The Role And Related Molecular Mechanism Of CircRNA＿0007843 In The Proliferation And Metastasis Of Prostate Cancer Cells

Prostate cancer is one of the most common malignant tumors in men worldwide.The molecular pathogenesis of PCa has not yet been elucidated.Finding new and critical factors about detection and therapeutic will contribute to improve the accuracy of clinical diagnosis and the efficiency of treatment of PCa.Circular RNA(Circular RNA,circ RNAs)is a kind of non-coding RNA(non-coding RNAs,ncRNAs)widely found in various tissues and cells of the human body.The abnormal expression of circ RNAs is closely associated with the pathophysiological process of many diseases.Because of their conservation,stability and other biological characteristics,more and more researchers turn their attention to circ RNAs in the field of oncomolecularbiology.Studying the role and mechanism of circ RNAs in the occurrence and development of PCa is of great significance for better elucidating the pathogenesis of PCa and finding more effective therapeutic targets.This study took circ＿0007843 as the research object to explore its role and mechanism in the proliferation and metastasis of PCa cells,as well as its potential value as a target for PCa diagnosis and treatment.Objective To study the role and molecular mechanism of circ＿0007843 in the proliferation and metastasis of PCa cells.Methods 1.Through literature search,bioinformatics analysis and qRT-PCR tests,the circular RNA circ＿0007843,which is abnormally expressed in PCa cells,was selected as the research object.2.The small interfering RNA of circ＿0007843(si-circ＿0007843)was synthesized and transfected into PCa cell lines PC3 and DU145.Clony formation,CCK8,scratch healing,transewell and flow cytometry were applied to detect the effects of circ＿0007843 silencing on proliferation,migration,invasion and cell cycles of PCa cells.3.Analyze and predict the downstream target miRNAs of circ＿0007843 by bioinformatics software.After silencing circ＿0007843,qRT-PCR was applied to select highly expressed miRNAs,and miRNA-3064-5p was choosen as a potential downstream target miRNA of circ＿0007843.4.Construct luciferase reporter plasmids containing circ＿0007843 or miRNA-3064-5p binding site(WT)/mutation binding site(Mut).Prove if there is targeting sites between circ＿0007843 and miRNA-3064-5p by the dual luciferase reporter assay.5.Use qRT-PCR to detect the expression of miRNA-3064-5p in RWPE-1 cell line and tumor cell lines.Synthesize miRNA-3064-5p mimic and miRNA-3064-5p inhibitor,then transfect them into PC3 cells and DU145 cells.Dectect the effects of miRNA-3064-5p on proliferation、metastasis、cell cycles of PCa cells by CCK8、colony formation、scratch healing、transwelll、flow cytometry assays.6.Predict the downstream targets of miRNA-3064-5p by bioinformatics analysis and screen out EIF5 which is related to cell cycle regulation as the research object.Test the targeting relationship between miRNA-3064-5p and EIF5 by western blot and dual luciferase report assays.7.Co-transfect PCa cells with si-circ＿0007843 and miRNA-3064-5p inhibitor,detect the effect on proliferation by CCK8、detect the effect on migration by scratch healing and detect the effect on invasion by transwell.Measure the expression of EIF5 protein using western blot.Results 1.qRT-PCR results showed that compared with normal prostate epithelial cell line RWPE-1,circ＿0007843 was significantly up-regulated in PCa cell lines PC3,DU145,22RV1 and LNCa P(P<0.05).2.The results of Colony formation,CCK8,scratch healing,flow cytometry and transewell assays make clear that in comparison with the nontarget control group,the proliferation,migration as well as invasion abilities of PCa cells and cell cycle progression were significantly inhibited after the transfection with si-circ＿0007843(P<0.05).3.The result of bioinformatic analysis shows that there is a potential target binding site between miRNA-3064-5p and circ＿0007843.qRT-PCR detection shows that the expression of miRNA-3064-5p were upregulated in circ＿0007843 silencing group.4.The results of the dual luciferase reporter experiment confirm that the luciferase activity of the group which is co-transfected miRNA-3064-5p and circ＿0007843-WT was significantly reduced in comparison with the nontarget control group(P<0.05).5.qRT-PCR results showed that,compared with the RWPE-1 cell line,the expression of miRNA-3064-5p in PC3,DU145,22RV1 and LNCa P cell lines were significantly down-regulated(P<0.05).Transfection of miRNA-3064-5p mimic can significantly inhibit the proliferation,migration as well as invasion of PC3 and DU145 cell lines,as well as the cell cycle progression,while the results after transfection of miRNA-3064-5p inhibitors are the opposite.6.The results of bioinformatics analysis show that EIF5 is a potential downstream target gene of miRNA-3064-5p.The dual luciferase reporter method confirmed that there is a binding site between EIF5 and miRNA-3064-5p.Transfection of miRNA-3064-5p mimic can significantly down-regulate the expression of EIF5 in PC3 and DU145 cells.7.The results of CCK8,scratch healing and transwell assays show that the co-transfection with si-circ＿0007843 and miRNA-3064-5p inhibitor significantly reverses the negative effect of si-circ＿0007843 on the proliferation,migration and invasion of PC3 and DU145 cell lines.The results of western blot indicate that the amount of EIF5 protein in PC3 and DU145 cell lines was significantly down-regulated when si-circ＿0007843 was transfected alone.Co-transfection with miRNA-3064-5p inhibitor can enhance the expression of EIF5.Conclusion 1.Circ＿0007843 is highly expressed in PCa cells.Silencing circ＿0007843can significantly inhibit the proliferation,migration and invasion of PCa cells,as well as cell cycle progression.2.Circ＿0007843 targets the miRNA-3064-5p and regulates its expression,silencing circ＿0007843 can promote the expression of miRNA-3064-5p.3.The content of miRNA-3064-5p in PCa cells was down regulated,overexpression of miRNA-3064-5p suppresses the proliferation,migration、invasion as well as cell cycle progression of PCa cell lines.4.MiRNA-3064-5p targets EIF5 and regulates its expression.5.Circ＿0007843participates in the adjustment of proliferation,migration and invasion of PCa cells and suppresses the amount of EIF5 protein by regulating miRNA-3064-5p.