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- 2015
sh-Set7/9表达载体的构建及其在HepG2细胞中的功能
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Abstract:
摘要：目的 构建sh-Set7/9表达载体并筛选稳定转染HepG2细胞株，考察其干扰效果，为后续研究Set7/9基因的功能及其在肝癌细胞系中的作用提供实验基础。方法 寻找靶向序列，设计siRNA片段，构建针对人Set7/9基因的shRNA和对照载体，将干扰载体和载体稳定转染肝癌细胞HepG2，Real-time PCR检测细胞中Set7/9基因的表达水平；同时利用Western blot方法在蛋白质水平进行检测，确定该基因的干扰效果。结果 成功构建载体Set7/9-shRNA；Real-time PCR结果显示该基因表达水平明显被抑制(P<0.05)，同样Western blot检测表明其蛋白表达也显著下调。此外，与其相关的Sirt1蛋白表达水平提高8.4倍，Suv39h1蛋白表达水平升高1.1倍。结论 构建稳定转染株后，可以显著下调Set7/9基因的表达，同时影响与其相关的Sirt1和Suv39h1蛋白表达水平，提示对肝癌HepG2细胞活性产生影响。
ABSTRACT: Objective To silence human gene Set7/9 and screen out stable transfection cell line in hepatocellular carcinoma cell line HepG2 so as to investigate the impact of down-regulation of Set7/9 in cell line HepG2 and provide experimental foundation for studies on the effect of set7/9 in HepG2. Methods The target oligo was designed and synthesized; shRNA interference vector and the control vector were constructed and transfected into HepG2 cells; the stable transfection cells were screened out. Then Real-time PCR and Western blot were performed to detect the silence of Set7/9 according to both gene expression and protein expression level. Results The shRNA interference vector was constructed and transfected into HepG2 cells successfully. Compared with that in the negative control group, the expression of Set7/9 was dramatically downregulated (P<0.05). Meanwhile, the expression of related protein Sirt1 and Suv39h1 was upregulated 8.4 folds and 1.1 fold, respectively.Conclusion Downregulation of Set7/9 expression can upregulate Sirt1 and Suv39h1, suggesting that Set7/9 may affect the activity of HepG2 cell lines
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