Study Of The Activation And Modulation Of IA Type PI3K/Akt Signaling Pathway In Gastric Adenocarcinoma

Gastric carcinoma is the most common and incurable digestive systerm tumor,which mortality is the highest.Studying its pathogenesis and trying to find more effective therapeutic modalities are the major research projects in medical field. Gastric carcinoma,just like tumors in other sites of the body,are involved in the processes of uncontrolled cell proliferation,cell dedifferentiation and dysregulation of cell apoptosis.With the development of molecular biology and molecular genetics,it has been found that some of the important oncogenes and tumor suppressor genes are related to the genesis of gastric carcinoma.However,up to date,it is still not clear which are the primary initiating molecular events and which are the secondary promoting molecular events.Therefore,seeking more genes associated with the genesis of gastric carcinoma,a comprehensive understanding of the molecular pathology of gastric carcinoma,and optimizing treatment strategies and developing novel therapeutic approaches for gastric carcinoma on this basis,have become an important research project for carcinoma.IA Type PI3K/Akt signaling pathway which regulates the proliferation and survival of tumor cells is closely related to the genesis and development of gastric adenocarcinoma.Its activity abnormality not only can induce cells malignant transfor -mation,but also influence the migration and adhesion of tumor cells,vascularization and degradation of extracellular matrix.The present study focused on the abnormal IA Type PI3K/Akt signaling pathway activity in gastric adenocarcinoma and siRNA targeting PI3Kp85αwas used to observe its inhibitory effect on the growth of human gastric adenocarcinoma SGC7901 cells and therapeutic efficacy of nude mice subcutaneous SGC7901 gastric adenocarcinoma model.The study was divided into 3 parts.In the first part,Using tissue microarray techniques and immunohistochemistry assay to survey the expression of PI3Kp85α,pAkt,Ki67,Bcl-2,MMP-2 in IA type PI3K/Akt signaling pathway during gastric adenocarcinoma malignant progression. In the second part,RNAi technology was used to observe its inhibitory effect on the growth of human gastric adenocarcinoma SGC7901 cells,siRNA targeting PI3Kp85αwas transfected into SGC7901 cells mediated by oligofectamine. PI3Kp85αmRNA expression were detected by realtime PCR after transfection.The expression of PI3Kp85αand other main downstream members including pAkt,Ki67, bcl-2,MMP-2 in IA type PI3K/Akt pathway was also studied by Western blotting and immunofluorescence staining after transfection.The phenotypic change of SGC7901 cells including proliferation,apoptosis and cell cycle after RNAi trasfection was studied by MTT assay,annexin V staining,flow cytometry;their motion and migra -tion ability was studied by scratch assay and 2-D Matrigel assay;the invasion ability was detected by Transwell analysis and 3-D Matrigel assay.In the third part,subcutaneous SGC7901 gastric adenocarcinoma model was established in nude mice.Every 4 days the mixture of oligofectamine and siRNA was injected into the tumors and the tumor volumes were measured.On 32^ndday after the first injection,tumors were resected.The expression of PI3Kp85α,pAkt,Ki67,Bcl-2, MMP-2 in IA type PI3K/Akt signaling pathway were studied by immunohistochemis -try.Apoptosis in tumors were detected by TUNEL method.Results:The first part:The expression of PI3Kp85α,pAkt,Ki67,Bcl-2,MMP-2 in IA type PI3K/Akt signaling pathway were overexpressed during gastric adenocarcinoma malignant progression.The second part:SGC7901 cells were transfected with siRNA targeting PI3Kp85αmediated by oligofectamine in vitro.SGC7901 mRNA expression were obviously knocked down after transfection with siRNA.SGC7901 ceils transfected with siRNA targeting PI3Kp85αshowed lowering proliferation activity by MTT, while the expression of PI3Kp85α,pAkt,Ki67,Bcl-2,MMP-2 were downregulated by Western blot analysis and immunohistochemical staining.The transfected cells had higher apotosis rate by annexin V staining and most cells arresting in the Go/G1phase by flow cytometry.The migration and invasive ability was attenuated by scratch assay,2-D Matrigel assay,Transwell analysis and 3-D Matrigel assay.The third part:Subcutaneous SGC7901 gastric adenocarcinoma model was established in nude mice for in vivo study.As compared with the control group,the tumors in mice treated with siRNA targeting PI3Kp85αgrew slowly and the different ce of tumor volumes became significant since the 20th day after the first time of siRNA therapy until the 32^ndday tumors were resected.PI3Kp85α,pAkt,Ki67, Bcl-2,MMP-2's expression were downregulated by immunohistochemistry. Meanwhile,cell apoptosis was increased by TUNEL method.Conclusion:1.PI3Kp85α,pAkt,Ki67,Bcl-2,MMP-2's overexpression can lead to IA type PI3K/Akt signaling pathway activation in gastric adenocarcinoma and result in cell proliferation and invasion.Meanwhile,the several proteins in this signaling pathway links to each other and maybe they are modulated with each other.2.Using RNAi technology,siRNA targeting PI3Kp85αmediated by oligofectamine efficiently knocks down the expression of PI3Kp85αin human gastric adenocarcinoma SGC7901 cells,inhibits activation of IA type PI3K/Akt signaling activity,results in decrease of cell proliferation activity and attenuateion of invasive and migration ability,and induces apoptosis.3.The established subcutaneous SGC7901 gastric adenocarcinoma models in nude mice are treated with siRNA targeting PI3Kp85α.The tumor growth is inhibited and cell apoptosis is induced.The findings of in vivo study is in accordance with those in vitro study4.Using RNAi technology to knock down the expression of PI3Kp85αin gastric adenocarcinoma is efficient in gastric adenocarcinoma therapy.PI3Kp85αcan be a candidate gene for gene therapy of gastric adenocarcinoma,which will have potential application prospects in clinics.