Preparation Of Human SIRT1 Polyclonal Antibody And Preliminary Study Of The Effect Of SIRT1 Gene On Hepatoma Cell Line HepG2

ObjectivesThe objectives of this study include:1) to prepare the rabbit anti-human SIRT1 polyclonal antibody;2) to establish a stable SIRT1 over-expression HepG2 cell line and a stable SIRT1 RNA interference HepG2 cell line;and 3) to investigate the effects of SIRT1 on HepG2 cell apoptosis,cell growth,as well as insulin receptor substrate-2(IRS-2) mRNA expression level.MethodsA pair of primers with BamH I and NotI restriction sites was designed according to the SIRT1 gene cDNA sequences in GeneBank.A gene fragment of SIRT1 coding region was cloned by PCR method with a pBabe-SIRT1 plasmid template containing the full-length SIRT1 cDNA sequence.The purified PCR product and pGEX-4T-1 vector were double-digested and purified,followed by a ligation of the purified double-digested products.The recombinant pGEX-4T-1-SIRT1 was then transformed into E.coli BL21(DE3).Induced by IPTG,the fusion protein GST-SIRT1 was expressed under optimized prokaryotic expression conditions.The expressed fusion protein GST-SIRT1 was purified through affinity chromatography,and the purified protein concentration was determined by Coomassie brilliant blue method.Two rabbits were routinely immuned with the purified fusion protein GST-SIRT1,and the serum titer and specificity of the polyclonal anti-serum were tested with ELISA and Western blot method.Mediated by the cationic polymer,the pBabe-SIRT1,pBabe,pSUPER-SIRTRNAi, and pSUPER vectors were transfected into HepG2 respectively.SIRT1 stable expression cell line and SIRT1 stable interference cell line were obtained after puromycin selection,and the expression level of SIRT1 was detected by the RT-PCR and Western blot methods.The apoptosis and cell cycle of these cell lines were analysed with flow cytometry.The effects of SIRT1 over-expression and interference on IRS-2 mRNA expression level were tested with RT-PCR,respectively.ResultsThe results of double digestion and sequencing showed that the recombinant prokaryotic expression vector pGEX-4T-1-SIRT1 was successfully constructed and a strain of BL21 with high level expression of GST-SIRT1 fusion proteins was obtained.The prokaryotic expression condition was optimized as follows:- the inducer IPTG concentration was 0.8mmol/L- the induced time was 4h- the induced temperature was 30℃Under such condition,the concentration of the expressed fusion protein was able to be as high as 2.427mg/mL,total volume was 5ml.The anti-serum titer reached 1:6400 measured by ELISA.Anti-serum specificity was fairly satisfactory by Western blot detection.RT-PCR and Western blot assay results proved that a stable SIRT1 over-expression HepG2 cell line and a stable SIRT1 interference HepG2 cell line were successfully establised.The flow cytometry results showed that the apoptosis of HepG2 cells was inhibited by SIRT1 over-expression and RNA interference separately.The differences were statistically significant (P＜0.01) compared with the transfected empty vector and non-transfected HepG2.SIRT1 over-expression led to a stagnation of S phase and G2 phase of cell cycle,However,no such effect were observed after the RNA interference with SIRT1.RT-PCR analysis showed that SIRT1 over-expression significantly increased the IRS-2 mRNA expression. Compared with pBabe transfected and non-transfected HepG2,the differences were statistically significant(P＜0.01);SIRT1 lower-expression by RNA interference significantly reduced the IRS-2 mRNA expression.Compared with pSUPER transfected and non-transfected cells,the difference was statistically significant(P＜0.01).Conclusions The anti-GST-SIRT1 polyclonal antibody with high titer and satisfactory specificity has successfuly been prepared;A stable SIRT1 over-expression HepG2 cell line and a stable SIRT1 interference HepG2 cell line have been succeesfully established;Both SIRT1 over expression and RNA interference can inhibit HepG2 cell apoptosis. SIRT1 over expression exerts a stagnant effect on cell cycle,but RNA interference has no significant effect on cell cycle;The SIRT1 expression level in HepG2 is positively correlated with IRS-2 expression level.