miRNA-138靶向调控细胞周期蛋白D3及波形蛋白影响肝癌细胞的增殖及迁移

Regulation of miR-138 on cyclin D3 and vimentin and its effect on proliferation and migration of hepatoma carcinoma cells

目的研究肝癌细胞miRNA-138(miR-138)通过调控细胞周期蛋白D3(CCND3)、波形蛋白对肝癌细胞的影响。方法利用RT-PCR对肝癌组织及癌旁组织(对照)、4种肝癌细胞系(HepG2、HHCC、HUH7、BEL-7402)及正常肝细胞系HL-7702(对照)的miR-138的mRNA表达进行检测;设计合成miR-138的模拟物(mimics)及抑制剂(inhibitor)。分别将空载体(对照)、miR-138的模拟物及抑制剂转染至HepG2细胞,与相应对照相比,分析miR-138对CCND3、波形蛋白表达及肝癌细胞活力的影响。应用CCK-8法、细胞创伤愈合试验和Transwell细胞迁移试验分别对转染空载体、miR-138 mimics 及inhibitor后的HepG2肝癌细胞增殖及迁移能力进行检测。Western blot检测上调或下调miR-138后miR-138相关靶蛋白CCND3及波形蛋白的表达。结果与相应对照相比,miR-138的表达水平在肝癌组织及肝癌细胞系中明显降低( P <0.01)。当miR-138 mimics转染肝癌细胞后,HepG2细胞的活力降低,创伤愈合能力较弱,迁移能力明显降低( P <0.01),经Western blot检测,波形蛋白和CCND3的表达水平降低( P <0.01)。转染miR-138 inhibitor 后,HepG2细胞的增殖能力升高,肝癌细胞的创伤愈合能力和迁移能力提升( P <0.01),与此同时,波形蛋白和 CCND3的表达水平升高( P < 0.01 )。结论通过调节波形蛋白和 CCND3的表达,miR-138在肝癌细胞的增殖活力、迁移能力的变化机制中发挥重要的作用。

Objective To study the influence of miR-138 on hepatocellular carcinoma(HCC) cells by regulating cyclin D3 (CCND3) and vimentin.Methods The expression of miR-138 in HCC tissues,adjacent liver tissues(control),four HCC cell lines (HepG2,HHCC,HUH7 and BEL-7402) and normal liver cell line HL-7702(control) was determined by RT-PCR.miR-138 mimics and miR-138 inhibitor were designed synthesis.Empty plasmid(control),miR-138 mimics and miR-138 inhibitor were transfected to HepG2 cells separately.The effects of miR-138 on cyclin D3,vimentin expression and HCC cell activity were analyzed compared with the corresponding controls.Cell viability was determined by CCK-8 and cell migration ability was detected by wound healing assay and Transwell cell migration assay after transfection.Western blot was used to detect the expressions of miR-138 related target protein cyclin D3 and vimentin after up-regulation or down-regulation of miR-138.Results Compared with the control group,the expression of miR-138 was significantly down-regulated in HCC tissues and HCC cell lines ( P <0.01).miR-138 mimics transfection significantly reduced the viability of HepG2 cells ( P <0.01).The expression levels of vimentin and CCND3 decreased by Western blot detection ( P <0.01).On the other hand,miR-138 inhibitor transfection significantly enhanced the viability of HepG2 cells,the ability of wound healing and migration of HCC cells obviously ( P <0.01).At the same time,the expressions of vimentin and CCND3 increased ( P <0.01).Conclusion miR-138 regulates vimentin and CCND3 expressions and then plays an important role in the proliferation and migration of HCC cells.