The Effect Of Akt2-sirna Interference To Human Adenocarcinoma Of Lung Cancer Cell A549

[Background and Objective]Cancer is one of the diseases which severely threaten human health. Lung cancer is a kind of primary tumor of the lung. In Recent years, lung cancer is becoming one of the malignant tumor which causes the highest morbidity and mortality because of smoking and a variety of environmental factors, and more than 50% of clinically diagnosed lung cancer is in advanced stage. Although the surgical treatment, radiotherapy and chemotherapy have made some progress, the survival rate of lung cancer has not been significantly improved. Therefore, there is an urgent need to find new diagnostic targets and effective treatment. With the improvement of molecular biology techniques, RNA interference(RNAi), as a new and powerful research tool, has broad application prospect in exploring gene function and the treatment of human disease.RNAi is the phenomenon of posttranscriptional gene silencing triggered by endogenous or ectogenesis double-stranded RNA(dsRNA). dsRNA is degraded to small fragments of RNA which has 21-23 pieces of nucleotides, that is small interfering RNA(siRNA). And using the siRNA as template, messenger RNA (mRNA) with homologous RNA paragraph is specially degraded to inhibit gene expression.Akt is one of gene coding serine/threonine protein kinase. It is named protein kinase B(PKB) because it has homologous subsequence with protein kinase A and protein kinase C. As the target protein of the downstream of phosphoinositide 3-kinase(PI3K), Akt is the key point in kinds of signal transduction pathway. Akt signal transduction pathway is activated in a large number of human tumors, moreover, the activation of Akt is related to the tumor prognosis. Downstream substrate of Akt involved in important biological functions, such as protein synthesis, cell cycle regulation, apoptosis. So Akt has become an important target for therapeutic intervention of the tumor. There are three Akt subtypes: Akt1, Akt2 and Akt3. It's found that three Akt isoforms participate in the activation of downstream substrates by researching Akt and its subtypes, of which Akt2 is the most important one. High positive rate of Akt2 was detected in pancreatic cancer, breast cancer, ovarian cancer and lung cancer.We take Akt2 as target point, use specific siRNA of Akt2 to inter -erence human adenocarcinoma of lung cancer cell A549, observe whether it can inhibition the expression of Akt2 or impact the chemosensitivity of the tumor cells, provide experimental basis for research on the gene function of Akt2 and lung cancer treatment.[Methods]Akt2-siRNA was transfected into A549 cells by liposome 2000. Stable cell lines were screeninged. The A549 cells were divided into three groups: Akt2-siRNA interference group, independent siRNA control group and untransfected group. Akt2 mRNA and protein expression were detected by reverse transcription PCR and western blot. The impact of siRNA interference, paclitaxel application to the apoptosis of A549 cells was observed by flow cytometry.[Results]1. The expression of Akt2 mRNA in Akt2-siRNA interference group was conspicuously lower than the independent siRNA control group and untransfected group. Grey scale scanning and statistical analysis appeared that the difference was significant (P<0.05) .2. Total Akt2 protein of three groups were extracted. Western blot detecting result appeared that the expression of Akt2 protein in siRNA interference group was conspicuously lower than the independent siRNA control group and untransfected blank group. Grey scale scanning and statistical analysis appeared that the difference was significant (P<0.05) .3. The apoptosis of A549 cells which firmly expressing Akt2-siRNA or induced bypaclitaxel for 48 hours was promoted obviously. The apoptosis effect of A549cells induced by paclitaxel was stronger than cells interferenced by Akt2-siRNA.If the Akt2-siRNA transfected A549 cells were induced by paclitaxel for 48 hours,the apoptosis effect was enhanced.[Conclusions]1. Akt2-siRNA can obviously reduce the Akt2 mRNA expression of human adenocarcinoma of lung cancer cell A549.2. Akt2-siRNA can obviously reduce the Akt2 protein expression of human adenocarcinoma of lung cancer cell A549.3. Akt2-siRNA can obviously promote the apoptosis of human adenocarcinoma oflung cancer cell A549. The stronger apoptosis effect is appeared in the cells whichtransfected by Akt2-siRNA and induced by paclitaxel.