miR-491-5p靶向FOLR1对乳腺癌细胞增殖及侵袭的影响

Effect of miR-491-5p targeting FOLR1 on proliferation and invasion of breast cancer cells

目的探讨miR-491-5p对乳腺癌增殖及侵袭的影响及相关机制。方法收集本科室30例有明确病理诊断的新鲜乳腺癌及相对应的癌旁正常组织,实时荧光定量PCR(qRT-PCR)检测miR-491-5p及叶酸受体1(folate-receptor 1,FOLR1)基因表达量。选取MCF-7细胞,分为:control组、NC-mimics组、miR-491-5p mimics组、NC inhibitor组和miR-491-5p inhibitor组,克隆形成实验检测细胞增殖能力,Transwell实验检测细胞侵袭能力,Western blot检测AKT/mTOR信号通路p-AKT、AKT、p-mTOR、mTOR蛋白表达情况。双荧光素酶实验检测分析miR-491-5p与FOLR1间的靶向关系。选取MCF-7细胞进行回复实验,分为NC inhibitor组、miR-491-5p inhibitor组、miR-491-5p inhibitor+si-NC组和miR-491-5p inhibitor+si-FOLR1组,克隆形成实验及Transwell实验分别检测细胞增殖及侵袭变化,Western blot检测AKT/mTOR信号通路p-AKT、AKT、p-mTOR、mTOR蛋白表达情况。结果与癌旁正常组织相比,癌组织中miR-491-5p的基因表达量明显降低(P<0.05),FOLR1基因表达量明显升高(P<0.05)。与control组及NC-mimics组相比,miR-491-5p mimics组细胞增殖及侵袭能力均降低(P<0.05),p-AKT和p-mTOR蛋白表达量减少(P<0.05);与control组及NC inhibitor组相比,miR-491-5p inhibitor组细胞增殖及侵袭能力均升高(P<0.05),p-AKT和p-mTOR蛋白表达量增高(P<0.05)。双荧光素酶报告基因检测证明FOLR1是miR-491-5p的靶标。回复实验表明,与miR-491-5p inhibitor组及miR-491-5p inhibitor+si-NC组相比,miR-491-5p inhibitor+si-FOLR1组细胞增殖及侵袭能力明显降低(P<0.05),p-AKT和p-mTOR蛋白表达量显著减少(P<0.05)。结论miR-491-5p靶向FOLR1抑制乳腺癌细胞增殖及侵袭,可能与改变AKT/mTOR信号通路相关蛋白表达具有一定相关性。

Objective To investigate the effects of miR-491-5p on the proliferation and invasion of breast cancer and the related mecha-nisms.Methods Thirty cases of fresh breast cancer with definite pathological diagnosis and corresponding adjacent normal tissues were collected.The expression levels of miR-491-5p and FOLR1 gene were detected by real-time fluorescence quantitative PCR(qRT-PCR).MCF-7 cells were divided into control group,NC-mimics group,miR-491-5p mimics group,NC inhibitor group,and miR-491-5p inhibitor group.Clonal formation assay was used to detect the cell proliferation and Transwell assay was used to detect the cell invasion.Western blot analysis was performed to detect the expressions of p-AKT,AKT,p-mTOR and mTOR protein in AKT/mTOR signaling pathway.The targeting relationship between miR-491-5p and FOLR1 was analyzed by double luciferase assay.MCF-7 cells were divided into NC inhibitor group,miR-491-5p inhibitor group,miR-491-5p inhibitor+si-NC group,and miR-491-5p inhibitor+si-FOLR1 group for the recovery experiment.Cell proliferation and invasion abilities were detected by clonal formation assay and Transwell assay,respectively.The expressions of p-AKT,AKT,p-mTOR and mTOR proteins in AKT/mTOR signaling pathway were detected by Western blot analysis.Results Compared with adjacent normal tissues,the gene expression level of miR-491-5p in cancer tissues was significantly decreased(P<0.05),while the gene expression level of FOLR1 in cancer tissues was significantly increased(P<0.05).Compared with control group and NC-mimics group,the proliferation and invasion abilities were decreased in miR-491-5p mimics group(P<0.05),and the protein expressions of p-AKT and p-mTOR were decreased(P<0.05).Compared with control group and NC inhibitor group,the cell proliferation and invasion abilities were increased in miR-491-5p inhibitor group(P<0.05),and the protein expression levels of p-AkT and p-mTOR were increased(P<0.05).Dual luciferase reporter assay demonstrated that FOLR1 was the target of miR-491-5p.The recovery experiment showed that compared with miR-491-5p inhibitor group and miR-491-5p inhibitor+si-NC group,the cell proliferation and invasion abilities were significantly decreased in miR-491-5p inhibitor+si-FOLR1 group(P<0.05),and the expression levels of p-Akt and p-mTOR protein were significantly decreased(P<0.05).Conclusion MiR-491-5p can inhibit the proliferation and invasion of breast cancer cells by targeting FOLR1,which may be related to the change of AKT/mTOR signaling pathway-related protein expression.