摘要
目的:探究长链非编码RNA母系表达基因3(LncRNA MEG3)调节miR-181b-5p/叉头框蛋白P1(FOXP1)轴对非酒精性脂肪性肝病(NAFLD)小鼠的作用机制。方法:以高脂饮食喂养C57BL/6J小鼠12周建立NAFLD模型,随机分为NAFLD模型组(尾静脉注射生理盐水)、LncRNA MEG3过表达组(尾静脉注射LncRNA MEG3过表达质粒)、LncRNA MEG3过表达+miR-181b-5p mimics组(尾静脉注射LncRNA MEG3过表达质粒和miR-181b-5p mimics)、阴性对照组(尾静脉注射空载质粒和miR-181b-5p mimics阴性对照),每组10只,另选10只小鼠喂养普通饲料12周作为正常对照组(尾静脉注射生理盐水)。各组小鼠均处理2周,每周2次。采用全自动生化分析仪测量各组小鼠血清脂代谢指标甘油三酯(TG)、总胆固醇(TC)、游离脂肪酸(FFA)及肝功能指标谷丙转氨酶(ALT)、谷草转氨酶(AST)水平;测量各组小鼠肝系数;HE染色检测各组小鼠肝组织病理形态;ELISA测量各组小鼠血清及肝组织炎症因子IL-6、IL-18水平;RT-qPCR检测各组小鼠肝组织LncRNA MEG3、miR-181b-5p表达;RT-qPCR与免疫印迹法检测各组小鼠肝组织FOXP1表达。采用双荧光素酶报告基因实验检测小鼠肝实质细胞中LncRNA MEG3对miR-181b-5p的靶向调节与miR-181b-5p对FOXP1的靶向调节。结果:与正常对照组相比,NAFLD模型组小鼠肝组织发生严重病理损伤,肝组织LncRNA MEG3表达、FOXP1 mRNA及蛋白表达显著降低(P<0.05),TG、TC、FFA、ALT及AST水平、肝系数、血清及肝组织IL-6、IL-18水平、肝组织miR-181b-5p表达显著升高(P<0.05)。与NAFLD模型组相比,LncRNA MEG3过表达组小鼠肝组织病理损伤减轻,肝组织LncRNA MEG3表达、FOXP1 mRNA及蛋白表达升高(P<0.05),TG、TC、FFA、ALT及AST水平、肝系数、血清及肝组织IL-6和IL-18水平、肝组织miR-181b-5p表达降低(P<0.05);阴性对照组小鼠各指标无明显变化(P>0.05)。与LncRNA MEG3过表达组相比,LncRNA MEG3过表达+miR-181b-5p mimics组小鼠肝组织病理损伤加重,肝组织FOXP1 mRNA及蛋白表达降低(P<0.05),TG、TC、FFA、ALT及AST水平、肝系数、血清及肝组织IL-6和IL-18水平、肝组织miR-181b-5p表达升高(P<0.05)。肝实质细胞中LncRNA MEG3可靶向下调miR-181b-5p表达,且miR-181b-5p可靶向下调FOXP1表达。结论:过表达LncRNA MEG3可通过下调miR-181b-5p增强FOXP1表达,从而改善NAFLD小鼠脂代谢,减轻炎症及肝组织损伤,最终修复其肝功能。
Objective:To investigate the mechanism of long non-coding RNA maternally expressed gene 3(LncRNA MEG3)on mice with non-alcoholic fatty liver disease(NAFLD)by regulating the miR-181b-5p/forkhead box protein P1(FOXP1)axis.Methods:NAFLD model was established in C57BL/6J mice by feeding high-fat diet for 12 weeks,which were randomly divided into NAFLD model group(tail vein injection of normal saline),LncRNA MEG3 overexpression group(tail vein injection of LncRNA MEG3 overexpression plasmid),LncRNA MEG3 overexpression+miR-181b-5p mimics group(tail vein injection of LncRNA MEG3 overexpressed plasmid and miR-181b-5p mimics)and negative control group(tail vein injection of empty plasmid and miR-181b-5p mimics negative control),with 10 mice in each group.Another 10 mice were fed with common diet for 12 weeks as normal control group(tail vein injection of normal saline).Each group of mice was treated for 2 weeks,twice a week.Levels of serum lipid metabolism indexes triglyceride(TG),total cholesterol(TC),free fatty acid(FFA)and liver function indexes alanine aminotransferase(ALT),aspartate aminotransferase(AST)were measured by automatic biochemical analyzer;liver coefficient of mice in each group was measured;pathological morphology of liver tissue of mice in each group was detected by HE staining;levels of IL-6 and IL-18 in serum and liver tissue of mice in each group were measured by ELISA;expressions of LncRNA MEG3 and miR-181b-5p in liver tissues of mice in each group were detected by RT-qPCR;expression of FOXP1 in liver tissues of mice in each group was detected by RT-qPCR and Western blot.Targeting regulation of miR-181b-5p and LncRNA MEG3,and targeting regulation of FOXP1 and miR-181b-5p in mice liver parenchyma cells were detected by double luciferase reporter gene assay.Results:Compared with normol control group,severe pathological injury occurred in liver tissue of mice in NAFLD model group,expressions of LncRNA MEG3,FOXP1 mRNA and protein in liver tissue were decreased obviously(P<0.05),levels of TG,TC,FFA,ALT and AST,liver coefficient,levels of IL-6,IL-18 in serum and liver tissue,and expression of miR-181b-5p in liver tissue were increased obviously(P<0.05).Compared with NAFLD model group,pathological damage of liver tissue in LncRNA MEG3 overexpression group was reduced,expressions of LncRNA MEG3,FOXP1 mRNA and protein in liver tissue were increased(P<0.05),levels of TG,TC,FFA,ALT and AST,liver coefficient,levels of IL-6,IL-18 in serum and liver tissue,and expression of miR-181b-5p in liver tissue were decreased(P<0.05);there was no obvious changes in each index of mice in negative control group(P>0.05).Compared with LncRNA MEG3 overexpression group,pathological damage of liver tissue in LncRNA MEG3 overexpression+miR-181b-5p mimics group was aggravated,expression of FOXP1 mRNA and protein in liver tissue was decreased(P<0.05),levels of TG,TC,FFA,ALT and AST,liver coefficient,levels of IL-6,IL-18 in serum and liver tissue,and expression of miR-181b-5p in liver tissue were increased(P<0.05).LncRNA MEG3 could target and down-regulate miR-181b-5p expression in liver parenchymal cells,and miR-181b-5p could target and down-regulate FOXP1 expression.Conclusion:Overexpression of LncRNA MEG3 can enhance the expression of FOXP1 by down-regulation of miR-181b-5p,thereby improving lipid metabolism,reducing inflammation and liver tissue damage,and ultimately repairing liver function in NAFLD mice.
作者
方金鸣
FANG Jinming(Wuhan Fourth Hospital,Wuhan 430033,China)
出处
《中国免疫学杂志》
北大核心
2025年第4期885-892,共8页
Chinese Journal of Immunology
基金
武汉市卫生健康委员会科研项目(WX19Y10)。
作者简介
方金鸣,男,博士,主治医师,主要从事代谢性疾病、肝病及发病机制研究,E-mail:fangjinming1983@163.com。
