miR-126对Hela细胞增殖的影响及其与CRKL的靶向关系

Effect of miR-126 on proliferation of human cervical carcinoma Hela cells and target relationship with CRKL

目的:探讨miR-126对宫颈癌Hela细胞增殖活性的影响及其与CRKL的靶向关系。方法:构建miR-126、CRKL野生型(CRKL-WT)和突变型(CRKL-MT)真核表达载体pcDNATM6.2-GW-pre-miR-126、pmirGLO-CRKLWT和pmirGLO-CRKL-MT。采用实时荧光定量PCR法检测未转染、pcDNATM6.2-GW转染和pcDNATM6.2-GWpre-miR-126转染Hela细胞miR-126和CRKL mRNA的表达水平,应用MTT法检测细胞的增殖活性,Western blot法检测细胞CRKL蛋白的表达水平。将细胞分别共转染pcDNATM6.2-GW和pmirGLO-CRKL-WT、pcDNATM6.2-GW和pmirGLO-CRKL-MT、pcDNATM6.2-GW-pre-miR-126和pmirGLO-CRKL-WT、pcDNATM6.2-GW-pre-miR-126和pmirGLO-CRKL-MT,采用双荧光素酶报告系统检测细胞荧光素酶活性。结果:与pcDNATM6.2-GW转染组细胞比较,pcDNATM6.2-GW-pre-miR-126组细胞miR-126 mRNA过表达(t=545.891,P<0.05),CRKL mRNA和蛋白表达水平均降低(t=135.755、180.661,P<0.05),同时细胞的增殖活性亦降低(P<0.05);共转染pcDNATM6.2-GWpre-miR-126和pmirGLO-CRKL-WT的Hela细胞荧光素酶活性下调(Fpre-miR-126=55.627,FCRKL=61.843,F交互=76.203,P均<0.001)。结论:miR-126的过表达能够显著抑制Hela细胞的增殖,并且与CRKL具有良好的靶向关系。

Aim：To assess the effect of miR-126 on proliferation of human cervical carcinoma Hela cells and target re-lationship between miR-126 and CRKL.Methods：Recombinant plasmids of pcDNATM 6.2-GW-pre-miR-126 targeted at miR-126 ,pmirGLO-CRKL-WT targeted at wide type CRKL gene and pmirGLO-CRKL-MT targeted at mutant type CRKL gene were constructed .qRT-PCR method was used to detect miR-126 and CRKL mRNA in cells transfected with pcD-NATM6.2-GW-pre-miR-126 or empty vector,and cells without transfection .Western blot method was used to detect CRKL protein.MTT method was used to detect the proliferation .The cells were divided into 4 group,and co-transfected with pcD-NATM6.2-GW and pmirGLO-CRKL-WT, pcDNATM6.2-GW and pmirGLO-CRKL-MT, pcDNATM6.2-GW-pre-miR-126 and pmirGLO-CRKL-WT,pcDNATM6.2-GW-pre-miR-126 and pmirGLO-CRKL-MT,respectively,dual-luciferase assay sys-tem was used to detect the luciferase activity in cells .Results：Compared with those of cells transfected with empty vector , the expression of miR-126 mRNA in the cells transfected with pcDNATM6.2-GW-pre-miR-126 increased（t=545.891,P〈0.05）,while CRKL mRNA and protein expression decreased （t=135.755,180.661,P〈0.05）,and the proliferation activ-ity decreased（P〈0.05）.Luciferase activities of the cells co-transfected with pcDNATM6.2-GW-pre-miR-126 and pmir-GLO-CRKL-WT decreased as well（FmiR-126 =55.627,FCRKL =61.843,Finteraction =76.203,P〈0.001）.Conclusion：Over-expression of miR-126 could suppress cell proliferation by targeting CRKL in Hela cells .