靶向人SIRT1基因的shRNA真核表达质粒的构建及鉴定

目的构建针对人SIRT1基因的shRNA真核表达质粒,并筛选出对胰腺癌细胞系PANC-1基因沉默效果最明显的shRNA质粒表达载体。方法针对SIRT1基因的mRNA序列设计,分别构建3个shRNA质粒表达载体和1个阴性对照质粒表达载体,经大肠杆菌扩增,酶切,PCR,测序鉴定,转染胰腺癌PANC-1细胞,实时荧光定量PCR和Western blot检测SIRT1 mRNA和蛋白的被抑制情况。结果经测序证实,成功构建SIRT1-shRNA真核表达质粒,插入的DNA片段的序列与设计序列完全一致。重组质粒转染PANC-1细胞后,SIRT1 mRNA和蛋白水平明显下调;其中以1号重组质粒效应最强。结论成功构建了携带以SIRT1为靶向的shRNA的重组质粒。其对胰腺癌PANC-1细胞SIRT1的表达具有显著抑制效应。该实验为进一步研究SIRT1的功能和肿瘤的基因治疗提供了实验基础。

Construction and identification of eukaryotic plasmid expression vectors encoding the short hairpin RNA targeting human SIRT1

Objective:To construct three short hairpin RNA (shRNA) interference expression plasmid vectors of human SIRT1 gene,  assay the expression of SIRT1 in pancreatic carcinoma PANC-1 cells after transfecting with recombinant plasmids, and detect the RNAi effect of shRNA.
Methods:Three plasmid expression vectors coding for shRNA targeting SIRT1 gene sequence and a control vector containing random DNA fragment were constructed. The recombinant plasmids were amplified in E.coli. DH5α was identified by restriction digestion, PCR and sequencing. The vectors were transfected into PANC-1 cells. SIRT1 expression was assayed with real-time quantitative PCR and Western blot.
Results:The successful construction of recombinant plasmids was confirmed by DNA sequencing of the inserted segments. Transfection of shRNA plasmids significantly down-regulated SIRT1 expression in PANC-1cells. Recombinant plasmid 1 had the strongest effect, with an inhibition ratio of 79.43% at the mRNA level and 83.27% at the protein level, which showed a significant difference from plasmids 2 and 3 (P＜0.05).
Conclusions:Plasmid vector expressing shRNA against SIRT1 has been successfully constructed and it can down-regulate SIRT1 expression after transfected into PANC-1 cells. This finding could facilitate further studies on SIRT1 function and its application in tumour gene therapy．