miR-484通过靶向SIRT1介导细胞凋亡参与非酒精性脂肪肝性肝病损伤

MiR-484 participates in nonalcoholic fatty liver injury by targeting SIRT1 to mediate cell apoptosis

背景目前非酒精性脂肪肝性病(non-alcoholic fatty liver disease,NAFLD)已经成为被严重低估的重大健康威胁.尽管NAFLD发病机理复杂,但是越来越多的证据表明microRNAs(miRNAs)在调控NAFLD的发生和发展中起着重要的作用.其中,miR-484是否参与NAFLD发生和发展仍有待阐明.目的研究miR-484参与肝脏脂肪变性的损伤作用机制.方法通过喂养高脂饲料建立小鼠高脂模型,采用RT-qPCR和Western blot方法测定肝组织中miR-484和SIRT1的mRNA和蛋白水平,再次构建miR-484基因敲除NAFLD小鼠模型,检测小鼠血清ALT、AST、TG、TC变化水平,通过油红O染色、HE染色和TUNEL染色检测脂肪变性程度和细胞凋亡水平.此外,通过游离脂肪酸构建NAFLD细胞模型,采用双荧光素酶报告基因实验验证miR-484和SIRT1的直接靶向关系;通过转染pc-DNA和pc-DNA SIRT1构建SIRT1过表达模型,通过油红O染色检测脂质积累和流式细胞学检测细胞凋亡水平.结果在NAFLD小鼠模型中,miR-484表达明显上调,而SIRT1表达下降;敲除miR-484后小鼠脂肪变性程度减轻,血清ALT和AST明显降低.在NAFLD细胞模型中,miR-484能够通过直接靶向SIRT1,过表达SIRT1显著降低细胞凋亡率,减轻肝细胞脂质积累.结论miR-484通过靶向SIRT1调控细胞凋亡,加重肝细胞脂质积累,表明miR-484可能是NAFLD的治疗靶标.

BACKGROUND Non-alcoholic fatty liver disease(NAFLD)has become a major health threat that is seriously underestimated.Although the pathogenesis of NAFLD is complex,more and more evidence shows that microRNAs(miRNAs)play an important role in regulating the occurrence and development of NAFLD.Whether miR-484 is involved in the occurrence and development of NAFLD remains to be clarified.AIM To explore the mechanism of miR-484 in the damage of liver steatosis.METHODS A mouse model of NAFLD was established by feeding mice a high-fat diet,and the expression levels of miR-484 and SIRT1 in liver tissues were measured by RTqPCR and Western blot.A miR-484 knockout NAFLD mouse model was constructed,the degree of steatosis and apoptosis were detected by oil red O staining,HE staining,and TUNEL staining,and the levels of serum ALT and AST were detected.In addition,a cell model of NAFLD was constructed through free fatty acid exposure.The dual luciferase reporter gene assay was first used to verify the direct targeting relationship between miR-484 and SIRT1,then an SIRT1 overexpression model was constructed by transfection with pc-DNA and pc-DNA SIRT1.Oil red O staining was used to detect lipid accumulation and flow cytometry was used to detect cell RESULTS In the mouse model of NAFLD,the expression of miR-484 was significantly up-regulated,while the expression of SIRT1 was decreased.The degree of steatosis was reduced and serum ALT and AST levels were significantly reduced in miR-484 knockout mice.In the cell model of NAFLD,miR-484 can directly target SIRT1.In addition,overexpression of SIRT1 significantly decreased the rate of apoptosis and alleviated lipid accumulation in liver cells CONCLUSION MiR-484 regulates cell apoptosis by targeting SIRT1 and aggravates lipid accumulation in liver cells,which suggests that miR-484 may be a therapeutic target for NAFLD.