| Objective: To explore the mechanism of TM6SF2 gene E167 K variant in the cultured hepatocyt.Methods:(1)The HEPA 1-6 cell lines were cultured in Dulbecco's Modified Eagle's Medium(DMEM)medium containing 100 U/m L penicillin,10% fetal bovine serum(FBS;Hyclone,USA)and 100 ?g/m L streptomycin(Gibco?,USA),and incubated at temperature 37 ? in a humidified atmosphere with 5% CO2.(2)HEPA 1-6 cells which could respectively overexpress TM6SF2 wild type and E167 K variant were cultured and HEPA 1-6 cells with zero load plasmids were used as matched control.(3)Flow cytometry was used to detect the cell cycles of these 3 type of HEPA 1-6 cells.(4)The three cell groups were compared to assess differences in triglyceride content(using oil red O staining)levels of triglyceride and cholesterol(using automated biochemical analyzer).(5)Realtime fluores-cence quantitative PCR and western blot were used to analyzed the expression of regulatory factors(Cyclin D1?p53?P16?P27?P21 and Rb)of cell cycle,inflammatory factor(TNF-??IL-2?IL-6 and IL-8)and SREBP-lc and FASN.T-test was used in statistical analysis.Results:(1)Cell cycle phase distribution was presented by the proportion of cells in each phases(%).While between variant type group and wild type group,G1 phase was significantly decreased(variant type group G1 phase36.26±0.31% VS wild type group57.63±0.28%,t was 88.607,P<0.05),S phase and G2/M phase were increased(variant type group S phase 28.41±0.31% VS wild type group 19.54±0.25%,variant type group G2/M phase 35.23±0.14% VS wild type group G2/M phase25.77±0.51%,t was 38.577,9.982,P<0.05).Compared with the control group,G1 phase was significantly decreased(the control group G1 phase 58.36±0.21%,t was 97.595,P <0.05),S phase and G2/M phase were increased(the control group S phase 19.31±0.25%,the control group G2/M phase 25.61±0.36%,t was 39.577,29.201,P<0.05).The cell cycle phase distribution had no differences between wild type group and the control group(P>0.05).Compared with control group,the relative expression of Cyclin D1?P53 and Rb m RNA in variant type group was significantly upregulated(2.03±0.01 VS 1.04±0.06,1.88±0.05 VS 1.37±0.03,1.29±0.06 VS 1.15±0.03,t was 28.190,15.152,3.615,respectivly,P<0.05)and P27 m RN A in variant type group was significantly downregulated(0.56±0.02 VS 0.85±0.05,t was 9.328,P<0.05).Compared with wild type group,the relative expression of C yclin D1?P53 and Rb m RNA in variant type group was significantly upregulated(wild type group 1.00±0.00,1.48±0.09,1.18±0.01,t was 178.532,6.729,3.132,respectivly,P<0.05)and P27 m RN A in variant type group was significantly downregulated(variant type group 0.82±0.05,t was 8.363,P<0.05).There was no statistical significance between wild type group and control group(P>0.05).P16 and P21 expression showed no statistical sigtfificance in any of these three groups(P>0.05).(2)Compared with control group,the relative expression of IL-2?IL-6 m RNA were increased in variant type group((1.24±0.07 VS 1.00±0.04,1.24±0.10 VS 1.01±0.04,t was 5.161,3.698,respectivly,P<0.05).Compared with wild type group,the relative expression of IL-2?IL-6 m RNA were increased in variant type group(wild type group: IL-2:1.00±0.05,IL-6:1.00±0.09,t was 4.829,3.089,respectivly,P<0.05).The relative expression of IL-8 had no statistical sigtfificance between variant type group and wild type group(P>0.05).(3)Cells expressing the TM6SF2 E167 K variant showed higher triglyceride content(0.54±0.02 VS 0.20±0.02 VS 0.24±0.02,t was 20.859,18.405,respectivly,P<0.01),higher SREBP-lc and FSAN m RNA expression(P<0.05)than wild type group and the control group.expression.The level of SREBP-lc was positively correlated with senlm triglyceride in the cells expressing the TM6SF2 E167 K variant(r=0.913,P<0.01).Conclusion:(1)E167K polymorphism of TM6SF2 gene affects cell cycles of HEPA1-6 cells via up-regulatating C yclin D1?P53 and Rb and down-regulatating P27.(2)The E167 K polymorphism of TM6SF2 gene can promote t he expression of IL-2 and IL-6,which may promote the development of liver injury.(3)The risk of liver disease associated with the TM6SF2 E167 K variant,which increases lipogenesis,may involve SREBP-lc and FSAN and a pathway that increases triglycerides. |
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