| Esophageal squamous cell carcinoma(ESCC)is a highly prevalent cancer with a poor survival rate and prognosis,drawing on many signal pathways during its metabolism,growth and transformation.A complex signaling network is formed by various signal pathways involved,which makes the therapeutic efficacy of ESCC is not as good as expected.It has been proved that it is of great significance to control the development of tumor by regulating metabolism as a therapeutic target.Different from normal cells,tumor cells obtain energy by glycolysis,no matter oxygen is rich or not.The pyruvate kinase M2 isoform(PKM2)catalyzes the last and physiologically irreversible step in glycolysis,the conversion of phosphoenolpyruvate(PEP)to pyruvate through the transfer of a phosphate group to ADP.The mechanism of PKM2 in tumor cells remains to be further studied.Substantial evidence from human malignancies indicates that the upregulation of PKM2 plays a crucial role not only in glucose metabolism but also in gene transcription and tumorigenesis.The mTOR(Mammalian target of rapamycin)pathway regulates an array of cellular functions,especially protein synthesis.The pathway is hyperactive in many cancers,and it can regulate transcription factors to enzyme formation.It is reported that mTOR can regulate the expression of PKM2 in liver and kidney tumors.Therefore,the study was designed experiments to observe whether mTOR signaling pathway can regulate the expression of PKM2 in ESCC,and whether it can affect the energy metabolism of tumor cells.An immunohistochemical staining assay with pathological ESCC sections determine the expression of mTOR and PKM2 in human tumor tissues.To make clear the effects of different expression levels of mTOR and PKM2 on metabolism of ESCC cell lines to prove that mTOR can regulate PKM2 and affect the aerobic glycolysis of ESCC cells to provide new targets and strategy for the treatment of ESCC.Objective1、Effects of PKM2 on aerobic glycolysis in ESCC cell line.2、Effects of mTOR signaling pathway on aerobic glycolysis in ESCC cell line.3、The mTOR pathway regulate PKM2 to affect aerobic glycolysis in ESCC cell line.Method Part 1: Data analysis of tissue samples of ESCC patients.To verify the expression of PKM2 and mTOR in ESCC,30 cases of ESCC and nontumoral tissue samples were selected.Immunohistochemical staining was used to detect the expression of PKM2 and mTOR in ESCC and non-tumoral tissues.Part 2: The expression of PKM2 affect the aerobic glycolysis of ESCC.1、ESCC cell line KYSE150 was transfected with shRNA,and detected the effect of down-regulation of PKM2 on glucose consumption and lactate production.2、Up-regulate PKM2 in ESCC cell line KYSE150 and detect the effect of PKM2 over-expression on glucose consumption and lactate production in ESCC cell.Part 3: Effects of inhibition or activation of mTOR signaling pathway on aerobic glycolysis in ESCC cell.1、Rapamycin,a specific mTOR inhibitor,acted on KYSE150 cell to observe the effect on mTOR signaling pathway and its effect on the aerobic glycolysis of ESCC.2、Effect of down-regulation of mTOR inhibitor PTEN on mTOR signaling pathway and aerobic glycolysis in ESCC cell.Part 4: mTOR pathway affects the aerobic glycolysis of KYSE150 cells by regulating PKM2 in ESCC cell line KYSE150.1、mTOR inhibitor rapamycin act on KYSE150 cells to affect the expression of PKM2.2、Effect of inhibit PTEN expression on PKM2 and the influence of glycolysis in cell KYSE150.3、Even PKM2 was knocked down,the inhibition of PTEN gene can not activate the glycolysis of ESCC cell.Results:1.The mTOR and PKM2 both overexpress in ESCC.2.The effect of PKM2 on the regulation of aerobic glycolysis in ESCC.3.The effect of mTOR on the metabolism of aerobic glycolysis in ESCC cell4.mTOR pathway can regulate the PKM2 to affect the aerobic glycolysis in ESCC.Conclusion:This study illustrates higher expression of PKM2 and mTOR in ESCC,mTOR can regulate PKM2 in ESCC,to affect aerobic glycolysis in ESCC,providing a new strategy for the treatment of ESCC. |
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