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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。
Objective To construct shRNA eukaryotic expression plasmid targeting human SIRT1 gene and screen the shRNA plasmid expression vector with the most obvious silencing effect on pancreatic cancer cell line PANC-1. Methods According to the sequence design of SIRT1 mRNA, three shRNA expression plasmids and one negative control plasmids were constructed respectively. The recombinant plasmids were amplified by PCR, identified by restriction enzyme digestion, PCR and sequencing, and transfected into pancreatic cancer PANC-1 cells. Fluorescence quantitative PCR and Western blot were used to detect SIRT1 mRNA and protein inhibition. Results Sequencing confirmed that the SIRT1-shRNA eukaryotic expression plasmid was successfully constructed. The sequence of inserted DNA fragment was exactly the same as the designed sequence. The recombinant plasmid was transfected into PANC-1 cells, the SIRT1 mRNA and protein levels were significantly down-regulated; among them, No. 1 recombinant plasmids had the strongest effect. Conclusion The recombinant plasmid carrying shRNA targeting SIRT1 was successfully constructed. It has a significant inhibitory effect on the expression of SIRT1 in pancreatic cancer PANC-1 cells. This experiment provides the experimental basis for further study on the function of SIRT1 and the gene therapy of tumor.