miRNA-574-5p通过靶基因高温需求因子A1调控肝癌细胞增殖、侵袭、迁移的分子机制

目的探讨微小RNA(mi RNA)-574-5p通过靶基因哺乳动物高温需求因子A1(HTRA1)调控肝癌细胞增殖、侵袭、迁移的分子机制。方法采用实时荧光定量聚合酶链反应(q RT-PCR)、蛋白质印迹法(Western blot)检测正常细胞株L02及肝癌细胞株Hep G2、SMMC-7721、BEL-7402中mi RNA-574-5p、HTRA1的表达水平,采用Western blot检测转染后Hep G2细胞中细胞周期蛋白D1(cyclin D1)、p21、p27、基质金属蛋白酶(MMP)2、MMP9、MMP14蛋白的表达水平,采用噻唑蓝(MTT)法检测细胞增殖情况,采用Transwell实验检测细胞迁移和侵袭能力,采用双荧光素酶实验检测mi RNA-574-5p对HTRA1活性的影响。结果与L02细胞系比较,肝癌细胞系Hep G2、SMMC-7721、BEL-7402中mi RNA-574-5p的表达水平均升高,HTRA1 m RNA和HTRA1蛋白的表达水平均下降(P﹤0.05)。anti-mi RNA-574-5p组Hep G2细胞中mi RNA-574-5p的表达水平明显低于anti-mi RNA-NC组(P﹤0.01)。与anti-mi RNA-NC组比较,anti-mi RNA-574-5p组Hep G2细胞48、72 h的增殖率及cyclin D1、MMP2、MMP9、MMP14蛋白的表达水平均明显降低,迁移、侵袭数目均明显减少,p21、p27蛋白的表达水平均明显升高(P﹤0.01)。pc DNA-HTRA1组Hep G2细胞中HTRA1蛋白的表达水平明显高于pc DNA组(P﹤0.01)。与pc DNA组比较,pc DNA-HTRA1组Hep G2细胞48、72 h的增殖率及cyclin D1、MMP2、MMP9蛋白的表达水平均明显降低,p21蛋白的表达水平明显升高,迁移、侵袭数目均明显减少(P﹤0.01)。双荧光素酶报告实验显示,mi RNA-574-5p+WT-HTRA1组Hep G-2细胞的荧光素酶活性明显低于mi RNA-NC+WT-HTRA1组(P﹤0.01)。mi RNA-574-5p+MUT-HTRA1组Hep G-2细胞的荧光素酶活性与mi RNA-NC+MUT-HTRA1组比较,差异无统计学意义(P﹥0.05)。与anti-mi RNA-574-5p+si-NC组比较,anti-mi RNA-574-5p+si-HTRA1组Hep G2细胞中HTRA1蛋白的表达水平降低,培养48、72 h的肝癌Hep G2细胞的增殖率均升高,cyclin D1、MMP2、MMP9蛋白的表达水平均升高,细胞迁移、侵袭数目均增多,p21蛋白的表达水平降低(P﹤0.05)。结论mi RNA-574-5p通过靶向负调控HTRA1基因调控肝癌细胞的增殖、侵袭、迁移。

Molecular mechanisms of miRNA-574-5p regulating the proliferation,invasion and migration of hepatocellular carcinoma cells via targeting high temperature requirement factor A1 gene

Objective To explore the molecular mechanism of microRNA(miRNA)-574-5p regulating the proliferation,invasion and migration of hepatocellular carcinoma cells through mammalian high temperature requirement factor A1(HTRA1).Method Real-time fluorescence quantitative polymerase chain reaction(PCR)and Western blot were used to detect the expression levels of miRNA-574-5p and HTRA1 in normal cell line L02 and HCC cell lines HepG2,SMMC-7721 and BEL-7402.The levels of cyclin D1,p21,p27,MMP2,MMP9 and MMP14 in transfected HepG2 cells were also detected by Western blot.Methylthiazolyl tetrazolium(MTT)was used to determine the cell proliferation.Transwell assay was utilized to investigate the cell migration and invasion.Dual luciferase reporter assay was applied to assess the effect of miRNA-574-5p on the activity of HTRA1.Result Compared with L02 cells,the expression of miRNA-574-5p in HepG2,SMMC-7721 and BEL-7402 cells increased significantly,while the expression of HTRA1 mRNA and proteins decreased significantly(P<0.05);miRNA-574-5p expression in HepG2 cells of anti-miRNA-574-5p group was significantly lower compared with anti-miRNA-NC group(P<0.01).Significantly decreased proliferation rate of HepG2 cells after culturing for 48 h and 72 h,reduced expression of cyclin D1,MMP2,MMP9 and MMP14 proteins,suppressed cell migration and invasion and higher expression of p21 and p27 proteins were observed in anti-miRNA-574-5p group versus anti-miRNA-NC group,the differences were of statistical significance(P<0.01).pcDNA-HTRA1 group had higher HTRA1 protein expression in HepG2 cells than in pcDNA group(P<0.01).As to the comparison between pcDNAHTRA1 group and pcDNA group,the proliferation rate of HepG2 cells after culturing for 48 h and 72 h decreased,protein expression of cyclin D1,MMP2 and MMP9 reduced,and p21 protein expression increased,while cell migration and invasion inhibited in the former group of cells(P<0.01).Dual luciferase reporter assay showed that miRNA-574-5p+WTHTRA1 group was with notably lower luciferase activity of HepG-2 cells compared to miRNA-NC+WT-HTRA1 group(P<0.01).No obvious difference was noted between miRNA-574-5p+MUT-HTRA1 group and miRNA-NC+MUTHTRA1 group with regard to the luciferase activity of HepG-2 cells(P>0.05).Compared with anti-miRNA-574-5p+si-NC group,anti-miRNA-574-5p+si-HTRA1 group demonstrated lower expression of HTRA1 protein,elevated proliferation rate of HepG2 cells after culturing for 48 h and 72 h,increased cyclin D1,MMP2,and MMP9 protein expression,promoted cell migration and invasion,and declined p21 protein expression,and the differences were of statistical significance(P<0.05).Conclusion miRNA-574-5p regulates the proliferation,invasion and migration of hepatocellular carcinoma cells by targeting HTRA1 gene.