The Effects Of AKT2 In The Pathogenesis Of Pancreatic Cancer

Tumors continuously grow because of their intrinsic unregulated growth program caused by genetic alterations. Therefore, tumors in general are always exposed to hypoxia and poor nutrition because of insufficient vascularization. Theoretically, there are two ways of adaptation to an insufficient supply of oxygen and nutrients that could be interlinked but distinct from each other. One way is by increasing the supply, an idea widely accepted. The other way is by tolerating insufficiency, austerity. A tumor grows beyond its ability to improve oxygen and nutrient supplies by angiogenesis, and this might be the reason why tumors are often associated with necrotic foci. Only the tumor cells that have acquired the ability to survive an unfavorable microenvironment might be malignant. Pancreatic cancer is one of the most aggressive cancers known; however, it is mostly hypovascular. The pancreatic cancer always infiltrates other organ and metastasizes when diagnosed. The rate of radical resection is low and the therapy efficacy is rather bad. So to research the mechanism of metastasis, may resolve the problem.In this study, we will explore the expression of AKT2 and HIF-1α protein in human pancreatic cancer, and to research the change of AKT2 and HIF-1α in pancreatic cancer cells which exposed to hypoxia and poor nutrition conditions, and to study the biological effects of inhibition the expression of AKT2 by RNAi. This study may provide a new fundamental and experimental data for exploring the mechanism of pancreatic cancer cell survival and metastasis. Part I Expression and Clinical Significance of AKT2 and HIF-1αin Pancreatic CancerObjective To investigate the expression and clinical significance of AKT2 and HIF-1α protein in human pancreatic carcinoma. Methods The expressions of AKT2 and HIF-1α protein were measured by using SABC immunohistochemical staining in specimens of 63 cases of pancreatic carcinoma and 23 cases of benign pancreatic diseases. The relationship of expressions of AKT2 and HIF-1α protein and clinicopathological factors in pancreatic carcinoma was also analyzed. Results The positive expression rate of AKT2(39.7%) and HIF-1α(63.5 %) in pancreatic carcinoma were significantly higher than that in benign pancreatic diseases(P<0.05), respectively. The expression of AKT2 was positively correlated with HIF-la protein(P<0.05). The expression level of Akt2 protein in pancreatic cancer showed an obvious difference in histological grade, lymph node metastasis, TNM stage(p<0.05, respectively), but not markedly correlated with patient's age, gender and tumor size. Conclusion Overexpression of AKT2 and HIF-1α were found in pancreatic carcinoma. AKT2 may induce the carcinogenesis and aggression in pancreatic cancer by regulating the transcription of HIF-1α gene.Part II Tolerance of Pancreatic Cancer Cells to Hypoxia and Nutrition Deprivation: Role of PI3K/AKT2 PathwayObjective To research the tolerance of pancreatic cancer cells to hypoxia and nutrition deprivation, and to explore the role of PI3K/AKT2 Pathway in this phenomenon. Methods The tetrazolium bromide(MTT) colorimetry was used to draw the growth curves of PANC1 and HepG2 cells under hypoxia and nutrition deprivation conditions. Before and after incubation in the PI3K inhibitor LY294002, the level of AKT2, HIF-1α mRNA and protein of PANC1 cell under hypoxia and nutrition deprivation conditions were examined by using RT-PCR and western blot. Results PANC1 was more resistant to the hypoxia and nutrition deprivation conditions than HepG2 cell. Western blot showed that the hypoxia and nutrition deprivation conditions activated AKT2, and the expression of HIF-1α protein was also increased. The PI3K inhibitor LY294002 blocked the activation of AKT2 and decreased the expression of HIF-1α protein in that condition. Conclusion The hypoxia and nutrition deprivation conditions increase the expression of HIF-1α protein via PI3K/AKT2 Pathway in pancreatic cancer cells.Part III Construction and Identification of shRNA Expression Vector Targeted to AKT2 GeneObjective To construct and identify short hairpin RNA(shRNA) expression vector targeted to AKT2 gene, and to study the effect of shRNA on the expression of AKT2 gene in pancreatic cancer cell line PANC1. Methods ShRNA template sequence of AKT2 was synthesized and linked into the pSIREN-DNR-DsRed-Express Donor Vector to generate the plasmid pSIREN-DNR-DsRed-Express-AKT2. The recombinant plasmid was transfected into PANC1 cells, and the transfection efficiency was detected. The expression of AKT2 gene was assayed by RT-PCR. Results DNA sequencing result showed that the shRNA express vector had been successfully constructed. The transfection rate was about 61% after 24 hours. The vecter-mediated shRNA induced RNA interfering(RNAi) and reduced the level of AKT2 mRNA significantly. Conclusion The RNA interference through shRNA can effectively inhibit the expression of target gene, and can be potentially useful in gene therapy of pancreatic cancer. Part IV shRNA Target to AKT2 Influence the Biological Behaviorof Pancreatic Cancer CellsObjective To study the inhibiting effect of shRNA on the expression of AKT2. To explore the regulation of PANC1 cell proliferation and apoptosis by AKT2 shRNA and expect to find a new way to conquer pancreatic cancer. Methods Recombinant plasmid pSIREN-DNR-DsRed-Express-AKT2 was transfected into the PANC1 cell mediated by lipofectamine. The transfected cells were examined the level of AKT2 protein by using western blot. The tetrazolium bromide(MTT) colorimetry was used to draw the growth curves. Cellular apoptosis was determined by hoechst33258 staining and flow cytometry(FCM). Results The vector-mediated AKT2 shRNA reduced the level of AKT2 protein. Compared with control group, the growth of transfected cells was inhibited. More transfected cells presented changes of apoptosis than control group. Conclusion AKT2 shRNA can inhibit proliferation and induce apoptosis of PANC1 cell. These results together indicate that suppression of AKT2 expression may be promising strategy that is selective for the pancreatic cancer therapy.