人环状RNA-0046366与肝细胞脂肪变性的相关性及其机制研究

摘要

目的 探讨人环状RNA-0046366(circRNA-0046366)表达变化对肝细胞脂肪变性的影响及其机制.方法 将肝HepG2细胞株随机分为正常对照组(n=3)和脂肪变性模型组(n=3),分别给予高糖DMEM、高脂培养基(油酸∶棕榈酸=2∶1,0.5 mmol/L)诱导24 h.采用油红O染色及甘油三酯(Triglyceride,TG)、丙二醛(Malonyldialdehyde,MDA)检测,观察肝细胞的脂肪变性及脂质过氧化.以实时荧光定量PCR(Real-Time PCR)法检测circRNA-0046366在脂肪变性前后的表达改变.在circBase数据库搜索、circRNA-miRNA互补序列分析的基础上,预测circRNA-0046366的靶miRNA及其下游基因. 通过Spearman相关性分析,揭示circRNA-0046366水平与靶miRNA表达、下游mRNA表达、肝细胞脂肪变性及脂质过氧化指标的相关性.结果 与正常对照组相比,脂肪变性模型组的肝细胞内脂滴、TG[(24.4±2.4) μmol/g vs (263.7±17.5) μmol/g,P<0.05]及MDA[(0.6±0.1) μmol/g vs (1.8±0.2) μmol/g,P<0.05]含量明显升高.circRNA-0046366表达水平在脂肪变性的肝细胞中则显著降低(P<0.05).circRNA-0046366可与过氧化物酶体增殖物激活受体α(peroxisome proliferator-activated receptor α,PPARα)竞争性结合miR-34a,从而阻断miR-34a对PPARα翻译抑制作用.相关性分析显示,肝细胞的circRNA-0046366与PPARα表达水平呈显著正相关(r=0.78,P<0.05),与miR-34a表达水平(r=-0.83,P<0.05)、TG(r=-0.72,P<0.05)及MDA(r=-0.69,P<0.05)含量则呈显著负相关.结论 肝细胞的circRNA-0046366水平与脂肪变性及脂质过氧化呈负相关.其机制可能与靶向结合miR-34a,从而解除PPARα的表达抑制相关.%Objective To investigate the association and related mechanisms between human circular RNA-0046366 (circRNA-0046366) and hepatocyte steatosis.Methods HepG2 cells were randomly divided into groups of normal control (n=3) and steatosismodel group (n=3), respectively.The normal control group was treated with DMEM, while the steatosis-model group was administrated with high-fat medium (OA∶PA=2∶1, 0.5 mmol/L) for 24 hours.Both oil red O staining and triglyceride (TG) concentration were employed to evaluate the hepatocyte steatosis.Hepatic level of malonaldehyde (MDA) reflected the lipid peroxidation, which occurred on the basis of hepatocyte steatosis.Real-time PCR exhibited the alternation of circRNA-0046366 expression during hepatic steatosis.Bioinformatic analysis demonstrated the target miRNA, and downstream mRNAs, of circRNA-0046366 by means of circBase searching and circRNA-miRNA complementation.Finally, the association among circRNA-0046366 level, miRNA, mRNAs, hepatocyte steatosis, and lipid peroxidation was assessed by Spearman correlation analysis.Results Compared with normal control group, steatosismodel group exhibited significant increase in lipid droplets, and hepatic contents TG [(24.4±2.4) μmol/g vs (263.7±17.5) μmol/g, P<0.05] and MDA [(0.6±0.1) μmol/g vs (1.8±0.2) μmol/g, P<0.05].The expression level of circRNA-0046366 underwent statistically decrease (P<0.05) during the hepatocyte steatosis.Both circRNA-0046366 and peroxisome proliferator-activated receptor α (PPARα) shared the sequence complementary to miR-34a, which may abolish the translational inhibition of miR-34a on PPARα by binding competition.In result, the circRNA-0046366 level correlated with PPARα expression (r=0.78, P<0.05), but negatively correlated with miR-34a level (r=-0.83, P<0.05), and concentrations of TG (r=-0.72, P<0.05) and MDA (r=-0.69, P<0.05) in the steatotic HepG2 cells.Conclusion circRNA-0046366 level negatively correlates with hepatic steatosis and lipid peroxidation.Abolishment of the PPARα inhibition by miR-34a may underlie its effect.