The Expression Of PKM2 In Ovarian Cancer And Its Effect On Glucose Metabolism, Proliferation And Migration Of Ovarian Cancer Cells

Ovarian malignant tumor(OMT)is a common malignant tumor of the female reproductive system.Because of its insidious onset,rapid progress and lack of effective early diagnostic methods,70%of patients are suffering in the late stage,and the five-year survival rate in patients with ovarian cancer is only 15%-30%.So it is important to investigate the mechanism that is critical to the development of the disease,and to identify novel targets for therapy to prolong the survival time of ovarian cancer patients.In recent years,with the research on the metabolism of tumor,more and more evidence shows that metabolic disorder is a core problem in the process of tumorigenesis and metabolic reprogramming is indispensably a genetic event which mediated malignant transformation.Pyruvate Kinase Isotype M2(PKM2)is a key rate-limiting enzyme in glycolysis pathway and plays an important role in the Warburg effect.PKM2 plays a crucial role in tumor glucose metabolism and proliferation makes it a potential therapeutic target for the treatment of various cancers.However,the mechanism of PKM2 in ovarian cancer still remains unclear.We performed the experiments in three parts:(1)The expression of PKM2 in ovarian cancer cells and tissues was assessed and correlated with clinicopathological parameters;(2)We investigated the effects of PKM2 in proliferation and metastasis by depleting its expression using short hairpin RNA(shRNA)in human ovarian cancer cell lines;(3)We further try to explore the regulating function of PKM2 in tumor glucose metabolism and to explore the regulation role of FSH on the expression of PKM2 and cell metabolism to affect cell proliferation.Part Ⅰ Expression of PKM2 in ovarian cancerObjective:To assess the expression of PKM2 in ovarian cancer cells and tissues and correlated with clinicopathological parameters.Methods:We used Real-time PCR and Western blot to screen ovarian cancer cell lines(C200,Caov3,SKOV3,HO8910 and OVCAR3)in which PKM2 was expressed.We detected 60 samples of ovarian carcinoma and 20 samples of benign ovarian tissue using immunohistochemistry method and calculated a composite histoscore to account for both stain intensity and uniformity.The expression of PKM2 in ovarian cancer tissues was assessed and correlated with clinicopathological parameters.Result:The expression of PKM2 in ovarian cancer tissue was significantly increased in EOC when compared with the benign ovarian tissue,but the expression level of PKM2 had no correlation with pathologic type(P=0.81).Further reseach showed PKM2 expression was associated with cell differentiation degree(P<0.01)and tumor size(P<0.05)in SOC.We also detected higher expression levels of PKM2 in human ovarian cancer cell lines and the expression of PKM2 in SKOV3 and OVCAR3 cells was significant higher.Conclusion:PKM2 is highly expressed in ovarian cancer cells and tissues and correlated with tumor size and cell differentiation,which suggest that PKM2 may promote the malignant progression of ovarian cancer.Part Ⅱ The effect of PKM2 on biological function of ovarian cancer cellsObjective:To investigate the biological function of PKM2 in ovarian cancer cells.Methods:PKM2 gene interference lentivirus vectors were built by miRNA transfection assay and we investigated the role of PKM2 in migration,invasion,proliferation and apoptosis using siRNA knockdown experiments in SKOV3 and OVCAR3 cell lines.Cell proliferation was determined by CCK-8.The transwell migration and invasion assays were performed to study the migratory and invasive ability of ovarian cancer cells.Cell cycle and apoptosis were examined by flow-cytometric analysis.Western blot and Real-time PCR were performed to measure the expression of the tumor-related genes E-cadherin,MMP2,MMP9,Bad,HIF1a,Caspase7 and VEGF after the down-regulation of PKM2。Results:siRNA-PKM2 treatment markedly inhibited cell proliferation,induced apoptosis and caused cell cycle arrested at G0-G1 phrase;Cell migration and invasion were also significantly suppressed by siRNA-PKM2 in SKOV3 and OVCAR3 cells.Further,the tumor-related genes caspsae7,bad and E-cadherin were up-regulated,while MMP2,HIF1a,VEGF and MMP9 were depressed by siRNA-PKM2.Conclusions:siRNA-PKM2 inhibited proliferation and metastasis of SKOV3 and OVCAR3 ovarian cancer cell lines and several tumor supressor genes and oncogenes were up and down regulated.Part Ⅲ Effect of down-regulation of PKM2 on the proliferation and glucose metabolism of ovarian cancer cells induced by FSHObjective:investigate the regulating function of PKM2 in tumor glucose metabolism and to explore the role of FSH on the expression of PKM2 and cell metabolism to affect cell proliferation.Methods:Lactate production and glucose consumption were measured to analyze the alteration of cell glycolytic rate after the down-regulation of PKM2 in SKOV3 and OVCAR3.Then the two cell lines were treated with different concentrations of FSH and the altered expression of PKM2 and glycolytic state were observed.The proliferation induced by FSH was further measured after down-regulation of PKM2.Results:1.Knockdown of PKM2 inhibited glycolysis rate in OVCAR3 and SKOV3 cells.2.FSH could upregulate the expression of PKM2 in mRNA and protein levels,and meanwhile,increase glucose consumption and lactate production in SKOV3 and OVCAR3.3.FSH has the effect of promoting the proliferation and the effect could be inhibited by the knockdown of PKM2,and the glucose consumption and lactate production was also decreased.Conclusion:Knockdown of PKM2 inhibits the aerobic glycolysis in ovarian cancer cells.FSH can induce the expression of PKM2 and promote aerobic glycolysis in ovarian cancer cells and the effect of FSH on promoting proliferation can be inhibited by knockdown of PKM2.