The Functions And Mechanisms Of CircRNA Hsa＿circ＿0000418 In Prostate Cancer

Background:Prostate cancer（PCa）is a malignant tumor of the prostate epithelium.In recent years,the morbidity of prostate cancer in China has been increasing year by year.At present,although the diagnosis and treatment of prostate cancer has made some progress,however,the mechanism of the development of prostate cancer is not completely clear.Circular RNAs（circular RNAs,circ RNAs）are a type of covalently closed circular long non-coding RNA,which does not have 3’poly（A）end and 5’cap end structure,and are mainly generated from introns or exons by reverse splicing or intron lariat.Circ RNAs are widely presented in eukaryotic cells and are stably expressed,with the characteristics of structural stability,tissue specificity and conservative evolution.In recent years,multiple lnc RNAs and miRNAs have been found to involve in the occurrence and development of tumors.There are more and more reports on circ RNAs in prostate cancer.Therefore,to explore the relationship and molecular mechanism of the abnormal expression of circ RNAs,as well as the relationship and the occurrence or development or prostate cancer,is a further expansion of the research on the mechanism of prostate cancer,which has positive significance for its clinical diagnosis and follow-up treatmentObjective:This study was designed to analyze the expression of hsa＿circ＿0000418 in prostate cancer cells,and explore its regulatory effects on the proliferation,migration and invasion of prostate cancer cells,and further explore the regulatory mechanism of hsa＿circ＿0000418 in prostate cancerMethod:1.The expression of hsa＿circ＿0000418 in human normal prostatic epithelial cell line RWPE-1 and prostate cancer cell line such as PCa cell line PC-3 and PC-3M-1E8 was detected by Q-PCR;then RNA was treated with RNase R,we detected the expression of hsa＿circ＿0000418 by Q-PCR,analysisd the stability of hsa＿circ＿0000418.2.Lipofectamine^TM2000 was used to transfect the interfering RNA（si RNA）of hsa＿circ＿0000418 to down-regulate the expression of hsa＿circ＿0000418 in PC-3 and PC-3M-1E8 cells.3.The effect of hsa＿circ＿0000418 on the proliferation ability of PC-3 and PC-3M-1E8 cells was analysed by CCK-8 assay as well as colony formation assay.4.The effect of hsa＿circ＿0000418 on the migration and invasion ability of PC-3 and PC-3M-1E8 cells was analysed by scratch test and Transwell invasion test.5.The potential binding miRNA of hsa＿circ＿0000418 was predict by online biological database,then it was verified by RNA pull-down assay.6.The target gene of the miRNA were predicted by online biological database,and the expression of the target gene and protein in si-hsa＿circ＿0000418 or si-miRNA PC-3 and PC-3M-1E8 cells were detected by Q-PCR and WB.7.The nude mice xenograft experiment was performed using sh-hsa＿circ＿0000418 stable transfected cells and control cells,in order to observed the effect of hsa＿circ＿0000418 on the proliferation of PCa cells in vivo.8.The effect of hsa＿circ＿0000418 on the expression of EMT-related genes and proteins in si-hsa＿circ＿0000418 PC-3 and PC-3M-1E8 cells was detected by Q-PCR and WB.Results:1.The Q-PCR results showed that the expression of hsa＿circ＿0000418 in prostate cancer cells was higher than normal prostate epithelial cell RWPE-1;Hsa＿circ＿0000418 can not easily degraded by RNase R and has good stability.2.The proliferation,migration and invasion ability were inhibited after knocked-down hsa＿circ＿0000418 of prostate cancer cells.3.The result of in silico prediction showed that miR-1304-5p was a potential binding miRNA of hsa＿circ＿0000418,and RNA pull-down assay indicated the hsa＿circ＿0000418 probe enriched miR-1304-5p in prostate cancer cells.4.Online biological database predictions showed that MYC gene was the potential target gene of miR-1304-5p,the expression of MYC was suppressed after knocked-down of hsa＿circ＿0000418 in prostate cancer cells,while knockdown of miR-1304-5p at the same time reversed the result.5.After knocked-down hsa＿circ＿0000418 in PC-3 cells,the tumorigenic ability in nude mice was reduced,and the tumor size and weight in knocked-down group were lower than in the control group.6.After knocking-down hsa＿circ＿0000418 in prostate cancer cells,the expression of EMT-related genes Snail was repressed,whereas the expression of E-cadherin was increased;Conclusion:1.Hsa＿circ＿0000418 is significantly up-regulated in PCa cells（PC-3 and PC-3M-1E8）,while down-regulate hsa＿circ＿0000418 could inhibit prostate cancer cells proliferation,migration and invasion.Down-regulation of hsa＿circ＿0000418inhibits PC-3 cell proliferation in nude mice.2.Hsa＿circ＿0000418 regulates EMT gene expression through miR-1304-5p/MYC axis to promote prostate cancer cell proliferation and invasion.