Study On The Effect Of TM6SF2 On Lipid Metabolism By Regulating APOB

Background and Purpose:Non-alcoholic fatty liver disease(NAFLD)is a clinicopathologic syndrome characterized by lipid accumulation in the liver.Transmembrane 6 superfamily member2(TM6SF2)has been demonstrated by numerous studies to play an important role in liver lipid metabolism.TM6SF2 E167 K polymorphism is related to the change of apolipoprotein B(APOB)expression level.The purpose of this study was to further determine whether TM6SF2 could affect the level of APOB and to investigate its effect on the accumulation of triglycerides.Methods(1)In this study,constructed plasmid transfection method was used to establish TM6SF2 overexpression cell models in liver cancer cell line Huh7 and intestinal epithelial cell line Caco-2.After the cell model was established,the cell protein was extracted,and the effect of TM6SF2 overexpression at the protein level was verified by western blot(WB).(2)Mouse models of intestine specific and liver specific knockout of TM6SF2 were established using Cas9/sg RNA system(c56bl/6).Normal mice of the same age were used as controls to verify the knockout effect of TM6SF2 at the protein level by WB.(3)High-fat diet and ordinary diet were dieted were fed for 16 weeks.The liver tissue and serum of mice were separated.GPO-PAP enzyme method was used to determine the levels of triglyceride in liver tissue,serum triglyceride and serum cholesterol in the intestine specific knockout group and the control group.The levels of triglyceride in liver tissue were measured in liver specific knockout group and control group.(4)Protein was extracted from intestinal tissues,liver tissues and cells,and the influence of TM6SF2 expression level on tissue and intracellular APOB expression level was investigated by WB at protein level.(5)Quantitative data were expressed in the form of mean ± standard deviation,and independent sample T-test was used for comparison between the two groups.P<0.05 was considered statistically significant.Result:(1)In cell lysis buffer,WB results showed both Caco-2 and Huh7 cells successfully overexpressed TM6SF2 protein.(2)In animal experiments,the modeling effect of gut-specific knockout of TM6SF2 in mice was verified at the protein level.WB results showed that the expression of TM6SF2 in the knockout group was significantly lower than that in the control group.Validation of TM6SF2 hepato-specific knockout mouse modeling at the protein level.The results of WB showed that TM6SF2 expression in liver tissue of knockout group was significantly lower than that of control group.(3)The determination of triglyceride levels in liver tissues of the TM6SF2 in gutspecific knockout group and the control group showed that: in the high-fat diet group,the triglyceride level in liver tissue of the TM6SF2 specific knockout group was significantly higher than that of the control group(P<0.05);There was no difference in triglyceride level between the knockout group and the control group in normal diet group(P>0.05).There was no difference in serum triglyceride level and total cholesterol level in TM6SF2-specific knockout group between high-fat diet group and ordinary diet group(P>0.05).The triglyceride level of liver tissue in the liver specific knockout group and the control group was determined: In the high fat diet group,the triglyceride level of liver specific knockout group was significantly higher than that in the control group(P<0.05).There was no difference in triglyceride level between the knockout group and the control group in normal diet group(P>0.05).(4)In order to further explore the mechanism of the effect of TM6SF2 on lipid metabolism at the cellular level,WB detection was performed on Caco-2 and Huh7 cell lysates,and no effect of TM6SF2 overexpression on APOB expression was found.(5)Further exploring the specific mechanism of TM6SF2’s effect on animal level triglycerides,it was found that the expression of APOB in gut and liver tissues of TM6SF2 gut-specific knockout mice was lower than that of the control group;The expression of APOB in liver tissue of TM6SF2 liver specific knockout mice was lower than that of control group.Conclusion:(1)Knockout of TM6SF2 in liver tissue can lead to decreased APOB expression in liver and increased triglyceride level in liver tissue.(2)Knockout of TM6SF2 in intestinal tissue can lead to decreased APOB expression in intestinal tissue,and lipid accumulation in liver.