Effect Of SiRNA-mediated AP4 Gene Silence On Apoptosis Of Human Lung Cancer Line A549 And Clinical Analysis Of Lung Cancer Cases

Objective: AP4 played an important role in the process of cell apoptosis and DNA renovate. Many studies in the world have shown that AP4 genes involved in a wide variety of tumor development, such as gastric cancer, hepatoma, colon carcinoma, and breast carcinoma. But there aree very few studies in lung cancer. In this research, we observed the morphological changes of A549 cell before and after transfection, tested the expression of AP4 protein and mRNA in A549 cell of lung neoplasm, which was transfection by siRNA of AP4, discussed the down-regulation of AP-4 expression affects apoptosis in lung cancer and apoptosis-related genes. provided a new theoretical basis for clinical treatment of lung cancer. By analysis of the three cases of patients with lung cancer, we understood that it was difficult to discover early stage of lung cancer because of the unconspicuous symptoms, an important approach of detecting early stage of lung cancer in time was lung cancer census(imageological examination).Methods: Human lung cancer cell line A549 cells were cultured and divided into four groups:Blank group(B); Mock group(M): treated with the transfection reagent only; SiRNA transfection group; Negative control group(N). Transient transfection of small interfering RNA(siRNA) against AP4 on human lung cancer cell lineA 549 cells were performed by Lipofectamine 2000. Observed the change of the cell morphology,then detected the AP4 gene mRNA and the expression of protein by the respectively using of Real-time PCR and Western blot detection; with the detected apoptosis rate of A549 cells after transfection by FCM, discussed the protein expression level of apoptosis-related genes, Bcl-2 and Caspase-9. Selected three cases of elderly patientswith lung cancer, analyzed those cases and contrasted the imaging data and postoperative pathological report of the patients, discussed the clinical significance of imaging examination for early detection and promptness diagnosis of lung cancer.Result: With the successful transfection into A549 cells by AP4 si RNA( the transfection efficiency could be a maximum of 95%), we could observed that, before the transfection, cytoplasm was satiation and well attached to cytoderm, most cells tend to have a spindle cell pattern, while compared with the cells after transfection, we could observed that the adhesion was poor and the cells were shrinking.(1)Respectively detected the expression quantity of A549 cells for AP4 siRNA24 after transfection and A549 cells which were 48 hours later by Real-time PCR. It turned out that the expression quantity of AP4 mRNA was no significant change in group B, M and N, while it was dramaticlly decline in the group siRNA(430 、 569)(P<0.05).(2)Respectively detected the expression quantity of A549 cells for AP4 siRNA 24 after transfection and A549 cells which were 48 hours later by Western blot. It turned out that the expression quantity of AP4 protein was no significant change in group B, M and N,while it was dramaticlly decline in the group si RNA(284 、 430 、 569)(P<0.05).(3)Used the Real time PCR and Western blot experiments to test the AP4 expression quantity of siRNA(284、430、569) three groups in A549 cell, which were silenced by Interference fragment, of lung adenocarcinoma A549 cell interference fragment silent AP4 expression, according to the results, we coule observed that AP4-siRNA(569) fragment silence the highest efficiency.(4)Used the flow cytometry instrument to detect apoptosis of lung adenocarcinoma A549 cells which were silenced by AP4 siRNA interference fragment, the results showed that, the total rate of apoptosis for Group B was5.573%±0.297%, for Group M was 8.896%±0.517%, for Group N was6.363%±0.066%,and for siRNA-AP4(569) transfection group was 28.047%±0.376%,so we could observed that the results of siRNA-AP4(569) transfection group had statistically significant comparing with the other groups(P < 0.05).(5)Tested the the protein expression level of Bcl-2 and Caspase-9 genes which were related to apoptosis furtherly, the results showed that the Bcl-2 protein level in the AP4 si RNA(569)transfection group was higher than that in group B, N, M, while the Caspase-9 protein levels was also higher than other groups, this difference was considered to be statistical significance.Especially elevated levels of Caspase-9 protein is more pronounced.Conclusion: It could induce its apoptosis by using RNA interference to silent the AP4 gene expression of human lung adenocarcinoma cells, which may be associated with the expression of apoptosis related gene. Therefore, the AP4 gene may promote the development of lung cancer, and it would become a new target gene in the therapy of lung cancer. Lung cancer census(imageological examination) could detect and treat early stage of lung cancer in time, meanwhile, it could effectively improve the survival rate of lung cancer and reduce the mortality.