短发卡RNA对人胃癌细胞STAT3基因的沉默作用

Silence effect of plasmid mediated shRNA on STAT3 gene in gastric cancer cells

目的探讨STAT3基因小发夹RNA(shRNA)表达质粒对胃癌MKN-45细胞STAT3基因的干扰作用。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),使用脂质体转染人胃癌细胞系(MKN-45细胞)。实验分为对照(A)组,psiRNA-H1转染(B)组和psiRNA-H1/STAT3转染(C)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。结果psiRNA-H1/STAT3经限制性酶切及部分序列分析证明基因插入正确,并经测序证实。将其成功转染MKN-45细胞后,该细胞的STAT3 mRNA和蛋白表达均明显下降(P<0.05)。结论将成功构建的针对STAT3基因的shRNA表达载体转染MKN-45细胞,能有效抑制该细胞的STAT3 mRNA和蛋白表达,为STAT3基因靶向治疗提供一定的实验依据。

Objective To study the interfering effect of short hairpin RNA shRNA ） expressing plasmid vectors on STAT3 gene expression of human gastric carcinoma cells. Methods Specific shRNA plasmids to STAT3 were constructed, and then transfected into MKN-45 cells by lipofectamine methods. Semi-quantitative RT-PCR and Western blotting were used to detect the expression of STAT3 mRNA and protein respectively. Cells were divided into three groups ： control group （ A group ） , psiRNA- H 1 group （ negative group, B group ） and psiRNA-H1/STAT3 group （C group ）. Results The synthesized strands of oligonucleotide contained correct and complete sequence of the shRNA, and the STAT3 specific shRNA was inserted into eukaxyotic expression. Compared with the A and B group cells, semi-quantitative RT-PCR and Western blotting showed the expression of STAT3 mRNA and protein was down-regulated in the C group （P 〈 0.05 ）. Conclusions The recombinant plasmid psiRNA-H1/STAT3 shRNA has been constructed successfully, and it can specificly inhibit not only the expression of STAT3 mRNA, but also the protein expression in gastric carcinoma cells in vitro. It provides certain experimental basis for STAT3 gene target therapy.