miR-199a-5p在肝脏组织中的表达及其作用的初步研究

The expression and role of miR-199a-5p in the liver

目的:探究不同营养状态下miR-199a-5p在肝脏中的表达水平,以及其对肝细胞甘油三酯(triglyceride,TAG)含量的影响及其潜在机制。方法:RT-qPCR检测高脂饮食小鼠肝脏组织中miR-199a-5p的表达;分别使用miR-199a-5p模拟物、抑制剂、阴性对照和pcDH-CD36-flag质粒转染Hepa1-6和AML12细胞,通过RT-qPCR和Western blot检测脂代谢相关标志物表达变化,采用试剂盒检测TAG含量;通过miRDB(microRNA Target Prediction Database)预测miR-199a-5p的靶基因并通过双荧光素酶报告实验进行验证。结果:高脂饮食和饥饿状态下C57BL/6J小鼠肝脏中miR-199a-5p的表达水平升高;过表达miR-199a-5p能够降低肝细胞内TAG,而miR-199a-5p抑制剂会增加细胞内TAG含量;miR-199a-5p通过作用于脂肪酸转位酶CD36基因的3′非翻译区(3′untranslated region,3′UTR)降低其蛋白表达。结论:在肝脏脂质积累过程中,miR-199a-5p的表达水平增加,并且miR-199a-5p可能通过靶向CD36降低肝细胞内TAG含量。

Objective:To investigate the expression levels of miR-199a-5p in the liver under various nutritional conditions and its effects on hepatic triacylglyceride(TAG)content,as well as the underlying mechanisms.Methods:RT-qPCR was used to detect the expression levels of miR-199a-5p in liver tissues of high fat diet mice.Hepa1-6 and AML12 cells were transfected with miR-199a-5p mimics,inhibitors,negative controls and pcDH-CD36-flag plasmid,respectively.The changes in biomarker expression related to lipid metabolism were detected by RT-qPCR and Western blot,and TAG content was detected by kit.The target gene of miR-199a-5p was predicted by microRNA Target Prediction Database(miRDB)and verified by the dual luciferase reporting assay.Results:The expression levels of miR-199a-5p in the liver of C57BL/6J mice were elevated under high-fat diet and fasting conditions.Overexpression of miR-199a-5p reduced TAG levels in hepatocytes,while inhibition of miR-199a-5p increased intracellular TAG content.miR-199a-5p decreased the protein expression of the fatty acid translocase CD36 by interacting with its 3′untranslated region(3′UTR).Conclusion:The expression level of miR-199a-5p increases during hepatic lipid accumulation,and miR-199a-5p may reduce TAG content in hepatocytes by targeting CD36.