The Functions And Mechanisms Of SLMI1 In Regulation Of Glycolysis In Melanoma Of Choroid

Objective1.To explore whether the SLIM1 gene regulating the growth function of melanoma of choroid is related to glycolysis.2.To investigate whether the SLIM1 gene inhibiting the growth of melanoma of choroid through the c-Myc gene mediated glycolysis.Methods1.MUM-2B cells at the stage of exponential growth were digested with trypsin and randomly divided into four groups.They were evenly spread into four 6 cm culture dishes and divided into four groups of ABCD.Group A was transfected with empty plasmid,group B was transfected with SLIM1 plasmid,group C was transfected empty plasmid and added 2-DG glycolysis inhibitor,group D was transfected with SLIM1 plasmid and added 2-DG glycolysis inhibitor.Cell Counting Kit-8(CCK-8)was used to detect the proliferation of cells in each group.The clonal formation analysis was used to verify the clonal formation status of cells in each group.Then,to explore the influence of SLIM1 gene on the growth of melanoma of choroid was related to glycolysis.2.MUM-2B cells at the stage of exponential growth were digested with trypsin and randomly divided into four groups.They were evenly spread into four 6 cm culture dishes and divided into four groups of ABCD.Group A was transfected with empty plasmid,group B was transfected with SLIM1 plasmid,group C was transfected with empty plasmid and c-Myc SiRNA,group D was transfected with SLIM1 plasmid and c-Myc SiRNA.CCK-8 assay was used to detect the proliferation of cells in each group.The clonal formation analysis was used to verify the clonal formation of cells in each group.Lactate,ATP and Glucose uptake assay were used to test the ability of cells in each group to produce lactate,ATP and glucose uptake.The extracellular acidification rate and oxygen consumption rate of cells in each group were detected by using the experimental method of extracellular acidification rate and oxygen consumption rate,so as to explore whether SLIM1 gene regulates glycolysis through c-Myc mediation and thereby inhibits the growth of melanoma of choroid.Results1.The results of CCK-8 growth curve experiment showed that compared with MUM-2B cells transfected with empty plasmid,MUM-2B cells overexpressing SLIM1 gene or added of 2-DG glycolytic inhibitor showed lower proliferation capacity.Compared with the difference of cell proliferation ability between the group transfected with SLIM1 plasmid and the group transfected empty plasmid,the difference of cell proliferation ability decreased after the addition of 2-DG glycolytic inhibitor.The results of clonal formation analysis showed that compared with MUM-2B cells transfected with empty plasmid,MUM-2B cells overexpressing SLIM1 gene or added of 2-DG glycolytic inhibitor showed lower clonal formation ability,which was manifested as smaller clone diameter and fewer clones.Compared with the difference of cell clonal formation ability between the group transfected with SLIM1 plasmid and the group transfected empty plasmid,the difference of cell clonal formation ability decreased after the addition of 2-DG glycolytic inhibitor,which means that the inhibition ability on the clonal formation ability of MUM-2B cells was weakened after the addition of 2-DG glycolytic inhibitor.2.The results of Western Blot showed that the expression level of c-Myc protein in MUM-2B cells was decreased in two groups after the knockdown of c-Myc.3.The results of CCK-8 growth curve experiment and clonal formation analysis showed that compared with MUM-2B cells transfected with empty plasmid,MUM-2B cells overexpressing SLIM1 gene showed lower proliferation and clonal formation capacity.Compared with the difference of cell proliferation and clonal formation ability between the group transfected with SLIM1 plasmid and the group transfected empty plasmid,the difference of cell proliferation and clonal formation ability decreased in two groups after the knockdown of c-Myc,which means that the proliferation and clonal formation ability of MUM-2B cells was reduced after the knockdown of c-Myc.4.The results of Lactate,ATP,glucose uptake,ECAR and OCR showed that compared with MUM-2B cells transfected with empty plasmid,MUM-2B cells overexpressing SLIM1 gene showed lower lactic acid,ATP production,glucose uptake and ECAR,and higher OCR.Compared with the difference of cell lactic acid,ATP production,glucose uptake,ECAR and OCR between the group transfected with SLIM1 plasmid and the group transfected empty plasmid,the difference was reduced in two groups after the knockdown of c-Myc.Conclusions1.SLIM1 gene inhibits the growth of melanoma of choroid through glycolysis.2.SLIM1 gene regulates glycolysis through c-Myc mediation,and inhibiting the growth of melanoma of choroid.