MicroRNA-219在脂毒性环境下对肝细胞脂质代谢、细胞增殖及凋亡的影响

The effect of microRNA-219 on lipid metabolism,cell viability and apoptosis of hepatocytes under lipotoxic environment

目的 研究在脂毒性环境下microRNA-219(miR-219)对肝细胞脂质代谢、细胞增殖及凋亡的影响。方法分别以低脂饲料(low fatty diet,LFD)与高脂饲料(high fatty diet,HFD)喂养小鼠,构建对照组及脂肪肝小鼠模型,HE、油红染色观察小鼠造模情况。使用两步胶原酶灌注法从C57BL/6J小鼠提取原代肝细胞(primary hepatocytes,PHC),200μmol棕榈酸(palmitic acid,PA)处理24 h以模拟脂毒性环境,检测AST和ALT水平判断肝细胞脂肪变性情况。Q-PCR检测脂肪肝小鼠肝组织与PHC中miR-219表达水平,过表达miR-219后脂质代谢因子[硬脂酰辅酶A去饱和酶1(stearoyl-CoA desaturase 1,SCD1)、脂肪酸合成酶(fatty acid synthase,FAS)]、增殖因子[增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、细胞周期蛋白D1(CyclinD1)]、凋亡相关因子[B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl-2)、半胱氨酸蛋白酶-3(Caspase3)]的mRNA表达;油红染色观察细胞脂质沉积;EDU检测细胞增殖情况。结果 非酒精性脂肪性肝病小鼠模型建模成功。相比对照组,在高脂小鼠和PA处理的PHC中miR-219的表达水平均明显下降,HFD组为0.455±0.028,LFD组为1.000±0.125(P<0.05);PA处理组为0.676±0.064,对照组为1.000±0.190(P<0.05)。在PA刺激的PHC中,相比对照组,过表达miR-219组PHC中脂质代谢因子SCD1、FAS的mRNA表达明显下降[SCD1(mi-miR219+PA:0.539±0.048,miR-NC+PA:1.000±0.033,P<0.01),FAS(mi-miR219+PA:0.722±0.036,miR-NC+PA:1.000±0.051,P<0.05)],油红染色也提示脂质沉积明显减轻;过表达miR-219后,PHC增殖因子(PCNA、Cyclin D1)的表达明显上升[PCNA (mi-miR219+PA:1.652±0.185,miR-NC+PA:1.000±0.203,P<0.05)和CyclinD1 (mimiR219+PA:1.791±0.154,miR-NC+PA:1.000±0.135,P<0.05)],EDU检测也提示细胞增殖明显增加。另外,促凋亡因子Casepase3的表达下降(mi-miR219+PA:0.574±0.054,miR-NC+PA:1.000±0.35,P<0.05),抑凋亡因子Bcl-2的表达上升(mi-miR291:1.535±0.109,miR-NC:1.000±0.208,P<0.05)。结论 在脂毒性环境下,miR-219可明显缓解肝细胞脂质沉积,提高肝细胞活性,促进增殖而抑制细胞凋亡,缓解NAFLD。

Objective To explore the effect of microRNA-219(miR-219)on lipid metabolism,cell viability and apoptosis of lipotoxic hepatocytes.Methods Mice were fed with low-fat diet(LFD)and high-fat diet(HFD)to construct control group and non-alcoholic fatty liver diseases(NAFLD)model.Hematoxylin-eosin(H&E)staining and oil red staining were used to observe the liver histology.Primary hepatocytes(PHCs)were isolated from wild-type mice and then incubated with 200uM palmitic acid(PA)for 24h to mimic a high-fat environment.Fatty degeneration of hepatocytes was judged by oil red staining,aspartate transaminase(AST)and alanine aminotransferase(ALT)levels.Quantitative polymerase chain reaction(q-PCR)was used to detect the expression of miR-219 in liver tissue and PHCs,and the mRNA expression of lipid metabolism factors[stearoyl-CoA desaturase 1(SCD1)and fatty acid synthase(FASN)],proliferation-related factors[proliferating cell nuclear antigen(PCNA)and CyclinD1]and apoptosis-related factors[B-cell lymphoma-2(BCL-2)and Caspase3]after overexpression of miR-219 through miR-219 mimic(mi-miR-219)or the negative control in PA-treated PHCs.Oil red staining was performed to observe cell lipid deposition.EDU was used to observe cell proliferation.Results The NAFLD mice model was successfully established.Compared with the relatively control group,miR-219 expression was significantly decreased in both the liver tissues of HFD group and PA-treated PHCs[HFD group:(0.455±0.028),LFD group:(1.000±0.125),P<0.05;PA-treated group:(0.676±0.064),Control:(1.000±0.190),P<0.05].After overexpressing miR-219,SCD1 and FASN mRNA expression were both significantly decreased in PA-treated PHCs[SCD1:mi-miR-219+PA:(0.539±0.048),negative control group(miR-NC)+PA:(1.000±0.033),P<0.001;FASN:mi-miR-219+PA:(0.722±0.036),miR-NC+PA:(1.000±0.051),P<0.05].Oil red staining indicated that the lipid deposition was also significantly alleviated in PA-treated PHC in mi-miR-219 group.Compared with the miR-NC group,the mRNA expression of proliferation-related factors was both obviously increased[PCNA:mi-miR-219+PA:(1.652±0.185),miR-NC+PA:(1.000±0.203),P<0.05;CyclinD1:mi-miR-219+PA:(1.791±0.154),miR-NC+PA:(1.000±0.135),P<0.05].And the more obvious red fluorescent was showed in mi-miR-219 PHCs group according to EDU assay.Besides,the mRNA expression of pro-apoptotic factor Caspase-3 was decreased[mi-miR-219+PA:(0.574±0.054),miR-NC+PA:(1.000±0.35),P<0.05],but the mRNA expression of anti-apoptotic factor BCL-2 was increased[mi-miR-291:(1.535±0.109),miR-NC:(1.000±0.208),P<0.05].Conclusion Under high-fat environment,miR-219 could significantly alleviate hepatocytes lipid deposition,promote hepatocytes viability and inhibit cell apoptosis to delay the progress of NAFLD.