Hepatic MIR20B promotes nonalcoholic fatty liver disease by suppressing PPARA

Abstract

Background:Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive lipid accumulation and imbalances in lipid metabolism in the liver. Although nuclear receptors (NRs) play a crucial role in hepatic lipid metabolism, the underlying mechanisms of NR regulation in NAFLD remain largely unclear. Methods:Using network analysis and RNA-seq to determine the correlation between NRs and microRNA in human NAFLD patients, we revealed that MIR20B specifically targets PPARA. MIR20B mimic and anti-MIR20B were administered to human HepG2 and Huh-7 cells and mouse primary hepatocytes as well as high fat diet (HFD)- or methionine-deficient diet (MCD)-fed mice to verify the specific function of MIR20B in NAFLD. We tested the inhibition of the therapeutic effect of a PPARα agonist, fenofibrate, by Mir20b and the synergic effect of combination of fenofibrate with anti-Mir20b in NAFLD mouse model. Results:We revealed that MIR20B specifically targets PPARA through miRNA regulatory network analysis of nuclear receptor genes in NAFLD. The expression of MIR20B was upregulated in free fatty acid (FA)-treated hepatocytes and the livers of both obesity-induced mice and NAFLD patients. Overexpression of MIR20B significantly increased hepatic lipid accumulation and triglyceride levels. Furthermore, MIR20B significantly reduced FA oxidation and mitochondrial biogenesis by targeting PPARA. In Mir20b-introduced mice, the effect of fenofibrate to ameliorate hepatic steatosis was significantly suppressed. Finally, inhibition of Mir20b significantly increased FA oxidation and uptake, resulting in improved insulin sensitivity and a decrease in NAFLD progression. Moreover, combination of fenofibrate and anti-Mir20b exhibited the synergic effect on improvement of NAFLD in MCD-fed mice. Conclusions:Taken together, our results demonstrate that the novel MIR20B targets PPARA, plays a significant role in hepatic lipid metabolism, and present an opportunity for the development of novel therapeutics for NAFLD. Funding:This research was funded by Korea Mouse Phenotyping Project (2016M3A9D5A01952411), the National Research Foundation of Korea (NRF) grant funded by the Korea government (2020R1F1A1061267, 2018R1A5A1024340, NRF-2021R1I1A2041463, 2020R1I1A1A01074940), and the Future-leading Project Research Fund (1.210034.01) of UNIST.

Optimized workflow to modify microRNA expression in primary human intravascular cells

“…Several studies explored the effects of specific miRNAs using miRNA inhibitors and mimics with different oligonucleotide chemistries and doses. Most of these studies, however, were performed in cell lines and used lipid-based carriers [ 23 – 25 ]. It is therefore unclear whether lipid-based carriers are essential to deliver miRNA inhibitors and mimics to cells.…”

“…Such vector-based miRNA inhibitors or mimics can provide efficient, sustained, and inducible expression in controlling target miRNAs. However, efficient delivery and strong expression of plasmid DNA vectors in primary cells and tissues are usually achieved by inserting these vectors into viral expression systems [ 25 , 38 ]. Production of high-titer virus particles and maintenance of a cell culture laboratory in accordance with biosafety regulations might decrease cost-effectiveness of this strategy.…”

“…Although functional dissection of the role of miRNA following upregulation using miRNA mimics can be performed in cell lines [ 23 – 25 ], studies of human primary cells are more relevant to delineate physiological roles of miRNAs. However, little work has been performed using miRNA mimics in human primary cells.…”

PPARα in the Epigenetic Driver Seat of NAFLD: New Therapeutic Opportunities for Epigenetic Drugs?

“…Some of these altered miRNAs target nuclear receptors, including PPARα [ 65 ]. For example, miR-200, miR-20b, miR181-a, miR-30a-3p, miR519d, miR-21 and miR-22 are elevated in NAFLD and directly target PPARα mRNA [ 66 , 67 , 68 , 69 , 70 , 71 , 72 ]. The working mechanism of these miRNAs leading to the aggravation of NAFLD is approximately the same.…”

“…They all bind to the 3′UTR of PPARα mRNA resulting in PPARα mRNA degradation, decreased protein expression and disturbed lipid metabolism, leading to the aggravation of an NAFLD phenotype. Moreover, the induced expression of specific miRNAs (miR-20b, miR181-a, miR-30a-3p and miR-22) in FFA-treated hepatocytes increased the intracellular lipid content upon reduction in PPARα mRNA levels and decreased protein expression [ 66 , 67 , 69 , 70 ]. Moreover, even in colorectal cancer-derived liver metastasis, deregulated PPAR targeting miRNAs have been observed [ 73 ].…”