Effects Of Survivin SiRNA On Proliferation Of Human Pancreatic Cancer Cells And Chemosensitivity To Gemcitabine

Pancreatic cancer is among the most. human malignant tumor for poor prognosis.Unfortunately the early symptom of pancreatic cancer is not clear, because of advancedstage when final diagnosis was taken, the 5-year survival rate remains less than 5%. Thecombined therapy including chemotherapy is critical to increase the survival rate ofpancreatic cancer patient, but this tumor is insensitive to most of the chemotherapeutics.Gemcitabine is a new-type miazines antimetabolite, and it has become the standardfirst-line chemotherapeutic agent for advanced and metastatic pancreatic cancer, withsignificant clinical benefit, but still has marginal survival advantage. Chemoresistance is themain reason for the failure of treatment. Thus, the development of an effective treatment tosolve the chemoresistance of pancreatic cancer remains an urgent task. With thedevelopment of molecular bioresearch, gene therapy targeting inhibitor of apoptosis proteingot much attention, and it possibly provide a new trend to effectively treat pancreaticcancer.Previous studies have shown that survivin is the newfound member of the inhibitors ofapoptosis protein (inhibitor of apoptosis protein, IAP) family, has dual function in theregulation of cell cycle and the inhibition of apoptosis. To date, the overexpression ofsurvivin has been reported in various human malignancies, but not or to be low in mostnormal adult tissues. Survivin is the smallest IAP which has been cloned hithreto, and islocated on chromosome 17q25. Its gene total length is 14.7kb, and is expressed specificlyin G2/M phase coding a 16.5kb protein. The expression of survivin has close correlation tothe prognosis, recurrence and chemoresistance of pancreatic cancer. Survivin primarylybind to mitotic spindle microtubule byαhelical structure in C-terminus, then its BIRstructure bind specificly to caspase-3 and caspase-7, and directly inhibit terminal effectorcaspase-3 and caspase-7 activity, so it can block various kinds of stimulus inducingapoptosis procedure. Survivin also can inhibit the cytochrome C release from mitochondria, breakdown the caspase activation, and so it can block the conduction of apoptosis signaland inhibit cell apoptosis at upstream. In the recent years, some reports showed thatblockdown of survivin can inhibit the growth of various tumor cells. Some researcher foundthat inhibition of survivin can enhance the radiosensitivity of pancreatic cancer cells.Therefore concerning the character of survivin, we think that the regulation of survivinexpression could be a possible new treatment for the chemosensitization of humanpancreatic cancer to Gemcitabine, yet such investigation has not been performed until nowin the world.In the present study, we firstly detect the expression of survivin and bcl-2 in pancreaticcarcinoma and analyze their significance, and then study the alteration of survivin geneexpression in pancreatic cancer cell after chemotherapy in virto. A short interfering RNA(siRNA) plasmid expression vector against survivin was constructed and transfected stablyinto Panc-1 and BxPC3 cells. The changes of survivin expression and cell cycle distributionfollowing RNAi and the role of siRNA in inducing tumor cell apoptosis and enhancing itschemosensitivity to gemcitabine were investigated. Together, these data provide strongevidence for the potential use of survivin-targeted RNAi as a novel way to chemosensitizehuman pancreatic cancer cells. Our study was divided into four parts below. Part One The expression of survivin and its relationship withbcl-2 in pancreatic carcinomaObjectiveTo investigate the expression and associativity of survivin and bcl-2 in tissues of humanpancreatic carcinoma, and study their role in the development of pancreatic cancer.MethodsThe expression of survivin and bcl-2 was detected by SP immunohistochemistry inpancreatic tissues from 50 patients with pancreatic carcinoma and that from 14 patientswith normal pancreas, and analyze their relation with clinicopathologic feature.ResultsSurvivin was expressed in 31 of 50 pancreatic carcinoma(62%). In contract, expressionof survivin in nomal pancreatic tissues was not detectable(p＜0.05). The survivin expressionwas correlated with histological grades and clinical stages(p＜0.05), and was not significantlycorrelated with sex, position of tumor or lymph node matastasis status(p＞0.05). Theexpression rate of survivin was 77.8% and 43.5% in positive and negative group of bcl-2expression respectively, and there was significant difference between two groups(p＜0.05).ConclusionA high expression of survivin in pancreatic carcinoma suggests that survivin may playan important role in the development of unfavourable prognosis. The expression of survivinhas positive correlation with bcl-2, and it may be a biological parameter to unfavourableprognosis. Part Two The alteration and significance of survivin expression inpancreatic cancer cell apoptosis induced by chemotherapyObjectiveTo study the alteration of survivin gene expression in pancreatic cancer cell afterchemotherapy(PA) in virto.MethodsCultivate the pancreatic cancer cell SW 1990 in virto, and give low concentration of PAto these cells, then alteration of survivin mRNA and protein expression was evaluated byPT-PCR and western-blot in pancreatic cancer cells which had been cultured with PA for24h,48h and control group respectively. Meanwhile, SW1990 cell apoptosis rate wasdetected by flow cytometry.ResultsThe apoptosis rate(%) of SW1990 cell of control group and in low concentration PAfor 24h,48h were 2.59±0.35,14.75±1.29,22.65±2.80( p＜0.05); Survivin mRNA levelwas increased 1.1 and 2.9 multiple(p＜0.05), Survivin protein level was increased 1.3 and3.6 multiple at 24h and 48h respectively(p＜0.05).ConclusionChemotherapy of PA can increase the expression of survivin in pancreatic cancer cell,which suggest that survivin may correlate with the chemoresistence of pancreaticcarcinoma cell. Part Three The construction and identification of siRNA eukaryoticexpression vector targeting survivin seneObjectiveTo construct and identify the siRNA eukaryotic expression vector targeting survivin gene,which could be applied to explore further gene therapy to pancreatic carcinoma.MethodsAccording to the cDNA sequence of survivin gene, two siRNA target sequence weredesigned on intemet, and then psiRNA1 and psiRNA2 were constructed respectively byeukaryotic expression vector psiRNA-hH1 neo. The constructed recombinant was identified byendonuclease digestion and DNA sequencing.ResultsThe results of endonuclease digestion and DNA sequencing suggested, the sequence ofinserted fragment was correct.ConclusionEukaryotic expression vector of siRNA targeting survivin was successfully constructedin this study, and should be a new effective vector for gene therapy of pancreaticcarcinoma. Part Four Effects of survivin siRNA on proliferation of humanpancreatic cancer cells and chemosensitivity to gemcitabineObjectiveTo construct the siRNA eukaryotic expression vector targeting survivin gene, andinvestigate the effects of survivin siRNA on proliferation of human pancreatic cancer cellsand chemosensitivity to gemcitabine.MethodsThe siRNA eukaryotic expression vector targeting survivin gene was constructed.Panc-1 and BxPC3 cells were transfected with constructed siRNA vector and then selectedby G418, and we got the stable transfected cells. The expression of survivin mRNA andprotein among the stable transfected cells and the untransfected cells was detected bysemiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Westernblot, respectively. The cell growth curve was drawn, and the cell cycle distribution wasmeasured by flow cytometry. The invasiveness of pancreatic cancer cells in vitro wasassayed using transwell cell culture chambers. Panc-1 and BxPC3 cells or transfected cellswere treated by Gemcitabine for 48h, and then the growth inhibition rates was measured byMTT assay and cell apoptosis rate was detected by flow cytometry.ResultsAfter the recombinant plasmid psiRNA-survivin transfected stably to pancreatic cancerline cells, survivin mRNA level were reduced by 68.52% and 64.32% respectively in stablytransfected Panc-1 and BxPC3 cells comparing with control group(P＜0.05), and survivinprotein level were reduced by 76. 68% and 74. 38% (P＜0.05) respectively, and the cellgrowth curve became much slower, many cells were blocked in the G0/G1 phase 68.72±3.21% (P＜0.05). The number of invasived cells in the experimental group was far less thanin control(P＜0.05). Further more, the growth inhibition rates and apoptosis of these stablytransfected cells were significantly increased after treatment by gemcitabine(P＜0.05).Conclusion The constructed siRNA eukaryotic expression vector targeting survivin could decreasesurvivin expression, inhibit the growth and cell invasiveness of pancreatic cancer cellssignificantly, and enhance the chemosensitivity to gemcitabine. Therefore, the inhibition ofsurvivin expression could be a possible new treatment for the chemosensitization of humanpancreatic cancer.