STAT3基因短发卡RNA表达质粒的构建及其对胃癌细胞生长和侵袭的抑制作用

Construction of shRNA Expression Vectors for STAT3 Gene and Its Inhibitory Effect on The Growth and Invasion of Gastric Cancer Cells

目的构建信号传导及转录活化因子3(STAT3)基因短发夹RNA(shRNA)表达质粒,探讨STAT3抑制后对胃癌MKN-45细胞增殖、凋亡和侵袭的影响。方法根据STAT3 mRNA编码序列,设计RNA干扰靶点,构建STAT3基因的特异性小RNA干扰质粒(psiRNA-H1/STAT3),使用脂质体转染MKN-45细胞,实验分为对照组、psiRNA-H1转染组和psiRNA-H1/STAT3转染组。通过RT-PCR和Western blot检测STAT3特异性小RNA干扰基因对胃癌细胞STAT3 mRNA和蛋白表达的影响;MTT比色法检测细胞的生长抑制率;采用流式细胞术检测转染后对MKN-45细胞凋亡的影响;采用Boyden小室侵袭实验检测转染后MKN-45细胞侵袭力的变化。结果成功转染重组体的MKN-45细胞中STAT3 mRNA和蛋白表达明显下降(P<0.05)。psiRNA-H1/STAT3转染组各时相均明显抑制MKN-45细胞生长,且MKN-45细胞凋亡率为34.26%,相比对照组(3.75%)和psiRNA-H1转染组(4.43%)明显升高(P<0.01)。另外,psiRNA-H1/STAT3转染组MKN-45细胞侵袭力较另外2组明显减弱(P<0.01)。结论成功构建了针对STAT3基因的shRNA表达载体,通过转染MKN-45细胞,在体外能有效抑制人胃癌细胞STAT3mRNA和蛋白表达,细胞增殖、侵袭能力减弱并且促进细胞凋亡,为STAT3基因靶向治疗提供一定的实验依据。

Objective To study the effect of knockdown of signal transducer and activator of transcription 3 （STAT3） expression by short hairpin RNA shRNA） on proliferation, apoptosis and invasion of human gastric cancer cell line MKN-45 in vitro. Methods Specific shRNA plasmids to STAT3 were constructed, and then transfected into MKN-45 cells by lipofectamine methods. Cells were divided into three groups： control group, psiRNA H1 transfected group as negative group and psiRNA-H1/STAT3 transfected group. Semiuantitative RT-PCR and Western blotting were used to detect the expression of STAT3 mRNA and protein, respectively. Proliferation and apoptosis of the transfected cells were observed by methyl thiazolyl tetrazolium （MTT） method and flow cytometry （FCM）, respectively. The invasion of the transfected MKN 45 cells was measured by Boyden chamber. Results Compared with the negative control cells, semi quantitative RT PCR and Western blotting showed that the expressions of STAT3 mRNA and protein were down-regulated in the psiRNA-H1/STAT3 transfected group （P〈0.05）. The suhcloned recombinant plasmid expressing shRNA effectively inhibited MKN-45 cell growth and proliferation while empty plasmid had no such specific effect. Cell apoptosis rate increased significantly in psiRNA-H1/STAT3 transfected group （P〈0.01）, and the invasion of MKN-45 cells was efficiently inhabited in psiRNA- H 1/STAT3 transfected group as compared with control group and psiRNA- H 1 transfected group （P〈0. 01）.Conclusion Recombinant plasmid psiRNA H1/STAT3 shRNA significantly inhibits the proliferation and invasion of MKN 45 cells and promotes their apoptosis.