MIA2慢病毒表达载体构建及其表达活性的研究

Construction of pLVX-IRES-ZsGreen1-MIA2 lentiviral expression vector and its expression activity

目的构建黑色素瘤抑制蛋白2(MIA2)慢病毒表达载体,并将其通过脂质体转染到HepG2细胞中,观察转染前后MIA2表达变化及其对细胞凋亡的影响。方法以pDONR223-MIA2为模板,PCR扩增MIA2,构建pLVX-IRES-ZsGreen1-MIA2慢病毒表达载体,体外瞬时转染肝癌细胞HepG2,荧光显微镜下观察MIA2的表达情况,用实时荧光定量PCR(RT-PCR)检测HepG2细胞中MIA2mRNA的表达情况,噻唑蓝(MTT)法和克隆形成实验检测细胞增殖的情况。结果成功构建pLVXIRES-ZsGreen1-MIA2慢病毒表达载体;RT-PCR结果显示,阴性对照组与实验组细胞MIA2mRNA表达水平差异有统计学意义(P<0.05);MTT检测发现,实验组细胞增殖数目较阴性对照组明显减少,差异有统计学意义(P<0.05);此外,实验组的克隆形成数和细胞迁移数均较阴性对照组明显减少,差异有统计学意义(P<0.05)。结论 pIRES2-ZsGreen1-MIA2可显著下调MIA2的表达水平进而抑制肝癌细胞HepG2的体外增殖及迁移能力。

Objective To construct an Lentiviral expression vector of pLVX-IRES-ZsGreen1-MIA2 targeting to MIA2 and investigate its effect on the expression of MIA2 and growth of HCC cell line HepG2 in vitro,observe MIA2 changes and the influence on apotheosis,thus to provide preliminary experimental fundament for successive researching on the role of MIA2 in the pathogenesis of HCC.Methods The sequence of pLVX-IRES-ZsGreen1-MIA2 was designed and synthesized.The pLVX-IRES-ZsGreen1-MIA2 Lentiviral expression vector was constructed and then transiently transfected into HepG2 HCC cells in vitro.The proportion of pLVX-IRES-ZsGreen1-MIA2 positive cells was observed under the fluorescence microscope.Then,the expression level of MIA2 was detected by real time PCR.Moreover,the proliferation of HepG2 cells was observed by MTT assay and colony formation assay.Finally,the migration of HepG2 cells in vitro was also determined by Scratch assay.Results pLVX-IRES-ZsGreen1-MIA2 Lentiviral expression vector was successfully constructed.Compared with control group（NC）,the expression level of MIA2 was significantly decreased in transfected groups（P〈0.05）;MTT assay showed that the proliferation of HepG2 cells was dramatically reduced in pIRES2-ZsGreen1-MIA2 transfected groups（P〈0.05）;furthermore,the number of both colony forming and migrating cells were also remarkably reduced in transfected groups（P〈0.05）.Conclusion The pIRES2-ZsGreen1-MIA2 can significantly reduce the expression level of MIA2 and inhibit the proliferation and migration of the HepG2 HCC cells in vitro.