SHP-2shRNA联合苦参碱对宫颈癌细胞增殖的影响

Effect of SHP-2 shRNA combined with matrine on proliferation of cervical cancer cells

目的应用RNA干扰技术构建SHP-2基因重组慢病毒载体,研究SHP-2shRNA联合苦参碱对宫颈癌Hela细胞生长增殖的影响。方法靶向构建SHP-2的shRNA序列慢病毒载体,将其转染至人宫颈癌Hela细胞以沉默SHP-2基因,用Real-time PCR(RT-PCR)测定Hela细胞SHP-2 mRNA,应用Western blot法测定SHP-2蛋白表达,CCK8法测定SHP-2基因沉默联合苦参碱对Hela细胞增殖的影响。结果设计并成功构建3组SHP-2基因的干扰序列(SHP-2 shRNA1、SHP-2 shRNA2和SHP-2 shRNA3)以及对照序列(NS),RT-PCR检测结果表明SHP-2 shRNA2慢病毒载体干扰效率最佳。筛选稳定细胞后荧光显微镜观察感染效率较高,Western blot结果显示细胞中SHP-2蛋白表达明显被抑制。CCK8提示细胞的生长增殖能力明显受限,且随着时间延长,联合组(SHP-2 shRNA2+苦参碱)增殖最慢,与对照组相比,差异有统计学意义(P<0.05)。结论成功构建出SHP-2基因的shRNA慢病毒干扰系统,SHP-2基因沉默联合苦参碱可影响宫颈癌细胞增殖。

Objective To investigate the effect of SHP-2 shRNA combined with matrine on proliferation of Hela bycontrsucting the lentiviral RNA interference vector of SHP-2 gene with interference technology.Methods The lentiviral vector of shRNA sequence targeting SHP-2 was constructed and transfected into human cervical cancer Hela cells to silence SHP-2 gene.The expression of SHP-2 mRNA and protein in transfected Hela cells was detected by Real-time PCR and Western blot respectively,and the effect of SHP-2 gene silencing associated with matrine on cell proliferation was detected by CCK8.Results The interference sequences of three groups of SHP-2 genes(SHP-2 shRNA1,SHP-2 shRNA2 and SHP-2 shRNA3)and the control sequence(NS)were designed and successfully constructed,and RT-PCR showed that the one with best interference effect was the SHP-2 shRNA2 sequence.After screening stable cells,fluorescence microscopy showed that the infection efficiency was high.Western blot demonstrated that the expression of SHP-2 in interference group was lower than that in control group.CCK-8 assay showed that the proliferation of Hela cells in the combined group was slower than that in the control group with time longer(P<0.05).Conclusion A cervical cancer cell model with stable SHP-2 down-regulation was successfully established,and SHP-2 gene silencing combined with matrine can affect cervical cancer cells proliferation.