CAJ | 학술논문

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-shR-NA；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。
목적：구건 sh-Set7/9표체재체병사선은정전염 HepG2세포주,고찰기간우효과,위후속연구 Set7/9기인적공능급기재간암세포계중적작용제공실험기출。방법심조파향서렬,설계 siRNA 편단,구건침대인 Set7/9기인적shRNA 화대조재체,장간우재체화재체은정전염간암세포 HepG2,Real-time PCR 검측세포중 Set7/9기인적표체수평；동시이용 Western blot 방법재단백질수평진행검측,학정해기인적간우효과。결과성공구건재체 Set7/9-shR-NA；Real-time PCR 결과현시해기인표체수평명현피억제(P <0.05),동양 Western blot 검측표명기단백표체야현저하조。차외,여기상관적 Sirt1단백표체수평제고8.4배,Suv39h1단백표체수평승고1.1배。결론구건은정전염주후,가이현저하조 Set7/9기인적표체,동시영향여기상관적 Sirt1화 Suv39h1단백표체수평,제시대간암 HepG2세포활성산생영향。
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.