Investigation Of Roles And Mechanisms Of Glycogen Synthase Kinase-3beta In Pancreatic Cancer Molecular Target Therapy

Objective To investigate the relationship between the expression of GSK-3βand clinical features in pancreatic cancer, and determine the role of GSK-3βin the development of pancreatic cancer.Methods The expression of GSK-3βin pancreatic cancer tissue and adjacent normal tissue was detected by SP immunohistochemical technique.Results GSK-3βwas expressed in 33 of 65 cases. In contrast, no expression of GSK-3βin adjacent normal tissue was detectable (P<0.01). Expression of GSK-3βhad no relationship with ages, sexes and tumor sizes of pancreatic cancer patient. But the expression was significantly correlated with differentiation level, metastatic status and JPS stage (P<0.05).Conclusion High expression of GSK-3βmay play an important role in carcinogenesis and development, invasion and metastasis of pancreatic cancer. Therefore, assessment of GSK-3βexpression might be useful for predicting the prognosis of pancreatic cancer patients.Objective To construct three shRNA interference expression plasmid vectors of human GSK-3βgene, detect the expression of GSK-3βin pancreatic carcinoma PANC-1 cells after transfection with recombinant plasmids, and transfect stably the most effective recombinant plasmid into PANC-1 cells.Methods Three plasmid expression vectors coding for shRNA targeting GSK-3βand a control vector were designed. The recombinant plasmids were amplified in E. coli. DH5a, and then treated with restriction enzymes, PCR and sequencing. PANC-1 cells stably expressing GSK-3βshRNA and control shRNA were screened with G418, as the experimental group and vector control group. The cells were not transfected as the blank control group. Cells transfection state was observed by fluorescence microscope and FCM. GSK-3βexpression was assayed with Real-time PCR and Western blot.Results 1.The successful construction of recombinant plasmids was determined by DNA sequencing.2 GSK-3βexpression was notably down-regulated after transfection of shRNA plasmids in PANC-1 cells. Recombinant plasmid 2 had the strongest effect.3. Compared with the control groups, the expression of GSK-3βshow a significantly down-regulation in experimental group.Conclusion Plasmid vector expressing shRNA against GSK-3βand PANC-1 cells with stably expressing GSK-3βshRNA were constructed, which could facilitate further studies on GSK-3βfunction and its application in tumor gene therapy.Objective To investigate the effects of GSK-3βRNA interference on pancreatic cancer cell proliferation, apoptosis, cycle and invasive capability and to explore the molecular mechanism involving the procedure and the possibility of targeting GSK-3βfor pancreatic cancer therapy.Methods PANC-1 cells stably expressing GSK-3βshRNA and control shRNA were the experimental group and the vector control group. The cells were not transfected as the blank control group. The growth curves of PANC-1 cells were designed by MTT assay. The cells apoptosis and cycle were analyzed by FCM. The invasive capability of PANC-1 cells was detected by Transwell. The expression levels of bcl-2,cyclin D1,VEGF,HIF-1αwere respectively assayed with Real-time PCR, Western blot and ELISA. NF-κB DNA binding activity was detected through EMSA analysis of nuclear extracts. Results Compared with control group, the growth rate and invasive capability of PANC-1 cells in experimental group decreased notably, the apoptotic number of cells increased obviously, and cell cycle was arrest at G0/G1 phase. The expression of bcl-2, cyclin D1, VEGF, and NF-κB DNA binding activity show a significantly down-regulation in experimental group. However, in GSK-3βRNAi PANC-1 cells, the HIF-1αprotein expression was not inconsistent with the gene expression.Conclusion GSK-3βmay play an important role in proliferation, survival and invasion of human pancreatic cancer. Blocking GSK-3βin pancreatic cancer cells may offer an avenue for gene therapy.Objective To study the effects of GSK-3βRNA interference on the growth and angiogenesis of human pancreatic cancer xenografts in nude mice.Methods PANC-1 cells were incubated and were inoculated subcutaneously in athymic nude mice to establish xenograft models. The mice were divided into blank control group, vector control group and experimental group. Tumor volume and weight was measured in nude mice bearing xenografts. The inhibitory rate was calculated according to the weights of xenografts. The proteins of PCNA and FⅧwere assessed by immunohistochemical method. SPF and MVD were respectively counted according to PCNA and FⅧstaining. The expression level of VEGF was detected by Real-time PCR and Western blot.Results Compared with control group, the growth rate of human pancreatic cancer xenografts of nude mice in experimental group decreased notably (P<0.05) and the inhibitory rate was 35.17%. The SPF index, MVD count and VEGF expression of xenografts were significantly decreased in experimental group than that in control group (P<0.05).Conclusion In vivo GSK-3βRNAi can inhibit the growth and angiogenesis of human pancreatic cancer xenografts in nude mice, which may be related to the downregulation of VEGF.Objective To investigate the sensitivity of pancreatic cancer cell to gemcitabine (GE) after GSK-3βRNA interference.Methods PANC-1 cells stably expressing GSK-3βshRNA and control shRNA were the experimental group and the vector control group. The cells were not transfected as the blank control group. Different dosages of GE were added into PANC-1 cells. The growth inhibition rates and the apoptosis rates were respectively measured by MTT and FCM.Results GE caused a greater increase of the growth inhibition rate and apoptosis rates of PANC-1 cells in experimental group than in control groups in a dose-dependent manner (P<0.05)Conclusion Our data demonstrate that GSK-3βRNAi inhibits the NF-κB DNA binding activity and the expression of bcl-2 and enhances the growth inhibition and the apoptosis induced by GE. Thus GSK-3βRNAi could sensitize pancreatic cancer cells to GE. Blocking GSK-3βin pancreatic cancer cells may offer an avenue for gene therapy.