<p>lncRNA DGCR5 Up-Regulates TGF-β1, Increases Cancer Cell Stemness and Predicts Survival of Prostate Cancer Patients</p>
Authors: Shuyun He
Zhirui Guo
Quan Liang
Huiling Zhou
Yanping Luo
Zhe Lin
Bin Li
Journal: Cancer Management and Research (2019)
Discipline: CLINICAL MEDICINE
Keywords: TGF-β1; lncRNA DGCR5; prostate cancer; stemness.
Affiliations: Department of Urology, The First Peoples' Hospital of Foshan, Foshan City, Guangdong Province 528000, People's Republic of China., Department of Radiology, The First Peoples' Hospital of Foshan, Foshan City, Guangdong Province 528000, People's Republic of China., Department of Infectious Disease, The First Peoples' Hospital of Foshan, Foshan City, Guangdong Province 528000, People's Republic of China., Department of Anesthesiology Surgery, The First Peoples' Hospital of Foshan, Foshan City, Guangdong Province 528000, People's Republic of China.

Abstract:  Background: Long non-coding RNA (lncRNA) DiGeorge syndrome critical region gene 5 (DGCR5) plays different roles in different types of human cancer, but its role in prostate cancer (PC) has not been reported. Methods: DGCR5 and TGF-β1 expression in paired tumor and adjacent healthy tissues from 64 PC patients was analyzed by performing RT-qPCR. A 5-year follow-up study was performed to analyze the prognostic value of DGCR5 for PC. The interaction between DGCR5 and TGF-β1 was analyzed by overexpression experiments. Cell stemness was analyzed by cell stemness assay. Results: In our study, we found that DGCR5 was down-regulated in tumor tissues than in adjacent healthy tissues of PC patients, but TGF-β1 was up-regulated in the tumor tissues. DGCR5 expression was not affected by clinical stages, but low DGCR5 level in the tumor was correlated with poor survival. DGCR5 and TGF-β1 were inversely correlated in tumor tissues but not in adjacent healthy tissues. DGCR5 over-expression resulted in down-regulation of TGF-β1, while TGF-β1 treatment did not significantly affect DGCR5 expression. DGCR5 over-expression led to decreased stemness of PC cells, but TGF-β1 treatment played a reverse role and attenuated the effects of DGCR5 over-expression. DGCR5 may decrease the stemness of PC cells by down-regulating TGF-β1. 

Retracted: No

PubPeer comments:
 I have several observations and questions about this paper. Most importantly, I trust that the authors will bring the paper into compliance with the publisher s data availability guidelines by depositing the RNA sequencing data with SRA and providing the associated analysis methods. Use of the term  exosome is not supported. Previous assumptions that exosomes (from the endosomal system) are always smaller than microvesicles (from the plasma membrane), can be separated by ultracentrifugation, or contain distinct cargo were often incorrect, and it is now known that the plasma membrane ectosomes can outnumber endosomal origin exosomes in certain cancer cell cultures. To show that isolation methods have yielded exosomes, it is important to show enrichment of true exosomal markers and depletion of true ectosomal markers. I am not sure that such markers have been established for the cells used in this study, and these steps are not taken in this study. Overall, there are few markers that are truly specific for one type or the other in any cell type. Specifically, CD9 and CD63 are not exclusive to exosomes, and showing that these proteins are detected does not tell the reader anything about the purity of the preparation. It is also unusual to see CD63 presenting as a distinct band. CD63 is usually a smear on the gel. The authors are encouraged to provide their full gel pics or scans, including the molecular weight markers, for confirmation, as many journals require. The cells in this study were grown in cell culture medium containing fetal bovine serum (FBS). FBS is a rich source of EVs including exosomes, and in many cell cultures, FBS EVs are more abundant than EVs released from the cells. Additionally, FBS contains many RNA-protecting particles other than EVs, such as lipoprotein particles and protein aggregates. These particles usually contain many more RNAs than EVs, and they will co-isolate with EVs when non-specific methods are used, as in this study. No evidence is presented in this manuscript that the RNAs such as miR-1246 are associated exclusively or mainly with EVs. FE, the results are consistent with greater cell death or activation in infected cultures, leading to the release of cellular RNAs like miR-1246. To underline the point, infection of cell cultures may result in activation processes and also cell death. Without a detailed understanding of cell reactions, not sought or described in this study, it may not be justified to attribute all apparent differences to  exosomes. The data availability statement is incorrect:  All data relevant to the study are included in the article or uploaded as supplementary information. Links to databases containing the raw RNA sequencing and proteomics data are not given. MicroRNA  reads are shown for certain miRNAs, but no analysis methods are provided, and it appears that only one sample per condition was sequenced, greatly diminishing confidence in the results. Additionally, a  normalized value is given for the microRNAs that does not always correspond with the raw counts and is not consistent with a  counts per type of normalization. No methods or explanations are given, or variance. Non-miRNA values, which are the majority of reads, are not given. The authors are encouraged to follow publisher policies and deposit the RNA sequencing and proteomics data with a public database. miR-1246 may be misannotated. It may be a fragment of a nuclear RNA. Are the authors sure that this fragment is processed and acts as a microRNA physiologically? The data as presented do not necessarily support the conclusion that  Fn infection increases the secretion of exosomes from CRC cells. Bacteria also release EVs. Can it be excluded that infected cultures contained bacterial EVs? Minor point, but NTA is not specific for exosomes or for EVs more broadly. It is a particle measurement technique that is best suited in most current applications to biological particles around 100-150 nm in diameter. , 
 #1 1.tThe analysis results of RNA sequencing data and proteomics data** (Table S5 and Table S7)** can be found in **Supplementary Materials and Methods**. 2.tDeposit the RNA sequencing data will be uploaded again to a public database in a few day. 3.tLibrary preparation, sequencing and bioinformatics analysis were conducted by Guangzhou RiboBio Co., Ltd **(Supplementary Materials and Methods)**. 4.tWe used Abcam Anti-CD63 antibody** (ab216130)** to detecte Evs . The predicted molecular weight is 26 kDa and present as a distinct band as the manufacturer s instructions. The observed molecular weight of CD63 may vary in the range of 25-65 kDa. It is no consensus molecular weight. Many manuscript uesd this antibidy (ab216130) to detecte exosomes (For example: PMID: 31949877; PMID: 31444335; PMID: 31696054; PMID: 32476275 PMID: 31733447). Certainly, the observed band size from some anti-CD63 antibody is 30-65 kDa which is a smear on the gel, such as abcam [EPR5702] (ab134045). 5.tFor exosome isolation from culture medium, cells were cultured in serum-free medium for 48 h and the culture supernatant was collected. 6.tExosomes were isolated by differential centrifugation. The cell supernatant was centrifuged at 16,500 g for 20 min and then passed through a 0.22 u03bcm filter **to remove shedding vesicles and the other vesicles with bigger sizes (See Supplementary Materials and Methods).** Differential ultracentrifugation was the first method used for exosome isolation and remains the gold standard for exosome isolation to date (PMID: 31311206). 7.tExosomes were isolated by sequential differential centrifugation and verified by transmission electron microscopy, NanoSight analysis and Western blotting (For example: PMID: 33163491; PMID: 28393839; PMID: 31416776; PMID: 33532041; PMID: 29769437). 8.tmiR-1246 92b-3p 27a-3p-enriched exosomes. To identify the small RNA components in exosome, total RNA extracted from exosome were analyzed using small RNA high-throughput sequencing. Furthermore, the expression of miRNAs were validated by RT-qPCR in exosomes. 9.t*F. nucleatum* is a strict anaerobe that can t survive to secrete EVs in aerobic cell culture medium. F. nucleatum is only survive inside the cells under aerobic cell culture. 10.t*F. nucleatum* is an specific oncobacterium and the infection of *F. nucleatum* can promotes the the proliferation and migration of host cells, but not activation processes or cell death. The exact mechanism is gradually clarified. , 
 The analysis results of RNA sequencing data and proteomics data** (Table S5 and Table S7)** can be found in Supplementary Materials and Methods. *- The exact analysis methods and results remain unavailable. Please deposit the complete **RAW** and **NORMALIZED** RNA and protein data to public databases.* Deposit the RNA sequencing data will be uploaded again to a public database in a few day. *-Unclear what is meant by  again : were these data already uploaded? I will look forward to the RNA sequencing and proteomics data both being available publicly.* Library preparation, sequencing and bioinformatics analysis were conducted by Guangzhou RiboBio Co., Ltd (Supplementary Materials and Methods). *-Yes, but no details are available. How can others replicate these results if the experiments have all been outsourced to a company that provides no methods or data?* We used Abcam Anti-CD63 antibody** (ab216130)** to detecte Evs . The predicted molecular weight is 26 kDa and present as a distinct band as the manufacturer s instructions. The observed molecular weight of CD63 may vary in the range of 25-65 kDa. It is no consensus molecular weight. Many manuscript uesd this antibidy (ab216130) to detecte exosomes (For example: PMID: 31949877; PMID: 31444335; PMID: 31696054; PMID: 32476275 PMID: 31733447). Certainly, the observed band size from some anti-CD63 antibody is 30-65 kDa which is a smear on the gel, such as abcam [EPR5702] (ab134045). *-That is interesting. Perhaps the specificity of this antibody could be investigated in light of the overall experience in the field? It would be useful to show some confirmatory data from an antibody that performs as expected, too.* For exosome isolation from culture medium, cells were cultured in serum-free medium for 48 h and the culture supernatant was collected. *-This is good to know, and I hope that the paper will be corrected to reflect this important point. However, was cell health and viability tested? Switching cells to serum-free medium abruptly can result in massive cell death, which will affect EV results significantly.* Exosomes were isolated by differential centrifugation. The cell supernatant was centrifuged at 16,500 g for 20 min and then passed through a 0.22 u03bcm filter to remove shedding vesicles and the other vesicles with bigger sizes (See Supplementary Materials and Methods). Differential ultracentrifugation was the first method used for exosome isolation and remains the gold standard for exosome isolation to date (PMID: 31311206). *-There may be some confusion about the definition of exosomes, which were at one time thought to always be smaller than microvesicles (about ten years ago). Exosomes come from the endosomal compartment. It is now known that they are often no different from particles from the plasma membrane in size or composition. Exosomes cannot be perfectly removed from other particles by filtration or centrifugation.* Exosomes were isolated by sequential differential centrifugation and verified by transmission electron microscopy, NanoSight analysis and Western blotting (For example: PMID: 33163491; PMID: 28393839; PMID: 31416776; PMID: 33532041; PMID: 29769437). *-Exosomes cannot be  isolated by UC or verified by EM, NTA, or Western blotting. The morphology of an exosome by EM is the same as the morphology of an ectosome. NTA  sees exosomes, ectosomes, proteins, lipoproteins, and more in the same way. CD63 can be found on ectosomes and exosomes. Was any attempt made in this study to distinguish exosomes from ectosomes, or even to distinguish EVs from other extracellular particles?* miR-1246 92b-3p 27a-3p-enriched exosomes. To identify the small RNA components in exosome, total RNA extracted from exosome were analyzed using small RNA high-throughput sequencing. Furthermore, the expression of miRNAs were validated by RT-qPCR in exosomes. *-miR-1246 is not a microRNA. It is a fragment of a nuclear RNA. It is likely in the media as a result of cell death.* F. nucleatum is a strict anaerobe that can t survive to secrete EVs in aerobic cell culture medium. F. nucleatum is only survive inside the cells under aerobic cell culture. *-This is very interesting, and it would be good to mention this in the correction to the paper and show evidence for it. Currently, I do not see such evidence or controls to account for what happened outside the cell before during infection.* F. nucleatum is an specific oncobacterium and the infection of F. nucleatum can promotes the the proliferation and migration of host cells, but not activation processes or cell death. The exact mechanism is gradually clarified. *-This is good to know, and again will be useful to clarify with data about activation and cell death in the correction to the current paper.* ,