the underlying mechanism, circ‐SMAD7 exhibits potential diagnostic merit in OSCC [Recent studies have highlighted the prevalence and highly tissue/cell‐type‐specific expression of circRNAs in OSCC through a combination of bioinformatics and molecular biological methods. For example, differential expression profiles of 861 circRNAs in different tumour and nodal stages were detected via microarray analysis and quantitative RT‐PCR, among which hsa_circRNA_100873 was the most significant candidate with a consistent expression pattern. Patients with early tumour stage, late nodal stage and relatively higher metastatic ability presented different hsa_circRNA_100873 levels compared with the healthy group and the group containing advanced tumour stage with early nodal stage. Moreover, miR‐522‐3p, miR‐1236‐3p, miR‐3064‐5p, miR‐6504‐5p and miR‐943 were predicted to bind to hsa_circRNA_100873. This discrepancy in hsa_circRNA_100873 expression confirmed its function in regulating the invasion and metastasis of OSCC . In addival (OS) . Moreoveval (OS) . circ‐SM in OSCC .in vivo and in vitro through promotion of cell‐cycle progression by sponging miR‐339‐5p. Moreover, cell division cycle 25A (CDC25A) was predicted and verified as a downstream targeting molecular signalling protein [Besides tissue/cell‐type‐specific expression, circRNAs also exhibit aberrant expression patterns in exosomes. Exosomes mediate cell‐to‐cell communication and reshape the tumour microenvironment by transporting biomolecules, including circRNAs, thus contributing to tumour growth and metastasis. Hence deciphering the role of exosomal circRNAs would also provide new insights into OSCC. A recent study reported that patients with OSCC with LNM have significantly higher serum exosome has_circ_0026611 levels than patients with OSCC without LNM. Higher expression of has_circ_0026611 is associated with clinical characteristics, such as T and N stages, and postoperative radiotherapy and chemotherapy [ protein . AlthougThe investigation of differentially expressed circRNAs between healthy and cancerous tissues and plasma, and between primary and secondary tumours, sheds light on the promising role of circRNAs as potential OSCC biomarkers and therapeutic targets. In addition, we discuss the clinical relevance of circRNAs and provide a reference point for future studies of OSCC metastatic characterization. Therefore, the aberrant expression of circRNAs has a tumour‐suppressive or oncogenic role that closely correlates with the clinicopathological appearance, prognosis and outcome of OSCC. However, despite the generally accepted correlation between circRNAs and OSCC, the integrated signalling pathways and molecular mechanisms associated with circRNA regulation remain unclear.Several studies have elucidated the versatile functional patterns of circRNAs, from sponging miRNAs to interacting with proteins to regulate host genes, transcription and encoding of proteins. Herein, we review the general biological roles of circRNAs involved in OSCC and summarize those worth investigating in other tumours, with the intent of providing new insights into OSCC research.It has been documented that circRNAs regulate gene expression via multiple approaches, among which the role of miRNA sponges has been reported most frequently in OSCC. Some circRNAs possess miRNA binding sites and serve as competitive endogenous RNAs to hamper direct base pairing between miRNA and untranslated regions of target mRNA, thus regulating miRNA‐inhibitory gene expression and up‐regulating target gene expression.In vitro, the ciRS‐7/miR‐876‐5p/MAGE‐A family axis was also corroborated to facilitate tumour growth and metastasis by increasing proliferation markers (Ki67 and PCNA) and metastatic markers  [in vitro in OSCC by sponging miR‐1301‐3p, further impacting the expression of collagen type I alpha 1 [Herein, we review the relevant circRNA–miRNA axis Table  and selend MMP9) . Notablynd MMP9) . Recent nd MMP9) . Overallnd MMP9) . Further alpha 1 . circRNA alpha 1 , hsa_cir alpha 1 , ciRS‐7  alpha 1  and circ alpha 1 , circLAP alpha 1  and circ alpha 1 , have be alpha 1 , circLAP alpha 1 , circula alpha 1  and circ alpha 1  all achiLuciferase reporter gene experiments and functional rescue validation have contributed to the identification of a growing number of circRNAs shown to function through a sponge mechanism. However, most circRNA mechanistic studies are relatively independent, neglecting the fact that various circRNAs and miRNAs exist in OSCC. Due to the complex interconnected networks of circRNAs and miRNAs, integration analysis between circRNAs may lead to new research directions for OSCC to better characterize the relationships between circRNAs. Nonetheless, the interconnected mechanism of circRNA–miRNA sponging confirms that this network plays a role in oesophageal oncogenic expression by interacting with a single common pathway or multiple pathways. Hence the potential regulatory role of the circRNA–miRNA target gene axis may serve as a promising tool for cancer diagnosis and therapy.Apart from sponging miRNAs, circRNAs can also interact with other cellular components, such as proteins, to activate or inhibit downstream signalling pathways that affect the malignant behaviour of tumours . Circulain vitro and repressed cell growth in vivo via the cir‐ITCH/Wnt network, the relationship between cir‐ITCH and ITCH requires further validation [Because most circRNAs are derived from protein‐coding genes and spliced cotranscriptionally , 46, it lidation . circRNAlidation . HoweverApart from sponging miRNAs, circRNAs can also target other cellular components, such as RNA‐binding proteins (RBPs) . ThroughDespite the earlier‐mentioned noncoding functions, increasing evidence indicates that circRNAs may not be a true class of noncoding RNAs, with at least a small subset of them being translatable , via seqBased on the aforementioned functional patterns and accumulating evidence of the distinct expression profile of circRNAs in OSCC, it can be deduced that circRNAs are implicated in OSCC development and metastasis‐related cellular events. Hence investigation of metastasis‐associated circRNAs may improve molecular and cellular insights into OSCC progression and metastasis. To date, hundreds of circRNAs have been implicated in metastasis‐related processes, including EMT. EMT, the transition from epithelial to mesenchymal states, has been identified as the main promoter and requisite for metastasis. EMT‐related markers can be detected at the invasive front of the tumour and further elaborated by mechanistic studies to drive the dissemination of tumour cells and initiate metastasis , 55, whiHerein, we review the current research on circRNAs related to the metastasis of OSCC while emphasizing the regulatory functions and mechanisms of circRNAs that contribute to the development of malignant phenotypes of OSCC.Metastasis, as the end product of a multistep cell biological process involving the dissemination of tumour cells from the primary site to distant sites, not only is the main cause of cancer‐related deaths but also represents a significant challenge to humans successfully fighting cancer . One hunIn view of the regulatory role of circRNA in metastatic pathways, it may be clinically applicable as a diagnostic and therapeutic tool. Indeed, features of circRNAs, such as abundance, conservation, stability, and tissue‐ and developmental stage‐specific expression, indicate the promising role of circRNA as a biomarker in cancer. Studies have shown that circRNA can be applied to evaluate the effect of diagnosis and treatment and to predict the prognosis and metastasis of many diseases, including OSCC , 80, 81.in vivo and in vitro. In addition, circRNA_100876 enhances migration, invasion and EMT processes, while eliminating cell‐cycle arrest and inhibiting apoptosis. Silencing circRNA_100876 increases the protein level of epithelial–phenotype E‐cadherin and reduces mesenchymal–phenotype N‐cadherin, vimentin and the EMT‐associated transcription factor SNAIL. Clinical data have further shown that high expression of circRNA_100876 strongly correlates with multiple metastatic indicators, such as tumour invasion depth (P = 0.017), LNM (P = 0.027) and vascular invasion (P = 0.036) [hsa_circRNA_100876 has been reported as up‐regulated in OSCC samples and contributes to the acceleration of tumour progression both = 0.036) .in vitro studies have revealed a mechanism underlying the regulatory role of circRNA in metastasis. Higher hsa_circ_0012563 expression was validated in OSCC in both clinical specimens and OSCC cell lines and was shown to be positively correlated with a lower survival rate and metastasis. In vitro, hsa_circ_0012563 positively affects cell proliferation, migration, invasion and apoptosis. Moreover, siRNA‐based experiments demonstrated that X‐ray repair cross‐complementing 1 (XRCC1) is the downstream molecule, and silencing of hsa_circ_0012563 increases the expression of XRCC1, consequently increasing the abundance of the EMT marker E‐cadherin and inhibiting N‐cadherin expression. Meanwhile, repression of XRCC1 also partly neutralizes the inhibitory effect on E‐cadherin protein expression and promotes the effect on N‐cadherin protein expression [In vitro, hsa_circ_0006948 promotes proliferation, migration and invasion capacity and induces EMT. In addition, miR‐490‐3p was found to bind to hsa_circ_0006948. The high mobility group AT‐hook 2 (HMGA2) was reported to facilitate EMT before it was verified to be a direct target of hsa_circ_0006948/miR‐490‐3p and affect the expression of vimentin, N‐cadherin and E‐cadherin. Thus, hsa_circ_0006948 could directly bind miR‐490‐3p to regulate HMGA2, further inducing EMT [La ribonucleoprotein 4 (LARP4) with a length of 1313 nucleotides. The functional relevance of circLARP4 is primarily exhibited in hindering cell proliferation and migration, driving apoptosis and impeding the EMT process by affecting the protein levels of E‐cadherin and N‐cadherin. circLARP4 inhibits miR‐1323 activity, thereby modulating the PTEN‐mediated PI3K/AKT pathway to impact the EMT process and other biological behaviours of OSCC cells [in vitro and promotes tumour growth in vivo. By sponging miR‐140‐3p, circNTRK2 further up‐regulated the expression of nuclear receptor‐interacting protein 1, which is related to various oncogenic pathways, thereby exerting its oncogenic role [Other pression . In addipression . Similarcing EMT . hsa_cirCC cells . circRNAnic role .Interestingly, research has shown that OSCC radioresistant cell lines possess stronger molecular characteristics with lower E‐cadherin expression and higher vimentin and SNAIL expression. The level of hsa_circRNA_100367 is also elevated in this radioresistant cell line and is positively associated with EMT. Meanwhile, silencing hsa_circRNA_100367 enhances the expression of EMT‐related molecules, such as vimentin and SNAIL, through the hsa_circRNA_100367/miR‐217/Wnt3 axis . Similarin vitro. Besides the alteration of EMT markers, circRNA can also directly facilitate the metastasis process in vivo. High expression of circ_0000654 was first detected in OSCC specimens and confirmed to be significantly associated with an increased T stage and local LNM in patients with OSCC. In vitro, circ_0000654 was found to accelerate OSCC progression by adsorbing miR‐149‐5p and indirectly activating the IL‐6/STAT3 signalling pathway. In addition, a lung metastasis model was constructed through vein injection, and hematoxylin and eosin staining revealed that knockdown of circ_0000654 significantly reduces lung metastasis with less OSCC cell infiltration [LPAR3 gene, could promote migration, invasion and metastasis in vivo and in vitro. However, simple alterations in protein levels of transfer‐associated molecules, such as MMP2 and MMP9, in the sh‐circLPAR3 group alone are not sufficient to account for the prometastatic effect of circLPAR3 in vitro, and more direct evidence is required to verify this inference.The studies discussed earlier focus on the relationship between circRNAs and metastasis by detecting the protein levels of EMT markers ltration . HoweverTreatments for OSCC have been developed to cure or delay the disease, including endoscopic therapy, surgical resection, chemotherapy, chemoradiotherapy and neoadjuvant chemoradiotherapy. Notably, the alliance of neoadjuvant chemoradiotherapy and surgery benefits patients with OSCC with curable potential ; neverthBecause circRNAs are expressed differently in drug‐resistant tissues and cell lines, it would be reasonable to speculate on their role in OSCC tumour resistance. In fact, a study reported the down‐regulation of circPSMC3 in OSCC tissues and gefitinib‐resistant cell lines. Vector overexpression of circPSMC3 significantly increased cellular sensitivity to gefitinib, with a lower survival rate of OSCC cells and corresponding gefitinib‐resistant cells, and lower half‐maximal inhibitory concentration value of gefitinib than the control group. circPSMC3 also accelerated apoptosis, with high cleaved caspase‐3 protein expression. The underlying mechanism of circPSMC3 was also explored, showing that circPSMC3 down‐regulates PTEN by acting as an miR‐10a‐5p sponge to suppress the progression of OSCC, meaning that the circPSMC3/miR‐10a‐5p/PTEN axis may be a reliable treatment strategy in the future . ciRS‐7 PRKCI gene, with 1484 bp in length. Flow cytometry and western blotting revealed that inhibited circPRKCI expression augments cell apoptosis with an increase in radiation dose, while colony formation assay results revealed that PARP9 enhancement overturns the circPRKC inhibition‐mediating effect on cell radiosensitivity. circPRKCI affects the anti‐radioresistant phenotypes in vitro by sequestering miR‐186‐5p and further activating PARP9 [circRNAs also have the potential to modulate the radioresistance of OSCC. circPRKCI is formed by cyclization of exons 15 and 16 of the ng PARP9 . Collectin vitro and in vivo.Elevated levels of circVRK1 attenuate cell proliferation, migration and EMT and reinforce cell sensitivity to radiotherapy. Overexpression of miR‐624‐3p and inhibition of PTEN reverse the radiosensitivity enhancement induced by circVRK1. Hence circVRK1 suppresses OSCC radioresistance via the miR‐624‐3p/PETN axis . A similOne of the common features of tumour cells is that they proliferate indefinitely. To sustain rapid growth and progression, altered metabolism is essential to provide more nutrition to the tumours . Emerginin vitro by down‐regulating pyruvate kinase. Loss‐of‐function experiments were conducted to further explore the relationship between hsa_circ_0006168, miR‐384 and RB‐binding protein 7 (RBBP7), and hsa_circ_0006168 was found to facilitate cell growth, migration, invasion and glycolysis by regulating miR‐384/RBBP7. In addition, the S6K/S6 pathway becomes activated by up‐regulating RBBP7 induced by hsa_circ_0006168 to modulate OC progression [Similarly, knockdown of hsa_circ_0006168 inhibits glucose consumption and lactate production gression . Lendinggression .Manifold literature has deepened our understanding of circRNAs and their relevance to various physiological and pathological processes. The intricate mechanisms underlying multitargeting circRNAs are associated with the initiation, metabolism, metastasis and other pathological OSCC processes. However, some obstacles still need to be overcome for future studies and application.From the detection aspect, the earlier‐mentioned studies have been based on second generation sequencing and focused on exon‐derived circRNAs. However, many issues are associated with short‐read RNA sequencing, in particular the inability to confirm the full‐length sequence and accurately quantify the circRNA. Meanwhile, novel detection methods, such as nanopore technology, catalogue full‐length circRNA with higher accuracy for circRNAs with low expression and mitochondrial circRNAs . Long‐rein situ hybridization to provide intracellular localization data in OSCC, whereas none of the earlier‐discussed studies provided in situ data to assess potential intratumour heterogeneity related to the expression of circRNAs. A recent study found that ciRS‐7 is abundantly expressed in tumour stromal cells within the tumour microenvironment and is completely absent in colon cancer cells [in situ hybridization could be applied to clarify the cellular expression patterns of circRNA more adequately in OSCC and exclude the influence of matrix on the results.From the validation aspect, it is important to clarify circRNA localization because its function is highly dependent on its space specificity. Select studies have leveraged fluorescence er cells . The impIn addition, predominant mechanistic studies of circRNAs in OSCC focus on their role as miRNA sponges in modulating downstream molecules, because most circRNAs are localized in the cytoplasm. However, one circRNA possesses multiple miRNA binding sites. Thus, focusing on one miRNA might neglect that some miRNAs have opposite downstream effects. It is thus important to determine whether circRNAs act through several miRNAs simultaneously, or through one primary miRNA when in conditions with very low levels of additional miRNA binding sites. Moreover, other general function patterns, such as the interaction between proteins and DNA, have rarely been investigated in OSCC and deserve more attention .Because circRNAs can alter the characteristics of tumours, several factors can lead to altered levels of circRNA. It was reported that ZEB1, as a key transcription factor, could bind the promoter region of hsa_circ_0001178 and increase its expression in colorectal cancer ; similarFurthermore, the development of OSCC is a complex process with multiple stages and successive activation of various cytokines and mechanisms. Investigations focusing on the aberrant expression of primary and metastatic OSCC tissue might provide more holistic evidence of the role of circRNAs in different periods of metastasis. In addition, although circRNAs are expected to assist in clinical diagnosis, prognosis and treatment according to current data, a single study may not be comprehensive and sufficiently in‐depth to verify whether circRNA could serve as a therapeutic target and as an upstream regulator. Further evidence, especially via clinical trials, is required.In summary, OSCC is a multistage and multifactorial disease with a mechanism that has not been fully elucidated. The lack of early typical clinical symptoms leads to late diagnosis and poor prognosis in patients with advanced OSCC, particularly in patients with distant metastasis. circRNAs as novel noncoding RNAs have been shown to mediate various biological events, including cell transformation, proliferation, migration, invasion, apoptosis and metastasis in OSCC. The aberrant expression of circRNAs was substantiated in OSCC and correlated with multiple clinicopathological features, including metastasis. In addition, various circRNAs play crucial roles in the development, metastasis and resistance to chemotherapy and radiotherapy in OSCC. In this regard, circRNAs exhibit promising value in clinical diagnostic, prognostic and therapeutic applications. Identifying an increasing number of circRNAs that affect OSCC is important, while exploring deeper triggers of alternative splicing that generate aberrant circRNAs might provide new insights into OSCC. Therefore, investigations on the regulation of OSCC by circRNAs are still in their infancy, and future mechanistic studies together with clinical research appear to be a future endeavour.The authors declare no conflict of interest.XF and SMS conceived and designed the project. XF acquired, analysed and interpreted the data. XF wrote the manuscript. XF, SMS and L‐HR made manuscript revisions. RS organized the research project, reviewed the manuscript and arranged necessary resources from the grants. All authors approved the final version of the manuscript.


Hepatocellular carcinoma (HCC) is one of the most common malignancies globally. Despite aggressive and multimodal treatment regimens, the overall survival of HCC patients remains poor.Circular RNAs (circRNAs) are noncoding RNAs (ncRNAs) with covalently closed structures and tissue- or organ-specific expression patterns in eukaryotes. They are highly stable and have important biological functions, including acting as microRNA sponges, protein scaffolds, transcription regulators, translation templates and interacting with RNA-binding protein. Recent advances have indicated that circRNAs present abnormal expression in HCC tissues and that their dysregulation contributes to HCC initiation and progression. Furthermore, researchers have revealed that some circRNAs might serve as diagnostic biomarkers or drug targets in clinical settings. In this review, we systematically evaluate the characteristics, biogenesis, mechanisms and functions of circRNAs in HCC and further discuss the current shortcomings and potential directions of prospective studies on liver cancer-related circRNAs.CircRNAs are a novel class of ncRNAs that play a significant role in HCC initiation and progression, but their internal mechanisms and clinical applications need further investigation. HCC was the sixth most common malignancy and the third leading cause of cancer-related death globally in 2020, accounts for 75–85% of primary liver cancer cases and has been ranked second in terms of mortality in men . ChronicEmerging evidence indicates that noncoding RNAs (ncRNAs) are involved in many cellular biological and physiological processes and even pathological disease processes . Among tDrosophila, mice and humans, and is also found in the hippocampus , 2316, 2Adenosine deaminase-acting on RNA (ADAR1) was found to be an RNA-editing enzyme and to play a suppressive role in circRNA formation. This function was correlated with its adenosine-to-inosine (A-to-I) editing process, which frequently occurs near the location of reverse complementary matches (RCMs), a conserved feature of circRNA biogenesis . InteresThe emerging roles of circRNAs emphasize the importance of sequencing these circular transcripts. In the past decade, RNA sequencing (RNA-seq) technology has developed rapidly and has become an indispensable tool for analyzing the differential expression of genes and differential splicing events of mRNA at the transcriptome level .Next-generation sequencing  is currently the main way to sequence circRNAs. In next-generation sequencing, the coordination of gene cluster replication decreases with increasing read length, resulting in a decline in the quality of base sequencing. Therefore, next-generation sequencing is a high-throughput and short-read technique. Since most circRNAs are derived from exons, this alignment-based method is unable to distinguish circular reads from the overlapping regions of corresponding linear transcripts. The biggest drawback is that next-generation sequencing’s relatively short-read capacity considerably limits its detection ability in structural variant detection and genome assembly .Emerging long-read sequencing technologies have become a powerful participant in genomics. Compared with short-read approaches, long-read technologies can generate continuous ultralong sequences directly from native DNA. However, oligo(dT) primers are not suitable to use for circRNA sequencing because of their lack of poly(A) tails, and as a result, long-read sequencing technology cannot be widely applied in circRNA studies .Strikingly, a recent report presented a novel algorithm (CIRI-long) for detailed analysis of full-length circRNAs using nanopore sequencing technology. This technology utilized full-length circular reverse transcription (which was performed with random primers and SMARTer reverse transcriptase) to amplify circRNAs by producing long complementary DNA (cDNA) molecules containing multiple copies of full-length circRNA sequences. Then, a nanopore approach was used to directly sequence full-length circRNA sequences, and a specific algorithm was applied to quantify circRNA expression and recognize full-length mutant transcript sequences. The data showed that compared with Illumina RNA-seq , nanopore sequencing can enhance the detection efficiency for circular reads (with a 20-fold increase) and provide a fivefold increase in identifying alternative circularization events, indicating its higher sensitivity for circular isoform identification. Furthermore, nanopore sequencing can recognize circRNAs at a relatively low abundance and capture nonclassical circRNAs more sensitively, such as a new type of intronic self-ligated circRNA .Although many studies suggest that some circRNAs have the potential to be novel diagnostic or prognostic markers, the key problem is how to detect circRNAs in body fluids more efficiently. Very recently, our group reported a fully integrated electrochemical point-of-care testing (POCT) platform based on Au nanoflower (AuNF)/peptide nucleic acid (PNA)-modified carbon-fiber microelectrodes (CFMEs) . The plaThese new technologies provide more possibilities for the study of circRNAs. Undoubtedly, an increasing number of new technologies will emerge in the future, opening uncharted territory in circRNA research.With the development of RNA sequencing technology, the whole genome transcriptional map of circRNAs in HCC has been reported in several studies , 36, 37.To identify the circRNAs involved in HCC tumorigenesis, a recent study detected differential circRNA expression between HCC tissues and adjacent noncancerous liver (ANL) tissues. In this study, 13,686 distinct circRNAs were identified in total, excluding those with very low abundance, and the expression levels of the identified circRNAs were further analyzed. The results showed that 236 circRNAs were differentially expressed in HCC compared with matched ANL tissues, of which 108 were upregulated and 128 were downregulated [Another study identified 220 circRNAs that were differentially expressed between patients who experienced postsurgical pulmonary metastasis and those who did not. Among the identified circRNAs, 144 were upregulated and 76 were downregulated .HCC is characterized by a clear gender disparity, and AR is thought to be critical for this bias . To veriThese sequencing and bioinformatics analysis statistics demonstrate the possible involvement of circRNAs in HCC tumorigenesis and development. Many studies have reported that circRNAs play an important role in HCC, although their specific functions and internal mechanisms are still under investigation , 39–60 .Table 1OWith further developments in the study of circRNAs, the mechanisms of circRNAs have been gradually revealed. Through pre-mRNA back-splicing, circRNAs gain high stability to exert their important regulatory functions. It is universally acknowledged that circRNAs play a vital role as competing endogenous RNAs (ceRNAs) to regulate downstream signaling pathways –63. MeanThe primary location of circRNAs is the cytoplasm, which is a precondition for them to exert posttranscriptional regulation functions . NumerouOngoing investigations have reported that some circRNAs can function as miRNA sponges in HCC. CircSMARCA5 is a circRNA derived from exons 15 and 16 of the SMARCA5 gene and is downregulated in HCC . In HCC A large amount of evidence has confirmed the existence of the circRNA-miRNA pathway. However, it can be seen from the examples above that circRNAs usually sponge more than one miRNA to exert their function , 37, 52.RBP is a general term for a group of proteins possessing RNA recognition motifs that function by binding RNA to regulate its metabolic processes . RBPs plSeveral groups have reported that circRNAs may combine with RBPs to regulate gene expression , 70, 71.As mentioned above, circRNAs can physically combine with specific RBPs to facilitate or inhibit their function. Interestingly, one study revealed that a specific circRNA can alter the subcellular localization of its target RBP, providing a novel pattern for RBPs to perform specific functions under unusual cellular localization . HoweverEmerging evidence has demonstrated that some circRNAs may act as dynamic scaffolding molecules to regulate protein functions. Ongoing studies have shown that circRNAs can bind corresponding proteins in specific subcellular locations and facilitate the colocalization of relevant proteins, which may modulate protein–protein interactions .An outstanding study revealed that circACC1 combines with the regulatory β and γ subunits of AMPK to form a ternary complex and thereby facilitates the stabilization and activity of the AMPK holoenzyme . In HCC,Recently, our laboratory demonstrated that circSORE could bind the master oncogenic protein YBX1 to prevent YBX1 nuclear interaction with the E3 ubiquitin ligase PRP19 and inhibit PRP19-mediated YBX1 degradation, thereby inducing sorafenib resistance . In contAll these circRNAs serve as protein scaffolds. However, they exhibit different biological functions, suggesting that circRNAs can play different roles as protein scaffolds. Recent years have witnessed excellent progress in understanding of this novel mechanism in HCC, to which our group has made a contribution . Given tCircRNAs are regarded as ncRNAs due to the lack of essential components for cap-dependent translation, including the 5′ cap and the poly(A) tail. However, emerging evidence indicates that circRNAs may have the potential to encode proteins in a cap-independent manner . circRNACircZNF609 is one of the few endogenous circRNAs that have been reported to act as translation templates. Structurally, circZNF609 contains a 753-nt open reading frame (ORF) spanning from the start codon of the host gene to a stop codon created 3 nt after the splice junction . MechaniRecently, a novel circRNA, circβ-catenin, was identified as a protein-encoding circRNA whose translation can promote HCC cell growth through activation of the Wnt pathway . The stuThese results broaden our understanding of the human proteome. Currently, there is only one report on the translational function of circRNAs in HCC, indicating that the internal mechanisms of encoding circRNAs lack in-depth study. Our group suggests using a bioinformatics database to predict whether ORFs, IRESs or m6A modification sites exist in candidates. CircRNAs possessing these basic conditions may have the potential to encode proteins, which requires subsequent experimental analysis for further verification. The abundance and subcellular localization of circRNAs may lead to inferior translation efficiency compared with their cognate mRNAs. Hence, it remains controversial whether circRNA-derived proteins could achieve observable effects on the development of HCC .Emerging evidence has indicated that circRNAs play vital roles in HCC tumorigenesis and progression and are involved in cell proliferation, tumor metastasis, immune escape and drug resistance Fig.  36, 37,, 51–53. Investigations have reported that several circRNAs are expressed at an extremely high level in the early stages of HCC, suggesting that these specific circRNAs may promote tumorigenesis. A previous study demonstrated that circCDYL can regulate tumorigenesis by promoting the stem-like properties of HCC cells and showed that circCDYL-transduced HCC cells present a higher expression level of stem cell-associated genes with a markedly increased percentage of EpCAM + cells. Moreover, overexpression of circCDYL increased the expression of Ki-67 and alpha-fetoprotein (AFP), indicating that circCDYL promotes the malignant proliferation of HCC cells . AnotherOn the other hand, emerging evidence indicates that circRNAs can regulate tumor cell apoptosis through specific mechanisms . ApoptosHence, the important role played by circRNAs in cell proliferation and tumorigenesis is becoming even clearer, providing insights for a deeper understanding of HCC pathogenesis.Epithelial-to-mesenchymal transition (EMT) refers to a biological process by which epithelial cells are transformed into cells with a mesenchymal phenotype, and EMT plays a vital role in the ability of malignant tumor cells derived from epithelial cells to obtain characteristics that enable invasion and metastasis . MechaniRecent advances have shown that some EMT-related circRNAs can affect the EMT process in HCC. For instance, circARFGEF2 was found to be overexpressed in portal vein tumor thrombus (PVTT) and HCC tissues and to promote the EMT process via a miR-143-3p/FOSL2 axis and PCBP1/CD44v6 axis , leading to a remarkable acceleration in intrahepatic and pulmonary metastasis . MoreoveConsequently, circRNAs are considered key factors with well-characterized functions in regulating the EMT process, cancer cell invasion and metastasis in HCC.Immune dysfunction plays an important role in HCC development. Experimental evidence has shown that the circRNA expression profile changes during viral infection, which can regulate the function of the immune system . SubsequFor instance, a recent study found that HCC cells secrete circUHRF1 through exosomes and that circUHRF1 can inhibit NK cells from secreting IFN-γ and TNF-α by upregulating the expression of TIM-3 in NK cells. This study showed that the level of plasma exosomal circUHRF1 was negatively correlated with the infiltration level of NK cells in tumors . ResearcThese examples confirm that circRNAs can affect HCC development and prognosis by regulating the immune system of HCC patients, which prompted us to wonder whether circRNAs will become an ideal immunotherapy target in the future.Statistics indicate that most HCC patients are diagnosed at an advanced stage. At present, the main drug treatment for unresectable HCC includes chemotherapy  and targeted therapy , but conventional systemic chemotherapy lacks survival benefits.Currently, multikinase inhibitors, monoclonal antibodies and immune checkpoint inhibitors are the main targeted molecular therapies approved for treatment of advanced-stage HCC; however, the effects of these treatments are not satisfactory , 94. TheIncreasing evidence has indicated that ncRNAs are critical for the development of sorafenib resistance in HCC , 98. OurMeanwhile, ongoing studies have shown that circRNAs play a vital role in the development of drug resistance . Our groIn summary, we must further clarify the mechanism underlying drug resistance and explore how circRNAs function in resistance to molecular targeted drugs. Identifying a specific circRNA that could be used as a new therapeutic target to avoid drug resistance would be an important step forward.“Secondary prevention”  can improve the prognosis of HCC patients. The poor prognosis of most patients with HCC is due to the diagnosis of advanced HCC, and the reaction of advanced HCC to all treatment regimens is not satisfactory . At presEmerging evidence has demonstrated that circRNAs hold great potential as a novel attractive class of diagnostic biomarkers for HCC due to their resistance to RNase R digestion and their stability during circulation. Recent studies have indicated that circRNAs can not only be detected in tumor tissues but also in exosomes, blood, saliva and urine –107. TheCircRNAs have gradually been recognized as possible prognostic biomarkers. For instance, circSLC3A2, which is elevated in HCC tissues, plays an oncogenic role by sponging miR-490-3p to modulate PPM1F expression and could serve as a prognostic biomarker due to its positive correlation with poor survival in patients with HCC . In contHowever, the clinical feasibility of using circRNAs still needs to be verified in a large cohort of HCC patient samples. Meanwhile, inventing efficient circRNA detection methods, such as the POCT platform, is also of great significance.Recently, an increasing number of studies have focused on the significance of circRNAs in HCC and their correlation with HCC tumorigenesis and development, and some authors have proposed that circRNAs have the potential to serve as therapeutic targets in HCC Table . For exaCurrently, the main treatment for high-grade (middle and advanced) HCC is chemotherapy and molecular targeted therapy; however, the development of resistance to HCC treatments is an unavoidable problem, including resistance to traditional chemotherapy and even first-line molecular targeted therapies, such as sorafenib . As prevRecently, circRNA-based therapy has attracted increasing attention. Compared with other drugs, such as monoclonal antibodies or small molecule inhibitors, circRNA-based therapy has several advantages. First, circRNA drugs have a long half-life , while traditional therapeutic drugs are unstable once they enter the circulatory system; therefore, with circRNA drugs, the patient dosing frequency can be decreased compared with that for antibodies or small molecules . Second,On the other hand, proteolysis-targeting chimeras (PROTACs) are a burgeoning and promising field and can modulate protein concentrations at a posttranslational level by coopting the ubiquitin–proteasome system . As prevGenerally, whether circRNAs have clinical value merits further study and discussion. Although circRNAs have the potential to serve as diagnostic markers, prognostic markers, therapeutic targets or novel drugs, these roles are based only on theoretical assumptions and predictions; thus, more substantive studies are needed to verify these conjectures Table .Substantial progress has been achieved in specific circRNAs. These findings not only reveal a previously unexpected complexity of cellular regulatory mechanisms but have also identified many circRNAs with important physiological and clinical significance. Although the progress is exciting, it has led to many more questions than answers, which require future investigation.6A modification of circRNAs has been proposed to facilitate the cytoplasmic export of circRNAs [First, most studies have focused on a specific circRNA and its downstream mechanisms, while the upstream mechanisms remain largely unknown. For example, how exactly are circRNAs formed? Under what circumstances do pre-mRNAs form circRNAs rather than mRNAs? Are there any factors that are specifically required for back-splicing but not for splicing? How are circRNAs exported from the nucleus to the cytoplasm? These issues are rarely mentioned in most current studies. However, research groups have tried to answer these questions, and mcircRNAs . HoweverSecond, current biological tools and methods applied in circRNA-related studies still have some obvious shortcomings. Due to the high degree of sequence similarity between circRNAs and their cognate mRNAs, off-target effects of circRNA knockdown are difficult to avoid or completely eliminate. Similarly, the same dilemma has been observed in circRNA FISH and circRNA pull-down experiments. To successfully overexpress circRNAs, special sequence elements that promote circularization are usually inserted at both ends of the linear circRNA sequence. Inevitably, a large number of “linear circRNAs” will be produced due to the low efficiency of circularization. Whether this by-product will affect experiments remains to be further studied. Furthermore, the CRISPR-Cas9 system may be restricted in knockout studies of circRNAs because deletion of the whole genome sequence could disturb the expression of cognate mRNAs and impact specific biological functions. These challenges highlight the need for more advanced technologies for circRNA interference, detection and other functional studies. Very recently, Chen and colleagues reported that the CRISPR-Cas13 technique combined with guide RNA targeting sequences spanning junction sites featured in circRNAs can be applied to knock down circRNAs. Furthermore, compared with shRNA-mediated knockdown, RfxCas13d–BSJ-gRNA-mediated circRNA knockdown showed higher efficiency and specificity and lower rates of off-target effects on cognate mRNAs .Finally, the number of circRNAs that truly have cellular and physiological functions remains to be explored. In particular, our research has shown that the expression of circRNAs in HCC is widely reduced, suggesting that low expression levels will lead to nonfunctionalization . Given tIn recent years, an increasing number of studies have begun to focus on circRNAs; thus, circRNA-related research is progressing at a steady and fast pace. In the following section, we propose several challenging and innovative future research directions in the field of circRNAs.First, the current research remains at the cellular and animal levels, and how to facilitate translation toward clinical application will become a hot topic in the future. Because circRNAs are stable and have been detected in many types of body fluids, further studies are warranted to confirm their potential as biomarkers, therapeutic targets or novel drugs.Second, whether a circRNA with relatively low abundance can achieve measurable effects remains controversial. Future studies should focus on certain types of circRNAs with the same properties instead of a specific circRNA. For example, a striking study reported that endogenous circRNAs with 16–26 bp that form imperfect RNA duplexes can act as inhibitors of double-stranded RNA (dsRNA)-activated protein kinase (PKR) related to innate immunity .Generally, HCC is a multistep, multistage and multifactor comprehensive hereditary malignancy, and the specific pathogenic mechanisms remain unclear. Despite the availability of a comprehensive treatment regimen, including surgery, chemotherapy, targeted therapy and immune therapy, the overall survival of patients with advanced HCC is still unsatisfactory. Notably, circRNAs are a novel class of ncRNAs that play a significant role in HCC initiation and progression. As described in this review, circRNAs are aberrantly expressed in HCC and associated with the clinicopathological features and prognosis of HCC patients. Unexpectedly, there is a high degree of heterogeneity among HCC-related circRNAs, and individual circRNAs may have prooncogenic or antioncogenic roles and function through various pathophysiological mechanisms. Given the stability and polyfunctionality of circRNAs, circRNAs might serve as diagnostic indicators, prognosis predictors, therapeutic targets or novel nucleic acid drugs for precise treatment of HCC, thus improving the quality of life and extending the survival of HCC patients.