Effects And Mechanism Of SIRT1 Gene Expression In Biological Behaviour Of Pancreatic Carcinoma

Objective:To investigate the expression and significance of SIRT1 in human pancreatic carcinoma and their association with the clinical pathologic characters. To detect the expression of SIRT1 in three human pancreatic carcinoma cell lines for experimental foundation of SIRT1 RNA interference. Taking human pancreatic carcinoma PANC-1 cells as investigative object, to construct three short hairpin RNA (shRNA) interference expression plasmid vectors of human SIRT1 gene, to assay the expression of SIRT1 in PANC-1 cells after transfecting with recombinant plasmids, and to detect the RNAi effect of shRNA. To investigate the effects and mechanism of SIRT1 gene expression inhibited by shRNA in proliferation, anchorage-independence, tumorigenic ability, chemosensitivity and invasion of PANC-1 cells.Methods:Immunohistochemistry staining and real-time quantitative polymerase chain reaction (PCR) were used to detect the expression of SIRT1 in 49 pancreatic carcinoma tissues and homologous nontumorous normal pancreatic tissues. The correlations among the variety of expression of SIRT1 and pancreatic carcinoma clinical pathologic characters were statistically analyze. Real-time quantitative PCR and western blot were were used to compare the expression levels of human pancreatic carcinoma cell lines PANC-1, AsPC-1 and BxPC-3. The cell line with the highest SIRT1 expression was chosed for RNA interference. Three plasmid expression vectors coding for shRNA targeting SIRT1 gene sequence, a positive control vector and a negative control vector were constructed. The recombinant plasmids were amplified in Ecoli. DH5 and identified by restriction digestion, PCR and sequencing. The vectors were transfected into PANC-1 cells. The green fluorescent protein (GFP) was observed by fluorescence microscope. SIRT1 expression was assayed with real-time quantitative PCR and western blot. The vector with the best interference effect and the negative control-shRNA vector were stable transfected into PANC-1 cells by G418 screening. The stable transfection cells of PANC-1-Negative cells and PANC-1-SIRT1-RNAi cells had been obtained. The growth curves of PANC-1 cells, PANC-1-Negative cells and PANC-1-SIRT1-RNAi cells were drawed by MTT method. The cell cycles were detected by flow cytometry. Colony forming efficiency in soft agar was used for anchorage-independent growth. The ratios of cell apoptosis were detected by TUNEL (TdT-mediated dUTP nick end labeling) test. The expressions of p53, FOXO3a, Bax and Bcl-2 were assayed by real-time quantitative PCR and western blot. Xenografts in BALB/c nude mice were used for evaluating the tumorigenic ability. The expression of SIRT1 in xenografts was detected by flourescence image formation in vivo and immunohistochemistry staining and the ratios of cell apoptosis in xenografts were detected by TUNEL test. The chemosensitivities of PANC-1 cells, PANC-1-Negative cells and PANC-1-SIRT1-RNAi cells to 5-FU and gemcitabine hydrochloride were detected by MTT, and each ICsowas calculated. Invasive abilities of PANC-1 cells, PANC-1-Negative cells and PANC-1-SIRT1-RNAi cells were observed with Transwell cell culture chambers, and the expressions of E-cadherin, MMP-2 and MMP-9 were assayed by real-time quantitative PCR and western blot.Results:The SIRT1 protein expression rate of the tumor tissues was 75.51%, significantly higher than that of homologous nontumorous normal pancreatic tissues (22.45%), which was coincident with the SIRT1 mRNA expression. The SIRT1 expression was significantly associated with patients'age, tumor size, TNM stage, nodes invasion and hepatic metastasis (P<0.01), and there was no rela tionship between SIRT1 expression and patients'sex, histologic grading, location of the tumor and blood vessel or nerves invasion (P>0.05). The SIRT1 expression of cell line PANC-1 was the highest in three human pancreatic carcinoma cell lines. 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. The stable transfections of recombinant plasmid of No.l and recombinant plasmid of negative control-shRNA were named PANC-1-SIRT1-RNAi cells and PANC-1-Negative cells, respectively. PANC-1-SIRT1-RNAi cells had SIRT1 inhibition ratio of 95.8% at the mRNA level and 86.0% at the protein level. The doubling generation time of PANC-1, PANC-1-Negative and PANC-1-SIRT1-RNAi cells was (42.76±1.28) h, (44.21±1.95) h and (80.32±6.44) h, respectively. Compared with PANC-1 and PANC-1-Negative cells, the proliferation of PANC-1-SIRTl-RNAi cells was inhibited with G0/G1 stage blocking, the anchorage-independence was weaken obviously, the ratio of cell apoptosis were raised up. Proapoptotic factors such as FOXO3a and Bax were up-regulated in PANC-1-SIRT1-RNAi cells, but p53 was not affectted. In the other hand, antiapoptotic Bcl-2 was down-regulated reciprocally. The expression of these factors was coincident in mRNA and protein levels. The tumorigenic ability of PANC-1-SIRTl-RNAi cells was decreased significantly. The apoptosis cell ratios of PANC-1, PANC-1-Negative and PANC-1-SIRT1-RNAi xenografts were (4.16±2.44)%, (5.23±1.41)% and (58.84±10.86)%, respectively (P<0.01). The 5-FU IC50s of PANC-1, PANC-1-Negative and PANC-1-SIRT1-RNAi cells were 117.42±31.19μg/mL,104.54±29.22μg/mL and 51.37±18.94μg/mL, respectively, and the gemcitabine IC50S were 78.32±15.49μg/mL, 72.68±20.11μg/mL and 21.47±7.53μg/mL, respectively. PANC-1-SIRT1-RNAi cells were much more chemosensitive to 5-FU and gemcitabine hydrochloride than PANC-1 cells and PANC-1-Negative cells (P<0.01). The Transwell invasive chambers found that the numbers of PANC-1 cells, PANC-1-Negative cells and PANC-1-SIRT1-RNAi cells were (59±13), (61±10) and (22±6), respectively. The invasive ability of PANC-1-SIRTl-RNAi cells decreased dramatically (P<0.01) through promoting expression of E-cadherin and inhibiting expressions of MMP-2 and MMP-9 in mRNA and protein levels. There was no difference between PANC-1 cells and PANC-1-Negative cells in above-mentioned bionomics (P>0.05).Conclusion:1. The SIRT1 expression was raised up in pancreatic carcinoma tissues, significantly associated with the patients'age, tumor proliferation, and could participate in invasion and metastasis of pancreatic carcinoma. SIRT1 expression may be regarded as a parameter of determining the degree of malignancy and prognosis of pancreatic carcinoma.2. The pancreatic carcinoma cell line PANC-1 was chosed for experiments of RNA interference. Plasmid vector expressing shRNA against SIRT1 has been successfully constructed and it can down-regulate SIRT1 expression after stable transfected into PANC-1 cells, which could facilitate further studies of SIRT1 functions and its application in tumour gene therapy.3. It could inhibit proliferation, weaken anchorage-independence, induce apoptosis and depress tumorigenic ability in PANC-1 cells by inhibitting the expression of SIRT1 gene stably.4. PANC-1-SIRT1-RNAi cells which the expression of SIRT1 gene were significantly inhibited were enhanced chemosensitivity to 5-FU and gemcitabine hydrochloride. Furthermore, the invasion of PANC-1-SIRT1-RNAi cells were inhibited obviously in vitro.