Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids ( IF 3.9 ) Pub Date : 2020-08-02 , DOI: 10.1016/j.bbalip.2020.158777 Smriti Mittal 1 , Shrirang Inamdar 2 , Jhankar Acharya 2 , Komal Pekhale 2 , Saurabh Kalamkar 2 , Ramanamurthy Boppana 3 , Saroj Ghaskadbi 2
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Aims
PPARγ is a crucial transcription factor involved in development of hepatic steatosis, an early stage of NAFLD. PPARγ is tightly regulated through various positive and negative regulators including miRNAs. In this study, we report for the first time miR-3666 as a negative regulator of PPARγ and its involvement in development of hepatic steatosis.
Methods
Binding of miR-3666 to regulate PPARγ was checked by luciferase assay and was confirmed by mutating PPARγ 3′UTR. Regulation of PPARγ was determined by overexpression of miR-3666 in HepG2 cells. Hepatic steatotic state in HepG2 cells was developed by exposure to excess palmitic acid and expression of PPARγ, miR-3666 and some PPARγ target and non-target genes was checked. Involvement of mir-3666 by regulating PPARγ in hepatic steatosis was also examined in liver of HFD fed mice.
Results
On overexpression of miR-3666, PPARγ expression decreased significantly in a dose-dependent manner in HepG2 cells. Binding of miR-3666 to PPARγ was confirmed as the luciferase activity using pMIR-REPORT with PPARγ 3′UTR decreased in PA treated HepG2 cells overexpressing miR-3666 and remained unchanged when PPARγ 3′UTR was mutated. In PA treated HepG2 cells during development of hepatic steatosis PPARγ was significantly up-regulated concomitant with down-regulation of miR-3666. Overexpression of miR-3666 in these cells decreased the extent of hepatic steatosis. Significant up-regulation of PPARγ and down-regulation of miR-3666 was also observed in liver of HFD fed mice indicating that miR-3666 regulates PPARγ in vivo.
Conclusions
miR-3666 negatively regulates PPARγ by binding to its 3′UTR during development of hepatic steatosis.
中文翻译：
miR-3666通过负调节PPARγ抑制肝脂肪变性的发展。
目的
PPARγ是参与肝脂肪变性（NAFLD的早期阶段）发展的关键转录因子。PPARγ通过多种正负调节剂（包括miRNA）受到严格调节。在这项研究中，我们首次报道了miR-3666作为PPARγ的负调节剂，并且参与了肝脂肪变性的发展。
方法
通过荧光素酶测定法检查miR-3666与调节PPARγ的结合，并通过突变PPARγ3'UTR确认。通过在HepG2细胞中过度表达miR-3666来确定PPARγ的调控。通过暴露于过量的棕榈酸使HepG2细胞的肝脂肪变性状态得以发展，并检查PPARγ，miR-3666和一些PPARγ靶标和非靶标基因的表达。还通过HFD喂养小鼠的肝脏检查了mir-3666通过调节PPARγ参与肝脂肪变性的过程。
结果
miR-3666过表达后，HepG2细胞中PPARγ表达以剂量依赖性方式显着降低。使用pMIR-REPORT的荧光素酶活性证实了miR-3666与PPARγ的结合，其中在过量表达miR-3666的PA处理的HepG2细胞中，PPARγ3'UTR的荧光素酶活性降低，而当PPARγ3'UTR突变时则保持不变。在PA处理的HepG2细胞中，肝脂肪变性期间PPARγ显着上调，而miR-3666则下调。这些细胞中miR-3666的过表达降低了肝脂肪变性的程度。在喂食HFD的小鼠肝脏中还观察到PPARγ的显着上调和miR-3666的下调，表明miR-3666在体内调节PPARγ 。
结论
在肝脂肪变性发展过程中，miR-3666通过与PPARγ的3'UTR结合来负调控PPARγ。
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