miR-21靶向抑制TIMP3表达促进肝细胞肝癌细胞侵袭

miR-21 targeted suppressor TIMP3 expression promotes the invasion of hepatocellular carcinoma cells

目的:探讨miR-21在肝细胞肝癌(hepatocellular carcinoma, HCC)细胞中的表达及其调控细胞增殖和侵袭的机制。方法:通过Real-time PCR检测HCC细胞株(Hep3B、HuH7、SNU398和Hep3G2)和人正常肝细胞(THLE2、THLE3)中miR-21的表达。使用miR-21-inhibitor及对照miR-NC分别瞬时转染Hep3B和HuH7细胞,CCK-8法检测细胞增殖,Transwell小室实验检测细胞侵袭能力。双荧光素酶实验检测miR-21与组织金属蛋白酶抑制因子3(tissue metalloproteinase inhibitor factor 3,TIMP3)的靶向结合关系;Western blot法检测TIMP3、基质金属蛋白酶2(matrix metalloproteinase 2,MMP2)和MMP9的表达。结果:Hep3B、HuH7、SNU398和Hep3G2细胞中miR-21表达均高于THLE2、THLE3细胞,差异有统计学意义(P<0.01)。Hep3B和HuH7细胞转染miR-21-inhibitor后细胞侵袭能力受到有效抑制(P<0.01),对细胞增殖没有显著影响。双荧光素酶实验结果显示,miR-21-mimic转染后,野生型质粒组荧光素酶活性显著降低,与miR-NC组相比,差异均有统计学意义(P<0.01);突变质粒组,miR-21-mimic对其荧光素酶活性没有显著影响,与miR-NC组相比,差异也不显著。miR-21-inhibitor转染组Hep3B和HuH7细胞中TIMP3表达均升高,MMP2和MMP9的表达降低,与miR-NC相比,差异具有统计学意义(P<0.01)。结论:miR-21能够通过靶向抑制TIMP3表达,进而上调MMP2和MMP9的表达与活化促进HCC细胞侵袭。

Objective:To explore miR-21 expression in hepatocellular carcinoma(HCC) cells and its mechanism of regulating cell proliferation and invasion.Methods:The expression of miR-21 was detected by Real-time PCR in HCC(Hep3 B,HuH7,SNU398,Hep3 G2) and human normal hepatocyte(THLE2,THLE3).Hep3 B and HuH7 cells were transfected with miR-21-inhibitor and miR-NC,respectively, then the proliferation of Hep3 B and HuH7 cells were detected by CCK-8 assay and the ability of invasion were detected by Transwell assay.Dual luciferase assay was used to detect miR-21 binding to TIMP3 3’-UTR to regulate the expression of TIMP3,and the expression of TIMP3,matrix metalloproteinase 2 and matrix metalloproteinase 9 were determined by Western blot.Results:miR-21 expression in Hep3 B,HuH7,SNU398 and Hep3 G2 cells was significantly higher than that in THLE2 and THLE3 cells(P<0.01).After transfected with miR-21-inhibitor, the invasion of Hep3 B and HuH7 cells were effectively inhibited(P<0.01).However, there’s no effect on proliferation.The results of double luciferase assay showed that the activity of luciferase was significantly lower in miR-21-mimic transfected +wild type plasmid group than miR-NC+wild type plasmid group(P<0.01).Compared with miR-NC+mutant plasmid group, the activity of luciferase was no significant changes in miR-21-mimic transfected+mutant plasmid group.After transfected with miR-21-inhibitor, the expression of TIMP3 was increased and the expression of MMP2 and MMP9 was decreased in Hep3 B and HuH7 cells, which were significantly different from miR-NC(P<0.01).Conclusion:miR-21 targeted inhibition the expression TIMP3 to promote the invasion of HCC cells by up-regulating the expression and activation of MMP2 and MMP9.