Studies On SIRT5 Regulation Of Glucose Metabolism And Proliferation In Colorectal Cancer Cells

ObjectiveTo study the effect of SIRT5 on the glucose metabolism and proliferation ability of colorectal cancer cells,and to preliminarily explore the mechanism of action and the expression of SIRT5 and HKⅡin colorectal cancer tissues and adjacent tissues,so as to clarify the role of SIRT5 and HKⅡin the occurrence and development of colorectal cancer,and to provide reliable basis for clinical treatment of colorectal cancer.Methods1.Human CRC cell lines-HCT116,SW480,SW620,HT29 and Lo Vo were cultured in vitro.Western blotting was used to detect the expression level of SIRT5 protein in these five cells,and two types of cells with the highest and lowest expression of SIRT5 protein were selected.2.SIRT5 plasmid was transfected into the cells with the lowest expression level to overexpress,SIRT5 si RNA was transfected into the cells with the highest expression level to knocked down SIRT5,and control cell lines were constructed respectively.3.The content of glucose in culture medium after transfection was determined by glucose oxidase method.4.The content of lactic acid in culture medium after transfection was determined by colorimetric method.5.The proliferation ability of transfected cells was detected by clonal formation assay.6.The CCK8 assay was used to detect the viability of transfected cells.7.The m RNA expression level of HK Ⅱafter transfection was detected by RT-PCR.8.The protein expression level of HK Ⅱafter transfection was detected by Western blot.9.SIRT5 si RNA and Flag-HK Ⅱ plasmid were co-transfected in HCT116 cells,and part 3-6 of the experiment was repeated.10.The expression level of SIRT5 and HKⅡ in colorectal cancer tissues was detected by immunohistochemical method.Results1.Western blotting results showed that SIRT5 expressed highest in HCT116 cells and expressed lowest in HT29 cells among the five colorectal cancer cell lines.2.Western blotting results showed that si RNA interference effect of SIRT5-si RNA#3 was the best.3.The results of glucose oxidase assay showed that the concentration of glucose in the culture medium increased significantly after SIRT5 knocking down,and the concentration of glucose in the culture medium decreased significantly after SIRT5 overexpressing,compared with the control group respectively.4.The results of the concentration of lactic acid in culture medium determined by colorimetric method showed that the concentration of glucose in the culture medium was significantly decreased after SIRT5 knocking down,while the concentration of glucose in the culture medium was significantly increased after SIRT5 overexpressing,compared with the control group respectively.5.The cell viability test results showed that the activity of cells was significantly decreased after SIRT5 knocking down,and the activity of cells was significantly increased after SIRT5 overexpressing,compared with the control group respectively.6.The results of the clone formation experiment showed that the proliferation ability of CRC cells was significantly reduced after SIRT5 knocking down,while the proliferation ability of cells was significantly enhanced after SIRT5 overexpressing,compared with the control group respectively.7.RT-PCR results showed that the expression level of HK Ⅱ m RNA was significantly decreased after SIRT5 knocking down,while the expression level of HKⅡ m RNA was significantly increased after SIRT5 overexpressing.8.Western blotting results showed that the expression level of HKⅡ protein was significantly decreased after SIRT5 knocking down,while the expression level of HKⅡ proteins were significantly increased after SIRT5 overexpressing.9.The results after co-transfecting of SIRT5 sh RNA and Flag-HKⅡ plasmid showed that SIRT5 promoted the consumption of glucose,the production of lactic acid and cell proliferation of colorectal cancer cells by regulating HKⅡ.10.Immunohistochemical results showed that the expression level of SIRT5 and HKⅡin colorectal cancer tissues was significantly higher than that in paracancerous tissues,and SIRT5 expression level was positively correlated with HKⅡ expression level.ConclusionsKnocking down SIRT5 can reduce the glucose consumption and lactic acid production in colorectal cancer cells,inhibit cell viability and clone formation ability,and inhibit the m RNA and protein expressions of HKⅡ.Overexpressing of SIRT5 promotes the glucose consumption and lactic acid production,inhibites cell viability and clonal formation ability,and promotes m RNA and protein expression of HK Ⅱ.Compared with adjacent tissues,the protein expression levels of SIRT5 and HK Ⅱ in colorectal cancer tissues are significantly increased,and the expression levels of SIRT5 and HK Ⅱ in colorectal cancer tissues are positively correlated.Therefore,our study confirmed that SIRT5 promotes aerobic glycolysis and cell proliferation of colorectal cancer cells by regulating HKⅡ.