miR-26b对宫颈癌细胞系迁移及上皮间质转化的影响

Effects of miR-26b on the migration and epithelial mesenchymal transition of cervical cancer cell line

目的探讨miR-26b对宫颈癌细胞迁移及上皮间质转化的影响及其作用机制。方法通过脂质体介导将miR-26b mimic转染C33A宫颈癌细胞系,细胞分为阴性对照组、无义对照组和miR-26b mimic组。用实时荧光定量PCR检测miR-26b及其靶基因的mRNA表达,细胞划痕实验检测细胞的迁移能力,Western blot检测E-cadherin和N-cadherin蛋白表达,用双荧光素酶基因报告载体检测miR-26b靶基因的表达。结果与正常宫颈上皮细胞相比,miR-26b mRNA在He La、Si Ha、Caski和C33A 4种宫颈癌细胞系中均低表达,其中以C33A细胞系最为明显;转染miR-26b mimic后,C33A细胞中miR-26b mRNA表达水平明显上升;过表达miR-26b可明显抑制C33A细胞的迁移能力,上调E-cadherin蛋白的表达,下调N-cadherin蛋白的表达,抑制上皮间质转化过程的发生;通过Target Scan、Pic Tar和miRDB软件预测显示,CXCL14、Smad4、TRAF5和Eph A2可能为miR-26b潜在的靶基因。双荧光素酶实验证实miR-26b可直接调控Smad4的表达。结论 miR-26b可能通过作用于Smad4的表达调节宫颈癌细胞的迁移和上皮间质转化过程的发生。

Objective To investigate the effect and mechanism of miR-26 b on the cell migration and epithelial mesenchymal transition of cervical cancer cells. Methods miR-26 b mimic was transfected into C33 A cervical cancer cell line by liposome-mediated method. The experiment was divided into negative control group,scramble control group and miR-26 b mimic group. The expression of miR-26 b and its target genes mRNA were detected by real-time fluorescence quantitative PCR. The cell migration ability was examined by scratch-wound assay. The expression of E-cadherin and N-cadherin proteins were detected by Western blot. Dual luciferase gene reporter assays were used to examine whether miR-26 b regulates the expression of Smad4. Results Compared with normal cervical epithelial cells,expression of miR-26 b mRNA was lower in Si Ha,He La,Caski and C33 A cell lines,among which C33 A cell line was the most obvious. mRNA expression levels of miR-26 b were significantly increased in C33 A cells by trans-fection of miR-26 b mimic. Over-expression of miR-26 b significantly inhibited the migration of C33 A cells,increased the expression of E-cadherin protein and reduced the expression of N-cadherin protein,and inhibited the process of epithelial mesenchymal transition. The potential target genes of miR-26 b were predicted by Target Scan,Pic Tar and miRDB software. Based on the annotated functions for these genes,CXCL14,Smad4,TRAF5 and Eph A2 were speculated to be the major potential target genes of miR-26 b. Dual luciferase gene reporter assays confirmed that miR-26 b can directly regulate the expression of Smad4. Conclusions miR-26 b may inhibit the migration of cervical cancer cells and the process of epithelial mesenchymal transition by regulating the expression of Smad4.