shRNA干扰STAT3基因表达对胃癌细胞MKN-45体内外生物学特性的影响

Effect of knockdown of STAT3 gene expression by shRNA on the characteristics of gastric cancer cell line MKN-45 in vitro and in vivo

目的:研究shRNA干扰STAT3基因表达对胃癌细胞MKN-45体内外生物学特性的作用.方法:应用本实验室已构建并鉴定的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3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基因mRNA和蛋白表达的影响;MTT比色法检测细胞的生长抑制率;流式细胞仪分析MKN-45细胞周期分布.用各组MKN-45细胞建立移植瘤裸鼠模型,筛选转染psiRNA-H1和psiRNA-H1/STAT3的MKN-45稳定细胞株观察裸鼠移植瘤的生长情况.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(0.612±0.074vs1.937±0.043,P<0.05;0.668±0.054vs2.005±0.064,P<0.01).G0/G1期细胞比例增高,另一方面S期细胞比例降低,细胞的增殖系数明显降低(25.42±3.48vs33.54±2.91,P<0.05).移植瘤裸鼠模型显示,psiRNA-H1/STAT3组瘤体在体积上明显减小(4.47cm3±0.18cm3vs13.65cm3±5.64cm3,P<0.05).结论:针对STAT3基因的特异性小RNA干扰质粒在体外能有效抑制人胃癌细胞系STAT3mRNA和蛋白表达,细胞增殖能力减弱,在体内明显抑制肿瘤生长,为STAT3基因靶向治疗提供一定的实验依据.

AIM： To determine the effect of knockdown of STAT3 expression by shRNA on the characteristics of human gastric carcinoma cell line MKN-45 in vitro and in vivo. METHODS： Specific shRNA plasmids to STAT3 were constructed and identified, then transfected into MKN-45. Cells were divided into three groups： control group, psiRNA-H1 group as the negative group and psiRNA-H1/STAT3 group. Semi-quantitative reverse transcriptionpolymerase chain reaction （RT-PCR） and Western blotting were used to detect the expression of STAT3 mRNA and protein respectively. MTT and flow cytometry were used to assess cell growth suppression and cell cycle distribution respectively. Models of xenograft in nude mice were established using the three-group cells, and the growth of stable ones was then observed. RESULTS： Compared with the negative control cells, semi-quantitative RT-PCR and Western blotting showed that the expression of STAT3 mRNA （0.612 ± 0.074 vs 1.937 ± 0.043, P 〈 0.05） and protein （0.668± 0.054 vs 2.005 ± 0.064, P 〈 0.01） was down-regulated in the psiRNA-H1/STAT3 group. The subcloned recombinant plasmid expressing shRNA effectively inhibited MKN-45 cell growth and proliferation （25.42 ± 3.48 vs 33.54 ± 2.91, P 〈 0.05） while empty plasmid had no such specific effect. The volumes of xenografts in the psiRNA-H1/STAT3 transfection group were larger than those in the other groups （4.47 cm^3 ± 0.18 cm^3 vs 13.65 cm^3 ± 5.64 cm^3, P 〈 0.05）. CONCLUSION： Recombinant plasmid psiRNA-H1/STAT3 shRNA can specifically inhibit not only the expression of STAT3 mRNA and protein in vitro, but also the growth of cell line MKN-45 in vitro and in vivo.