To Evaluate The Role Of PKM2on Proliferation And Apoptosis In Gastric Adenocarcinoma Cells

Gastric cancer (GA) is one of the most common malignancies in worldwide，especially in the south-east coastal areas of China. Based on a large number of literature and the previous work of our laboratory, in this study, immunohistochemistry, eukaryotic PKM2expression vector transfection, Western blot, MTT, flow cytometry, ELISA and real-time fluorescent quantitative PCR assays were applied to evaluate the role of PKM2on proliferation and apoptosis in gastric adenocarcinoma and preliminary mechanism with the purpose to provide a foundation for discovering the possible therapeutic target of drug.In the first part of this study, the expression of PKM2and IL-6in113patients with GA was investigated by employing immunohistochemistry, and the relationship between PKM2overexpression and the clinical pathological characteristic of patients was then analyzed. We showed that PKM2and IL-6were overexpressed at61.9%（70/113）,61.1%（69/113）ratio respectively in gastric adenocarcinoma (GA) tissues compared to normal tissues of the same patients. PKM2overexpression level was closely associated with tumor size, TNM stage, invation beyond the serasa, lymph node metastasis and poor survival of GA patients（survival，P=4.78x10-4). By using COX model and K-M survival analysis, we reported that death risk of GA patients was higher with the increase of PKM2overexpression level. Compared to GA patients of no PKM2overexpression, death risk was sharply increased in GA patients had PKM2overexpression with1+,2+and3+level of cancer tissues（HR，2.66，10.01，18.74respectively）and median survival time(MST) was dramicly decreaded from67.0months to35.0months(Log Rank P=1.05×10-5) in GA patients with PKM2overexpression. PKM2overexpression level was closely correlated with IL-6overexpression level in GA tissues(R=0.678，P=2.05×10-5).In the second part of this study, RT-PCR was used to amply PKM2cDNA obtained by first reverse transcript total RNA from GA tissue then PCR. AKT3 cDNA was inserted into eukaryotic expression vector, pDsRed-Express-C1, by the BamHⅠrestriction sites. The recombinant vectors were confirmed by DNA sequencing, then transfected into AGS and MKN-45cells. Followed, western blot was applied to analyze the expressed effect of PKM2expression vectors in these two GA cells. The results demonstrated that the PKM2eukaryotic expression vector were successfully constructed and western blot confirmed that PKM2had a good expression in these two GA, providing foundations for the third part’s study.In the third part of this study, the activity of cell proliferation untransfected and transfected with PKM2gene was measured by MTT assay. The apoptostic change of normal and PKM2overexpressed cells were tested by flow cytometry. Concentration of IL-6in the cells culture supernatant was detected via ELISA assay. In addition, expression level of mRNA of IL-6was determined by QPCR. The proliferation activity of AGS and MKN-45cells transfected with PKM2gene was significantly increased （AGS，P=0.031；MKN-45，P=0.038）and the apoptosis was dramaticlly decreased（AGS，P=4.06x10-4；MKN-45，P=0.001）compared to those cells transfected with pDsRed-Express-C1only (control groups). Intrestingly, the same effect was showed in IL-6stimulation groups. The results suggested that the overexpression of PKM2gene results to the changes by up regulation of IL-6mRNA and protein secretion.