Exosomal LINC00161 promotes angiogenesis and metastasis via regulating miR-590-3p/ROCK axis in hepatocellular carcinoma
Authors: Wu-Kui Huang
Heng Zhang
Wen-Jia Guo
Yi Hao
Qing Tong
Lin Xu
Qiao Zhang
Li-Na You
Qin-Wen Tai
Journal: Cancer Gene Therapy (2021)
Discipline: CLINICAL MEDICINE
Keywords: Hepatocellular carcinoma; HCC; exosomal LINC00161; biomarker; therapeutic treatment
Affiliations: Department of Traditional Chinese Medicine, the Fifth Affiliated Hospital of Xinjiang Medical University, Urumqi, 830011, PR China., Traditional Chinese Medicine Gynecology, Maternal and Child Health Hospital of Xinjiang Uyghur Autonomous Region, Urumqi, 830011, PR China., Department of General Surgery, ShenZhen Hospital of Southern Medical University, Shenzhen, 518000, PR China., Department of Hepatobiliary& Pancreatic Surgery, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital & Shenzhen Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Shenzhen, 518000, PR China., Department of Ultrasound Medicine, ShenZhen Hospital of Southern Medical University, Shenzhen, 518000, PR China., Cancer Institute, Cancer Hospital Affiliated to Xinjiang Medical University, Urumqi, 830011, PR China., Department of Pulmonary Medicine, Cancer Hospital Affiliated to Xinjiang Medical University, Urumqi, 830011, PR China., Department of Hepatobiliary and Pancreatic Surgery, Cancer Hospital Affiliated to Xinjiang Medical University, Urumqi, 830011, PR China., Department of General Surgery, ShenZhen Hospital of Southern Medical University, Shenzhen, 518000, PR China. ., InterventionaL Diagnosis and Treatment Department, Affiliated Tumor Hospital of Xinjiang Medical University, Urumqi, 830011, PR China. .

Abstract:  Hepatocellular carcinoma (HCC) is a lethal malignancy with few effective options for therapeutic treatment in its advanced stages. While exosomal LINC00161 has been identified as a potential biomarker for HCC, its regulatory function and clinical values remain largely unknown. LINC00161 expressions in serum-derived exosomes from HCC patients and HCC cells were determined by qRT-PCR. The ability of proliferation, migration, and angiogenesis in HUVECs was assessed by MTT, Transwell, and tube formation. Luciferase reporter assay and AGO2-RIP assay were conducted to explore the interactions among LINC00161, miR-590-3p, and ROCK2. The level of ROCK signal-related proteins was examined by Western blotting and immunohistochemistry (IHC) assay. Subcutaneous tumor growth was observed in nude mice, in which in vivo metastasis was observed following tail vein injection of HCC cells. High levels of LINC00161 were detected in both serum-derived exosomes from HCC patients and the supernatants of HCC cell lines and were significantly associated with poor survival. Functional study demonstrated that exosomal LINC00161 derived from HCC-cells were significantly associated with enhanced proliferation, migration, and angiogenesis in HUVECs in vitro, all of which were effectively inhibited when LINC00161 was sliced with shRNA in HCC-cells. In vivo experiment showed that LINC00161 loss inhibited tumorigenesis and metastasis of HCC. Mechanistic study revealed that exosome-carried LINC00161 directly targeted miR-590-3p and induced its downstream target ROCK2, finally activating growth/metastasis-related signals in HCC. Exosome-carried LINC00161 promotes HCC tumorigenesis through inhibiting miR-590-3p to activate the ROCK2 signaling pathway, suggesting that LINC00161 may be used as potential targets to improve HCC treatment efficiency. 

Retracted: No

PubPeer comments:
 The basis for this study is questionable: CD63 immunoblots should have a smeared appearance, which is not seen in Figure 1. The EM data claiming to be exosomes could be anything as long as there is no immunostaining of exosomal markers. In addition, the exosome ultrastructure is not very convincing.  , 
 The basis of this study is questionable for several even more important reasons, not that Indigofera Tanganyikensis s points are to be ignored. First, the methodologies are not specific to exosomes (extracellular vesicles from the endosomal compartment) or even to extracellular vesicles (EVs) in general. Polyethylene glycol (PEG) precipitation was used to concentrate material from patient blood samples and from cell culture. This method concentrates a very wide range of extracellular particles and yields mostly non-EV particles. There is no way to draw conclusions about EVs, much less the exosome subtype, when such methods are used. Overexpressing an RNA or silencing an RNA can lead to many unforeseen effects beyond altering levels of an RNA in secreted EVs. Since nothing else was considered or measured, the experiments unfortunately have little basis and the results are largely uninterpretable. Second, what about the lncRNA? Why was just one lncRNA chosen for investigation?  A previous study demonstrated that exosomal [sic] LINC00161 was up-regulated in HCC cells . This is incorrect. The cited publication (Sun, et al, J Cancer, 2018, PMID: 30087703) was not a study of HCC cells or their exosomes or other EVs. Sun et al instead measured levels of eight lncRNAs in PEG-precipitated material from HCC patient serum. In that study, only one EM image was shown, depicting what appear to be lipoprotein particles, not EVs or exosomes. A single sample was run at two concentrations for Western blot of CD63, which appears as a single band (not a smear). Finally, a  nanosight plot is shown for one sample (HCC? control?), with particles ranging from 5 nm to 6 microns in diameter. This range is implausible for nanoparticle tracking analysis (NTA). More importantly, since we already know that PEG precipitation does not yield exosomes or EVs,  There was no statistically significant correlation between the LINC00161 and clinical pathological information such as age, gender, chronic hepatitis, cirrhosis, tumor size and tumor number (P 0.05). The difference between HCC and control was  significant only at an uncorrected p of 0.011. The RT-qPCR methodology in the cited paper was also vague, since an  internal (GAPDH) and  external (cel-miR-39) control were mentioned for the ddCt correction, but the exact spike-in strategy and measurement were not disclosed, nor was it stated why GAPDH was chosen for a general serum reference gene. In other words, the study that was the cited basis for the selection of LINC00161 was itself ambiguous at best. Why did Sun, et al, in the cited basis of the current study, select LINC00161 as one of the 8 lncRNAs in their investigation that was published in 2018? Each of their eight lncRNAs was chosen based on a single previous publication. For LINC00161, it was a publication in Oncotarget in 2017: Xu, et al, PMID: 28915581. Xu et al focused exclusively on LINC000161 expression in  HCC tissues and adjacent non-tumor liver tissues. They found a largely overlapping but nevertheless reportedly significant expression difference between the adjacent tissues. However, the magnitude of the difference is not reported (Figure 1A does not indicate what the y-axis represents), and there is no investigation of compositional cellular differences between the HCC and adjacent tissues. Not so strong. Why did Xu, et al, in turn decide to focus exclusively on LINC00161 in their study? They cited a single publication by Wang, et al, Cancer Letters, November 2016, PMID: 27609068. Wang et al were said to have found that LINC00161 played  an important role in osteosarcoma. But the devil is in the details, and this vague language concealed them. Aside from the unclear bridge between osteosarcoma and HCC, in fact, Wang et al did not report that LINC00161 was a  bad guy in tumor progression. They found that it sensitized cells to chemotherapy via the  miR-645-IFIT2 axis. But the Wang et al study was also not particularly strong. The paper examined two cell lines only. The microarray analysis that identified LINC00161 was done with just n=1 per treatment time point and with only one of the two cell lines used in the study. RT-qPCR verification of the results is stated and shown, but the methods do not reveal the details for the LINC00161 verification. To summarize: Wang et al PMID: 27609068 publish in late 2016 some rather weak evidence that LINC00161 acts on balance against osteosarcoma in one or two cell lines. By early 2017, Xu et al PMID: 28915581 have submitted a study, accepted one month later, that LINC00161 appears to be ever so slightly higher in HCC tissue compared with adjacent normal tissue, but with vague methodology. Sun et al, PMID: 30087703, publish findings in 2018 that precipitated RNA from HCC patient serum has levels of LINC00161 that correlate weakly with a few HCC patient characteristics, but with major experimental design caveats. You et al, PMID: 33414518 (this study), based on the fragmented and weak evidence above, focus exclusively on LINC00161 in an extensive, expensive study with animal experimentation and possibly flawed experimental design. Importantly, no attention is paid to the study that started this  chain of evidence, which suggests an anti-tumour rather than pro-tumour effect of the lncRNA. In setting up a new  miR-590-3p ROCK axis, should it not be an important point to also revisit the previously proposed  miR-645-IFIT2 axis since it might act contrariwise? Third and finally, where does miR-590-3p come from? After 20 years of miRNA research, we are at the point where any randomly picked miRNA can likely be found associated with one cancer or another in a few publications. The support for miR-590-3p is probably stronger than that for LINC00161 in this paper, but it is still weak as the basis for a presumably multi-year study. ,