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

肝脏 ›› 2022, Vol. 27 ›› Issue (8): 920-924.

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

徐梓馨, 廖红雨, 李慧一, 徐铭益

200080 上海交通大学附属第一人民医院消化内科(徐梓馨);西藏山南扎囊县卫生服务中心消化内科(廖红雨);同济大学附属东方医院消化内科(李慧一,徐铭益) 共同第一作者:廖红雨

收稿日期:2021-11-28 出版日期:2022-08-31 发布日期:2022-09-30
通讯作者: 徐铭益,Email:xumingyi2014@163.com
基金资助:
国家自然科学基金面上项目(81770597)

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

XU Zi-xin, LIAO Hong-yu, LI Hui-yi, XU Ming-yi

1. Department of Gastroenterology, the first people's Hospital Affiliated to Medical College of Shanghai Jiaotong University, Shanghai 200080, China;
2. Department of Gastroenterology, Health Service Center of Zha'nang County, Shannan, Tibet 850804, China;
3. Department of Gastroenterology, Dongfang Hospital Affiliated to Tongji University, Shanghai 200120, China

Received:2021-11-28 Online:2022-08-31 Published:2022-09-30
Contact: XU Ming-yi,Email:xumingyi2014@163.com

摘要/Abstract

摘要： 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),200 μmol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 (mi-miR219+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。

关键词: MicroRNA-219, 脂质代谢, 细胞增殖及凋亡, 非酒精性脂肪性肝病

Abstract: 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.

Key words: MicroRNA-219, Lipid metabolism, Cell viability and apoptosis, Non-alcoholic fatty liver diseases

徐梓馨, 廖红雨, 李慧一, 徐铭益. 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[J]. 肝脏, 2022, 27(8): 920-924.

XU Zi-xin, LIAO Hong-yu, LI Hui-yi, XU Ming-yi. The effect of microRNA-219 on lipid metabolism, cell viability and apoptosis of hepatocytes under lipotoxic environment[J]. Chinese Hepatolgy, 2022, 27(8): 920-924.

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