Runx3-shRNA表达载体的构建及其干扰作用的检测

Construction and interfering effect of Runx3-shRNA expression vector

目的构建Runx3基因shRNA重组表达质粒,并检测其干扰作用。方法人工合成靶向Runx3基因的shRNA序列,克隆至pGenesil-1.1表达载体上,构建重组表达质粒Runx3-shRNA,转染人耐药肝癌HepG2细胞;采用半定量RT-PCR及Western blot法检测重组表达质粒对HepG2细胞中Runx3基因的转录和表达水平的影响。结果重组表达质粒Runx3-shRNA经酶切及测序证明构建正确;其转染人耐药肝癌HepG2细胞后,细胞Runx3基因的转录和蛋白表达水平均明显降低,与空白对照组和阴性对照组比较,差异均有统计学意义(P<0.05)。结论已成功构建了Runx3-shRNA表达质粒,为进一步研究Runx3基因在肿瘤耐药细胞中的作用奠定了基础。

Objective To construct recombinant shRNA expression vector for Runx3 gene and observe its interfering effect.Methods The shRNA sequence targeting Runx3 gene was synthesized and cloned into expression vector pGenesil-1.1.HepG2 cells were transfected with the constructed recombinant plasmid Runx3-shRNA and determined for transcription level of Runx3 mRNA and expression level of Runx3 protein by semi-quantitative RT-PCR and Western blot respectively.Results Restriction analysis and sequencing proved that recombinant plasmid Runx3-shRNA was constructed correctly.Both the transcription level of Runx3 mRNA and expression level of Runx3 protein in HepG2 cells transfected with the plasmid decreased significantly as compared with those in blank and negative control groups（P 0.05）.Conclusion Runx3-shRNA expression vector was successfully constructed,which laid a foundation of further study on role of Runx3 gene in tumor-resistant cells.