miR-219-5p对人肾癌细胞增殖和迁移、侵袭的影响及其作用机制

Effects of miR-219-5p on Proliferation,Migration and Invasion of Human Renal Carcinoma Cells and Its Mechanism

目的探讨微小RNA-219-5p(miR-219-5p)对人肾癌细胞增殖、迁移、侵袭的影响及其作用机制。方法采用实时荧光定量PCR(qPCR)和蛋白质印迹法(Western blot)检测miR-219-5p和NIMA相关激酶6(NEK6)蛋白在肾癌组织和肾癌细胞786-O、ACHN中的表达。建立过表达miR-219-5p的细胞,观察其对肾癌细胞786-O、ACHN增殖、迁移和侵袭的影响。生物信息学预测结合双荧光素酶报告实验分析miR-219-5p与NEK6的靶向关系。在786-O、ACHN细胞中转染si-NEK6,噻唑蓝(MTT)和细胞克隆实验检测细胞存活和克隆数,Transwell检测细胞迁移和侵袭,Western blot检测细胞周期蛋白D1(Cyclin D1)、P21、E-钙黏蛋白(E-cadherin)、基质金属蛋白酶-2(MMP-2)、NEK6蛋白表达。共转染miR-219-5p和pcDNA3.1-NEK6,观察过表达NEK6对miR-219-5p诱导的786-O和ACHN细胞增殖、迁移和侵袭的影响。结果与癌旁组织或肾上皮细胞比较,肾癌组织或肾癌细胞786-O、ACHN中miR-219-5p表达量明显减少,NEK6蛋白表达量显著增加(P<0.05)。过表达miR-219-5p明显减低786-O、ACHN细胞存活率、克隆数、迁移细胞数、侵袭细胞数、Cyclin D1、MMP-2蛋白表达量,提高P21、E-cadherin蛋白水平(P<0.05)。miR-219-5p靶向调控NEK6表达。抑制NEK6明显减少786-O、ACHN细胞中NEK6、Cyclin D1、MMP-2蛋白表达量、细胞存活率、克隆数、迁移细胞数和侵袭细胞数,显著提高P21、E-cadherin蛋白水平(P<0.05)。过表达NEK6逆转了miR-219-5p对786-O、ACHN细胞增殖、迁移、侵袭、Cyclin D1、MMP-2蛋白表达的抑制作用,以及逆转了其对P21、E-cadherin蛋白表达的促进作用。结论miR-219-5p通过靶向调控NEK6表达抑制肾癌细胞的增殖、迁移和侵袭。

Objective To study the effects of miR-219-5p on the proliferation,migration and invasion of human renal carcinoma cells and its mechanism of action.Methods The expression levels of miR-219-5p and NIMA-related kinase 6(NEK6)protein in renal cell carcinoma and renal carcinoma ACHN and 786-O cells were detected by qPCR and Western blot.Cells overexpressing miR-219-5p were established,and the effects of miR-219-5p on the proliferation,migration and invasion of 786-O and ACHN cells were observed.Bioinformatics prediction and dual luciferase reporter assay were performed to analyze the targeting relationship between miR-219-5p and NEK6.The 786-O and ACHN cells were transfected with Si-NEK6,and cell survival and number of clones were measured by MTT assay.Cell migration and invasion were determined by Transwell assay,and the expression of Cyclin D1,P21,E-Cadherin,matrix metalloproteinase-2(MMP-2)and NEK6 was determined by Western blot.In addition,the effects of NEK6 overexpression on miR-219-5p-induced proliferation,migration and invasion of 786-O and ACHN cells were investigated by co-transfection with miR-219-5p and pcDNA3.1-NEK6.Results Compared with paracancer tissue or renal epithelial cells,miR-219-5p expression decreased but NEK6 protein expression increased in renal cancer tissue or 786-O and ACHN cells(P<0.05).The overexpression of miR-219-5p reduced the survival rate,number of clones,number of migrated cells,number of invasive cells and expression of Cyclin D1 and MMP-2 proteins,and increased the levels of P21 and E-cadherin proteins in 786-O and ACHN cells(P<0.05).The miR-219-5p was targeted to regulate NEK6 expression.The inhibition of NEK6 decreased the expression of NEK6,Cyclin D1 and MMP-2 proteins,viability of cells,number of clones,number of migrated cells and number of invading cells,and increased P21 and E-cadherin protein levels in 786-O and ACHN cells(P<0.05).The overexpression of NEK6 reversed the inhibitory effect of miR-219-5p on the proliferation,migration,invasion and Cyclin D1 and MMP-2 protein expression,as well as the promoting effect on P21 and E-cadherin protein expression in 786-O and ACHN cells.Conclusion miR-219-5p can inhibit the proliferation,migration and invasion of renal cancer cells by targeting NEK6 expression.