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．