| Objective:Gastric cancer is the most common malignant tumor in digestive system,which doesn't have special symptom in early stage,and most of which is advanced stage when discovered,patients will die in a short time.Although there are many methods to treat it,such as radiotherapy,chemotherapy and so on,except early gastric cancer can be healing well by surgery,others are not satisfied.In recent years, With the development of molecular biology and molecular genetic,it has been found a new therapy,that is to say gene therapy,and it has been a focus to study.Akt is the key gene in PI3K/Akt survival signal pathway,which regulate the cell growth,survival,proliferation,invasion and apoptosis through phosphorylate downstream substrate,and abnormal activation of Akt closely relates to the genesis and development of tumor.Methods:The present study focused on the abnormal expression of Akt signal pathway in gastric adenocacinoma,and RNA interference technology was used to detect the effect of malignant phenotype of gastric cancer when it is abnormally actived.The study was divided into 3 parts.In the first part,using tissue microarray and immunohistochemistry to detect the expression of Akt1 and the members in the signal pathway,found the abnormal expression of MMP2,Ki-67,Bcl-2 and PTEN.In the second part,RNAi technology was used to observe its inhibitory effect on the growth of human gastric cancer cells SGC7901.SiRNA targeting Akt1 was transfected into SGC7901 cells mediated by oligofectamine.Akt1 expression were detected by realtime PCR and western blotting technology after transfection.The expression of other main members in Akt1 patheway were also studied The phenotypic change of SGC7901 cells including proliferation,apoptosis and invasive ability was studied by MTT assay,annexin V staining,flowcytometry and Matrigel 3D growth experiment.In the third part,subcutaneous SGC7901 gastric cancer model was established in nude mice.The mixture of oligofectamine and siRNA was injected into the tumors and the tumor volumes were measured.And the expression of Akt1 and other members in Akt1 pathway were studied by immunohistochemistry after resected. Apoptosis in tumors were detected by TUNEL method..Results:The first part:The expression of Akt1,MMP2,Ki-67,Bcl-2 in gastric carcinoma were upregulated,and,high expression was observed in poor differentiation. gastric carcinoma.However,the expression of PTEN was reversed.The second part:The expression of Akt1 was obviously knocked down after transfection,and the expression of other members were also decreased.The result of flowcytometry revealed that apoptosis cells increased,cell cycle was arrested in G0/G1phase.MTT assay indicated that cell proliferation rate was inhibited obviously,matrigel experiment and transwell assay showed that cell invasive ability was attenuated,cell immigration decreased dectected by scrach assay.The third part:The tumors in mice treated with siRNA targeting Akt1 grew slowly The expression of Akt1 and other members were decreased detected by immunohistochemistry.Meanwhile,cell apoptosis was increased.Conclusion:1.Abnormal expression of Akt1 and other members in the pathway lead to cell proliferation and invasion,block the apoptosis of cell.2.Using RNAi technology,siRNA targeting Akt1 mediated by oligofectamine efficiently knocks down the expression of Akt1,inhibits activation of Akt1 pathway, results in decrease of cell proliferation activity and invasive ability,arrests cell cycle and induces apoptosis.3.The established subcutaneous SGC7901 gastric cancer models in nude mice are treated with siRNA targeting Akt1.The tumor growth is inhibited and cell apoptosis is induced.4.Akt1 can be the candidate genes for gene therapy of gastric cancer.RNAi is a new gene silencing technology with high efficiency,which will have potential application prospects in clinics. |
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