miR-125a-5p对卵巢癌细胞的增殖迁移和侵袭能力的影响

目的探讨miR-125a-5p对卵巢癌细胞增殖、迁移和侵袭的影响及其作用的靶基因。方法购入正常人卵巢上皮细胞HOSEpi C,卵巢癌细胞SKOV3、A2780、ES2和HO8910各1株,通过实时定量聚合酶链式反应(RT-PCR)检测人卵巢上皮细胞HOSEpi C以及卵巢癌细胞SKOV3、A2780、ES2和HO8910中miR-125a-5p的表达水平。利用生物信息学方法预测miR-125a-5p的靶基因,构建预测靶基因3’端非编码区(3’-UTR)的野生型(WT)和突变型(mut)荧光素酶报告基因质粒,通过双荧光素酶报告基因法验证miR-125a-5p与预测靶基因的靶向作用。利用慢病毒感染的方法建立稳定过表达miR-125a-5p的SKOV3细胞,使用荧光定量PCR和Western blot检测靶基因的表达水平。利用细胞计数试剂盒-8(CCK-8)分析过表达miR-125a-5p的SKOV3细胞增殖能力的变化,通过Transwell迁移和侵袭试验分析过表达miR-125a-5p的SKOV3细胞迁移和侵袭能力的变化。结果正常人卵巢上皮细胞HOSEpi C中miR-125a-5p的表达水平高于卵巢癌细胞SKOV3、A2780、ES2和HO8910中miR-125a-5p的表达水平,其中SKOV3中miR-125a-5p的表达水平最低,差异均有统计学意义(P <0. 05),选择SKOV3进行后续实验。生物信息学分析显示,miR-125a-5p能够靶向结合Rab3D的3’-UTR;双荧光素酶报告基因分析证实miR-125a-5p能够靶向作用于Rab3D的3’-UTR。筛选稳定表达miR-125a-5p和si-Rab3D及相应对照的SKOV3细胞,分别命名为SKOV3/miR-125a-5p组,SKOV3/si-Rab3D组,SKOV3/miR-NC组和SKOV3/si-NC组。在SKOV3/miR-125a-5p组中,Rab3D的表达水平低于SKOV3组和SKOV3/miR-NC组,差异均有统计学意义(P <0. 05)。细胞增殖能力检测结果显示,与SKOV3组和SKOV3/miR-NC组相比,SKOV3/miR-125a-5p组在72小时和96小时细胞增殖能力降低,差异有统计学意义(P <0. 05)。Transwell迁移和侵袭试验结果显示,SKOV3/miR-125a-5p组分别与SKOV3组和SKOV3/miR-NC组相比,细胞迁移和侵袭能力均显著下降,差异有统计学意义(P <0. 05)。结论 miR-125a-5p能够靶向作用Rab3D,抑制卵巢癌细胞的增殖、迁移和侵袭。

miR-125a-5p inhibits proliferation, migration and invasion of ovarian cancer cells by targeting Rab3D

Objective To identify miR-125a-5p involved in regulation of the specific gene expression in ovarian cancer and to define their biological function of proliferation, migration and invasion. Methods The expression levels of miR-125a-5p in normal human ovarian epithelial cells HOSEpiC, ovarian cancer cells SKOV3, A2780, ES2 and HO8910 were detected by real-time PCR. The target genes of miR-125a-5p were predicted by bioinformatics. The wild-type (WT) and mutant (mut) luciferase reporter plasmids predicting the target gene 3''-UTR were constructed. The targeting effect of microRNA-125a-5p and the predicted target gene was verified by double luciferase reporter gene method. SKOV3 cells (SKOV3/miR-125a-5p) with stable overexpression of miR-125a-5p were established by lent virus infection. The expression of target genes was detected by fluorescence quantitative PCR and Western blot. Cell proliferation was analyzed by CCK-8 kit. Migration and invasion ability was analyzed by Transwell migration and invasion assay. Results The expression level of miR-125a-5p in normal human ovarian epithelial cells HOSEpiC was significantly higher than that in ovarian cancer cells SKOV3, A2780, ES2 and HO8910. SKOV3 was selected for subsequent experiments. Bioinformatics analysis showed that miR-125a-5p could target 3''-UTR binding to Rab3D, and double luciferase reporter gene analysis confirmed that prediction. The expression of miR-125a-5p in SKOV3/miR-125a-5p cells with stable overexpression of miR-125a-5p was significantly higher than that in SKOV3 cell group (P<0.01) and control group (SKOV3/miR-NC) (P<0.01), while the expression of Rab3D was lower than that in SKOV3 (P<0.01) and SKOV3/miR-NC (P<0.01). Cell viability test showed that compared with SKOV3 group (P<0.05) and SKOV3/miR-NC group (P<0.01), cell viability in SKOV3/miR-125a-5p group decreased significantly at 72 h and 96 h. The results of Transwell migration and invasion test showed that the migration and invasion ability of SKOV3/miR-125a-5p group decreased significantly compared with SKOV3 group and SKOV3/miR-NC group (P<0.01). Conclusion miR-125a-5p can inhibit the proliferation, migration and invasion of ovarian cancer cells by targeting Rab3D.