miR-599表达变化对卵巢癌细胞增殖侵袭能力的影响

Effect of miR-599 expression changes on proliferation and invasion of ovarian cancer cells

目的探讨微小RNA(miRNA,miR)-599在卵巢癌中的表达及其对卵巢癌细胞增殖和侵袭的影响及机制。方法实时荧光定量聚合酶链反应(qPCR)检测miR-599在36例卵巢癌及癌旁组织、卵巢癌细胞株及正常卵巢上皮细胞中的表达。将miR-599抑制物(实验组)和阴性对照miR-NC(对照组)分别转染至卵巢癌细胞株A2780,qPCR检测转染效率。利用生物信息学技术预测筛选miR-599的候选靶基因,建立双荧光素酶报告基因分析系统,验证miR-599对候选靶基因的直接靶定作用。qPCR和Western blot检测靶基因的mRNA和相关蛋白表达变化。细胞计数法(CCK-8)和Transwell侵袭实验分别检测miR-599对A2780细胞增殖和侵袭能力的影响。结果qPCR结果显示,卵巢癌组织miR-599表达水平高于癌旁组织(P<0.01)。卵巢癌细胞株miR-599表达水平高于正常卵巢上皮细胞(P<0.05)。阿片样物质结合蛋白(OPCML)是miR-599的候选靶基因,双荧光素酶报告基因实验证实miR-599可直接靶定OPCML基因的3′-非翻译区(3′-UTR)(P<0.01)。下调miR-599表达后,A2780细胞OPCML的mRNA和蛋白表达水平降低(P<0.01),细胞周期调控蛋白如CDK4和Cyclin D2的表达降低;细胞侵袭负向调控蛋白E-cadherin蛋白表达升高,正向调控蛋白N-cadherin表达降低,A2780细胞增殖及侵袭能力均降低(P<0.05)。结论miR-599在卵巢癌组织和细胞株中高表达,下调卵巢癌细胞A2780中的miR-599表达可通过升高OPCML基因表达抑制卵巢癌细胞增殖和侵袭能力。

Objective To investigate the expression of microRNA(miR)-599 in ovarian cancer and its effect on the proliferation and invasion of ovarian cancer cells.Methods Quantitative real-time quantitative polymerase chain reaction(qPCR)was used to detect the expression of miR-599 in 36 cases of ovarian carcinoma and paracancerous tissues,ovarian cancer cell lines and normal ovarian epithelial cells.The miR-599 inhibitor(experimental group)and the negative control miR-NC(control group)were transfected into the ovarian cancer cell line A2780,respectively.qPCR was used to detect the transfection efficiency.Bioinformatics technology was used to predict and screen candidate target genes of miR-599,and dual luciferase reporter gene analysis system was established to verify the direct targeting effect of miR-599 on candidate target genes.qPCR and Western blot were used to detect the mRNA and protein expression changes of target genes.Cell counting(CCK-8)and transwell invasion assays were used to detect the effect of miR-599 on proliferation and invasion of A2780 cells.Results qPCR results showed that the expression of miR-599 in ovarian cancer tissues was higher than that in paracancerous tissues(P<0.01).The expression level of miR-599 in ovarian cancer cells was higher than that in normal ovarian epithelial cells(P<0.05).The opioid binding protein(OPCML)is a candidate target gene for miR-599.The dual luciferase reporter assay confirmed that miR-599 can directly target the 3′-untranslated region(3′-UTR)of the OPCML gene(P<0.01).After down-regulation of miR-599 expression,the mRNA and protein expression levels of OPCML in A2780 cells decreased(P<0.01);the expression of cell cycle regulatory proteins such as CDK4 and cyclin D2 was decreased;the expression of negative regulatory protein E-cadherin was increased,and the expression of positive regulatory protein N-cadherin was decreased;the proliferation and the invasion ability of A2780 cells decreased(P<0.05).Conclusions miR-599 is highly expressed in ovarian cancer tissues and cell lines.Down-regulation of miR-599 expression in ovarian cancer cells A2780 can inhibit the proliferation and invasion of ovarian cancer cells by increasing OPCML gene expression.