Expression Of Transcription Factor AP-4 In Gastric Cancer And Its Biological Function And Mechanism In Gastric Cancer Cell

PartⅠThe expression of transcription factor AP-4 in gastric carcinoma and its clinical significanceObjectives:To evaluate the expression of transcription factor AP-4 in gastric cancer, explore the AP-4 role and relationship between the AP-4 expression the clinical pathological data in gastric cancer.Methods:98 gastric carcinoma specimens and 66 normal tissues which were adjacent cancer tiusses were obtained from patients between 2005 and 2007, the expression of AP-4 was detected in different tiusse with immunohistochemistry and western blotting in protein level and with real time quantitative PCR in mRNA level and these data were examined for correlation with histology, pTNM stage, and prognosis.Results:The AP-4 expression rate was 83.67% in a total of 98 gastric cancer tissues, which was significantly higher than 40.91% in non-neoplastic tissues, there was significant difference(P<0.0001), and AP-4 expression has a significantly positive correlation with the depth of tumor invasion (P<0.0001), degree of tumor differentiation (P=0.0058), lymph node metastasis (P=0.0255), and pTNM stage (P=0.001); AP-4 relative expression shows a significant difference between gastric cancer and normal tissues with real time PCR and western blotting. Survival analysis showed that AP-4-positive patients' median survival time was significantly shorter than that of AP-4-negative patients.Conclusion:The AP-4 was significantly overexpression in tumor tissues, and AP-4 expression in gastric cancer is associated with clinicopathological parameters of gastric cancer, such as differentiation, lymph node metastasis, depth of invasion and pTNM stage. What's more, AP-4 overexpression indicated a worse prognosis for patients. So AP-4 gene may be an oncogene and play a important role in development of gastric cancer. PartⅡEffect of down-regulation of AP-4 expression for proliferation and chemotherapy in gastric cancer cellsObjective:To investigate the down-regulation of AP-4 expression affects gastric cancer cell proliferation and effect to chemotherapy.Methods:AP-4-specific-siRNAs were transfected into AGS cells, the AP-4 expression was detected with real time PCR and western blotting at diferent time points after transfection, the control siRNA and the untransfected AGS cells served as controls. Forty-eight hours post-transfection, the cell proliferation was evaluated by MTT. Six hours post-transfection, we replaced the medium with the madium containing different concentrations chemotherapy drugs, and detect cell proliferation after 48 hours.Results:AP-4 specific siRNAs were transfected into AGS cells and suppression of AP-4 expression started at 24 hours, lasted 96 hours. The most efficient timepoint were 48 h and 72 h in suppressing the AP-4 expression. Forty-eight hours post-transfection, cells relative growth rate:negative control group was 98.52±7.32%, AP-4-siRNA-1 group was 66.28±1.79%, AP-4-siRNA-2 group was 61.55±2.33%, the difference was statistically significant between them. Effect of siRNA combined with chemotherapy drugs was evaluated, the relative inhibitory rate:5-Fu(10μg/ml) groups:blank control group was 27.93±4.59%, negative control group was 30.70±0.87%, AP-4-siRNA-1 group was52.23±3.71%, AP-4-siRNA-2 group was56.74±3.66%; 5-Fu(20μg/ml) groups:blank control group was 51.77±2.82%, negative control group was 49.03±2.31%, AP-4-siRNA-1 group was 73.80±4.64%, AP-4-siRNA-2 group was 75.11+2.67%; cisplatinum(0.5μg/ml) groups:blank control group was 20.65±4.71%, negative control group was 18.30±3.13%, AP-4-siRNA-1 group was 50.66±3.05%, AP-4-siRNA-2 group was 61.54±3.64%; cisplatinum(1.0μg/ml) groups:blank control group was 45.24±0.77%, negative control group was 48.82±3.43%, AP-4-siRNA-1 group was 70.03±1.48%, AP-4-siRNA-2 group was 71.49±3.99%; doxorubicin(0.4μg/ml) groups:blank control group was 28.43±3.92%, negative control group was 27.52±4.68%, AP-4-siRNA-1 group was 55.95±1.67%, AP-4-siRNA-2 group was 63.70±1.47%; doxorubicin(0.6μg/ml) groups:blank control group was 53.88±2.55%, negative control group was 55.23±3.26%, AP-4-siRNA-1 group was 77.36±4.35%, AP-4-siRNA-2 group was 79.57±3.69%, the difference was statistically significant between AP-4 specific siRNAs group and blank control group, negative control group.Conclusion:Down-regulation of AP-4 can inhibit cell proliferation, increased inhibition ratio of chemotherapeutic agents. So the transcription factor AP-4 may promote gastric cancer development and increase resistance to chemotherapeutic drugs. PartⅢEffect of down-regulation of AP-4 expression for cell cycle arrest and apoptosis in gastric cancer cellsObjective:To study the effect of AP-4 for cell cycle arrest and apoptosis in gastric cancer cells, and explore its mechanism.Methods:48 hours post-transfection, cell cycle arrest and apoptosis were detected with flow cytometry, and cell cycle regulators, p21, p53 and cyclin D1, and apoptosis-related genes, Bcl-2, Bcl-xL, Bax, Caspase-8 and Caspase-9, expression was examined with real time PCR in mRNA level.Results:48 hours post-transfection, cell cycle distribution:blank control group:G0/G1 phase of 48.44±2.35%, G2/M phase was 18.91±0.52%, S phase was 32.65±1.02%; negative control group:G0/G1 phase was 50.38±2.47%, G2/M phase was 18.44±0.43%, S phase was 31.235±0.98%; siRNA-1 group:G0/G1 phase was 61.92±3.86%, G2/M phase was 13.72±0.34%, S phase was 24.36±0.96%; siRNA-2 group:G0/G1 phase was 63.67±3.31%, G2/M phase was 13.42±0.37%, S phase was 22.91±0.89%; AP-4-specific-siRNAs can cause cell cycle arrest in G0/G1. The down-regulation of AP-4 can promote the expression of p21 and p53, and inhibition of cyclin D1 expression. Post-transfection, the cells apoptosis rate was significantly increased. AP-4-siRNAs could suppress the Bcl-2 and Bcl-xL expression in mRNA levels, while Bax, Caspase-8 and Caspase-9 expression was up regulation.Conclusions:Silencing of AP-4 can cause cell cycle arrest and trigger apoptosis, which may be related to cell cycle regulators and apoptosis related genes.