Study On The Expression And Modulation Of PI3K/Akt Signaling Pathway In Human Ovarian Carcinoma

Ovarian cancer is a common solid tumor and is the lead cause of death from gynaecological malignant tumor.Disease progression and patient decline is typically due to locoregional peritoneal dissemination and its consequence rather than due to visceral metastatic disease.The prognosis of the patients with ovarian cancer has not been improved in recent years,with 5-years survival of 25%-30%,even though combined therapeutic modalities including surgical resection,chemotherapy and radiotherapy are currently available.As the knowledge of tumor biology and molecular genetics increased,it has been shown that the development of ovarian cancer,just the same as the tumors in the other site of the body,results from the activation of proto-oncogenes and inactivation of tumor suppressor genes,and involves multiple genetic and molecular alterations.A number of studies showed that PI3K/Akt signaling pathway is closely related to the genesis and development of tumor.AKT play a central role in the promotion of cell proliferation and invasion.However,as to our knowledge,the expression of PI3K/Akt and its role in ovarian cancer has not been fully investigated,so the present study aims at better understanding of the role of AKT and inactivation of IA Type Pi3K/Akt pathway in the prevention of progression of ovarian cancer.The study was divided into 3 parts:In the first part,using tissue microarray techniques and immunohistochemistry assay to survey the expression of PI3Kp85α,AKT1,AKT2 and Ki67 in ovarian cancer as well as the relationship among the expression level of PI3Kp85α,AKT1,AKT2,Ki67 and their correlation with degree of malignancy of ovarian cancer.In the second part,RNAi technology was used to observe its inhibitory effect on the growth of human ovarian carcinoma SKOV3 cells,siRNA targeting PI3Kp85αand AKT1 were transfected into SKOV3 cells mediated by oligofectamine.The expression of PI3Kp85αand AKT1 mRNA were detected by realtime PCR after transfection.The expression of PI3Kp85α,AKT1,AKT2,Ki67 was also studied by Western blotting and immunofluorescence staining after transfection.MTT methods and annexin V staining were used to evaluate cell proliferation and apoptosis.Flow cytometry was used for cell cycle analysis.Tumor invasion was examined by Transwell analysis.In the third part,siRNA targeting AKT1 were transfected into human ovarian carcinoma SKOV3 cells,and differentially expressed genes were examined by using human Oligo genechip containing 35035 genes.ResultsThe first part:Expression analysis of PI3Kp85α-AKT1-AKT2-Ki67PI3Kp85α,AKT1,AKT2,Ki67 were overexpressed in ovarian cancer tissue.The expression of PI3Kp85α,AKT1,AKT2 and Ki67 were increased correspondingly to the ascending of tumor grade,clinical stages malignant progression.In addition,the expression of PI3Kp85α,AKT1,AKT2 and Ki67 correlated positively with each other. Such results imply that,PI3K/Akt pathways might be important for the progression of ovarian cancer,and activation of this pathway may contribute to tumor proliferation and invasion,which are the most important phenotypes of ovarian cancer.Moreover, interruption of the PI3K/Akt pathway suppose to be a key strategy in prevention the progression of ovarian cancer,PI3Kp85α,AKT1,AKT2,might be a candidate for gene therapy of ovarian cancer.The second part:In vitro studySKOV3 cells were transfected with siRNA targeting PI3Kp85αand AKT1 mediated by oligofectamine in vitro.SKOV3 mRNA expression were obviously knocked down after transfection with siRNA.As compared to control and nonsense siRNA transfected cells,SKOV3 cells transfected with siRNA targeting PI3Kp85αand AKT1 showed lowering proliferation activity by MTT,while the expression of PI3Kp85α,AKT1,AKT2,Ki67 were downregulated by Western blot analysis and immunofluorescence staining.The transfected cells had higher apotosis rate by annexin V staining and most cells arresting in the Go/G₁ phase by flow cytometry. The migration and invasive ability was attenuated by Transwell analysis.The third part:Relevant gene expression profiles changes by silencing AKT1 with siRNASilencing AKT1 gene with small interfering RNA in vitro,the difference of gene expression profile were analyzed by genchip.There were 37 common genes showed difference greater than 2 fold in gene expression array,including 21 genes down,and 16 up regulated.There were 38 pathways significant correlated by searching from Kegg,BioCarta and GenMAPP data base.15,15 and 18 genes were significant correlated to molecular function,biological process and cellular component respectively.Conclusion:1.PI3Kp85α,AKT1,AKT2 and Ki67 were overexpression in ovarian cancer.The expression of PI3Kp85α,AKT1,AKT2 and Ki67 were increased correspondingly to the ascending of tumor grade and clinical stages.Suggested that IA type PI3K/ Akt signaling pathway might be important for the progression of ovarian cancer, and activation of this pathway may contribute to tumor proliferation and invasion.2.Silencing PI3Kp85αand AKT1 genes with small interfering RNA blocked ovarian carcinoma endothelial cell proliferation and migration,arrested cell cycle and induced apoptosis in vitro.3.Silencing AKT1 gene with siRNA in vitro,the difference of gene expression profile were analyzed by genchip.There were 37 common genes showed difference greater than 2 fold in gene expression array,including 21 genes down, and 16 up regulated.There were 38 pathways significant correlated by searching from Kegg,BioCarta and GenMAPP data base.15,15 and 18 genes were significant correlated to molecular function,biological process and cellular component respectively.The information would be analyzed by system-level understanding,the approach advocated in systems biology forward.4.Manipulating the PI3K/Akt pathway will be likely to provide rich therapeutic benefits ultimately.PI3Kp85αand AKT1 can be the candidate genes for gene therapy of ovarian cancer.PI3K and AKT inhibitors will therefore find indications once successfully integrated with chemotherapy.