Obesity has emerged as a host of metabolic disorders, such as non-alcoholic fatty liver disease (NAFLD). Many reports have demonstrated that 90% of obese patients in the United States have NAFLD ranging from hepatic steatosis to much more severe forms of non-alcoholic steatohepatitis (NASH), which can induce fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) (Corey and Kaplan, 2014). NAFLD is associated with hepatic metabolic reprogramming that leads to excessive lipid accumulation and imbalances in lipid metabolism in the liver (de Alwis and Day, 2008). Hepatic lipid homeostasis is appropriately described as a complex machinery involving signaling and transcriptional pathways, as well as targeted genes associated with fatty acid (FA) oxidation and lipogenesis (Fabbrini et al., 2010). Although the pathogenesis of NAFLD has been widely studied for years, the molecular mechanisms underlying its complicated disorder are still being investigated.

Nuclear receptors (NRs) are a superfamily of ligand-activated transcription factors that regulate biological homeostasis (McKenna et al., 2009). Recent evidence suggests that NRs are key regulators in the progression of diverse hepatic diseases associated with glucose and lipid metabolism, inflammation, bile acid homeostasis, fibrosis, and cancer development in the liver (López-Velázquez et al., 2012). Among them, growing evidence suggests a link between PPARα and obesity-induced NAFLD. Hepatic PPARα plays an important role in energy homeostasis by regulating the expression of genes required for FA uptake, FA oxidation, and triglyceride (TG) hydrolysis in the liver (Chakravarthy et al., 2005). The decreased expression of PPARα is significantly associated with severity in NAFLD patients (Francque et al., 2015). Therefore, understanding the molecular mechanism underlying PPARα regulation is critical for understanding the pathogenesis of NAFLD.

MicroRNAs (miRNAs) are short, non-coding RNA molecules with a length of 18–25 nucleotides that play an important role in regulating the expression of target genes in a post-transcriptional manner by targeting base-pairing with the 3’UTR of specific target mRNAs, inhibiting translation, or mRNA degradation (Bartel, 2004). These miRNAs contribute to the regulation of a wide variety of cellular functions and metabolic homeostasis, including fatty acid metabolism. Recent studies have suggested that miRNAs significantly regulate the pathogenesis of NAFLD by targeting the nuclear receptors (López-Sánchez et al., 2021). Previous report has demonstrated that MIR20B, a member of the MIR17 family, presents in the circulating plasma of NAFLD patients and has been highlighted as a novel biomarker of NAFLD and type 2 diabetes mellitus (T2DM) for the diagnosis and risk estimation of NAFLD (Ye et al., 2018). However, the mechanisms underlying the involvement of MIR20B in the occurrence and progression of NAFLD remain unknown. In this study, we analyzed the regulatory networks of miRNAs for NR genes and RNA-seq data in NAFLD patients, which prioritized MIR20B as a key regulator in NAFLD.

Obesity has been widely demonstrated to be central to the pathogenesis of NAFLD. Among other peripheral tissues, the liver plays a dominant role in the regulation of lipid homeostasis (Pawlak et al., 2015). Although abnormal regulation of metabolic homeostasis in the liver has been recognized in diabetes and NAFLD, the underlying molecular mechanisms remain to be elucidated. Growing evidence has demonstrated that MIR20B levels are significantly upregulated in the plasma miRNA profiles of NAFLD patients (Jin et al., 2012). Moreover, plasma MIR20B levels were highly elevated in T2DM/NAFLD patients compared to those in T2DM patients (Ye et al., 2018). However, the molecular mechanism through which MIR20B regulates NAFLD progression remains unknown. In this study, we demonstrated that MIR20B promotes NAFLD progression by modulating lipid metabolism, including FA β-oxidation, FA uptake, and TG synthesis, as well as ATP production by mitochondrial biogenesis. Our data clearly showed the regulatory mechanism of PPARα by MIR20B, and MIR20B may serve as a novel biological marker in NAFLD.

Decreased PPARα is contributed to development of NAFLD (Francque et al., 2015). A few miRNAs were reported to regulate PPARA expression in NAFLD. miR-34a targets PPARA and SIRT1, associating with FA oxidation and cholesterol synthesis. However, the effect of MIR34A on inflammation and fibrosis is not clear (Ding et al., 2015). MIR21 also decreases the expression of PPARA in NASH, however, activated PPARα by MIR21 suppression reduces inflammation, liver injury, and fibrosis without improvement in FA β-oxidation and lipid accumulation (Loyer et al., 2016). In the present study, we demonstrate the novel miRNA which has different mode of action. MIR20B showed the deteriorating effects on FA oxidation, steatosis, inflammation, and fibrosis in HFD- or MCD-fed mice. How these miRNAs targeting PPARA have different regulatory mechanisms should be further studied.

A previous study demonstrated that upregulated MIR20B levels in obesity-induced metabolic disorders such as T2DM were considered to prevent several targets, such as STAT3, CD36, and PTEN, which are involved in glucose and lipid homeostasis. The MIR20B/STAT axis, which is involved in the insulin signaling pathway, alters glycogen synthesis in human skeletal muscle cells (Katayama et al., 2019). Moreover, MIR20B directly targets PTEN involved in PI3K/Akt/mTOR signaling pathway that modulates glucose metabolism in gastric cells (Streleckiene et al., 2020). Hosui et al., 2017 proposed a model in which the fatty acid transporter CD36 is also a potential target of MIR20B, which crucially regulates hepatic lipid metabolism in STAT5 KO mice models. But, it has been reported that CD36 and FABP1 are direct PPARα target genes (Rakhshandehroo et al., 2010). In this study, we observed that MIR20B downregulated PPARA and suppressed the expression of CD36 and FABP1. Thus, PPARA is the primary target of MIR20B in regulating hepatic lipid metabolism.

MIR20B was induced by OA, but FA uptake was decreased by overexpression of MIR20B, and was accompanied by a considerable decrease in CD36 expression (Figure 4B and J). However, other lipid transporters such as FATPs, except for PPARα target FATP1, were not significantly altered (Figure 4—figure supplements 1 and 5), suggesting that FA uptake is continued by these transporters. The expression of CD36 is relatively low in normal hepatocytes, and the molecule may not be the primary fatty acid transporter in these cells (Wilson et al., 2016). Furthermore, the decrease in FA uptake upon CD36 KO is modest even during a HFD (Wilson et al., 2016). In addition, we observed that the expression of MIR20B is induced and increased for up to 24 hr by OA treatment (Figure 4—figure supplement 6). This is followed by a slight decrease, remaining at a constant elevated level. Together, the findings indicated that other fatty acid transporters contributing to FA uptake account for the entry of OA into cells and MIR20B induction.

Hepatic steatosis could be affected by adipose tissue through FFA release and hepatic uptake of circulating FFAs (Rasineni et al., 2021). Our results showed that the epididymal adipose tissue of HFD-fed mice was enlarged upon AAV-Mir20b treatment; however, the serum FFA levels in these mice were comparable to those in mice treated with the AAV-Control (Figure 5—figure supplement 4B). Of note, the expression of genes related to lipolysis did not change in adipose tissues, and that of hepatic FA transporter, CD36, was decreased by AAV-Mir20b treatment (Figure 5Q and Figure 5—figure supplement 4A). In addition, excess hepatic TGs are secreted as very low-density lipoproteins (VLDLs), and the secretion rate increases with the TG level (Fabbrini et al., 2008). VLDLs deliver TGs from the liver to adipose tissue and contributes expansion of adipose tissue (Chiba et al., 2003). Together, these reports suggest that adipose tissue is also remodeled by the liver in HFD-fed mice and patients with NAFLD. Therefore, the increase of hepatic TGs by AAV-Mir20b is unlikely affected by AAV-Mir20b on epididymal adipose tissue, and the increase in fat content (Figure 5—figure supplement 3) may be a consequence of increased hepatic TG levels.

The expression of glycerolipid synthetic genes, including AGPATs, GPATs, and DGATs, was increased by OA treatment, but MIR20B overexpression did not influence the expression of these genes except for that of DGAT1. However, treatment with anti-MIR20B significantly reduced the expression of glycerolipid synthetic genes, including GPATs and DGATs, under OA treatment (Figure 4C and N). These results suggested that MIR20B is necessary but not sufficient to induce the expression of glycerolipid synthetic genes under OA treatment. We have shown that OA induces the expression of MIR20B (Figure 1C and D), which can explain why MIR20B overexpression did not show an additional enhancement under OA treatment. The increase in DGAT1 expression induced by MIR20B might contribute to the increase in TG formation and capacity to store OA. This could change the flux of oleyl-CoA to TG synthesis, not β-oxidation with reduced expression of lipid oxidation-associated genes (Figure 4B). Thus, we can expect that the decrease in OA uptake and increase in TG formation induced by MIR20B resulted in reduced amounts of OA or oleyl-CoA inside the cell. However, as lipid consumption through FA oxidation is decreased by MIR20B, free OA or oleyl-CoA might be maintained at a stably increased level compared to that of OA-untreated MIR NC or MIR20B condition, and the impact of the changes in OA or oleyl-CoA levels on the transcriptional phenotype might not be significant as found in a constant elevated level of MIR20B by OA.

Recent reports suggest that some transcription factors regulate metabolic homeostasis by directly mediating the expression of miRNAs (Yang and Wang, 2011). E2F1, which is a member of the E2F transcription factor family, regulates myoblast differentiation and proliferation via the auto-regulatory feedback loop between E2F1 and Mir20b in muscle (Luo et al., 2016). Both the hepatic expression and activity of E2F1 are increased during obesity. E2f1 deficiency protects against obesity- and diabetes-induced liver steatosis in mouse models (Zhang et al., 2014). Additionally, E2F1-induced chronic inflammation and hepatic lipid metabolism during NAFLD development (Denechaud et al., 2016). Consistent with these results, we observed that the expression of E2F1 was significantly increased in the fatty liver of both mice and humans, and its expression was positively correlated with that of MIR20B (Figure 10). These results suggest that E2F1 may be an upstream regulator of MIR20B in the liver, and this upregulation of MIR20B regulates lipid metabolism in the pathogenesis of NAFLD.

Figure 10

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E2F1 is upregulated in both NAFLD patients and mice model.

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NAFLD patients can develop NASH, which is characterized by hepatic steatosis complicated by chronic hepatocellular damage and severe inflammation with fibrosis, potentially developing into cirrhosis and HCC (Corey and Kaplan, 2014). This indicates that suppression of NAFLD progression is the primary option for preventing the development of HCC. Recent reports suggest that Ppara KO mice fed an MCD developed much more severe NASH than wild-type mice, and the expression of Ppara in HCC tissue was significantly lower than that in normal liver tissue (Montagner et al., 2016). PPARα activation contributes to the inhibition of HCC cell proliferation Thus, hepatic PPARα plays a crucial role in tumorigenesis in the liver (Lefebvre et al., 2006). Interestingly, it has been reported that upregulated Mir20b highly regulates cancer cell proliferation and promotes proliferation of H22 hepatocellular carcinoma cells (Peng et al., 2019; Xia et al., 2020). Thus, plasma MIR20B can be a promising target in liver cancer development. Indeed, we observed that the level of MIR20B was increased in NAFLD patients, but even robustly increased in the NASH stage. Furthermore, we observed that the hepatic function of Mir20b dramatically regulates the genes involved in inflammation and fibrosis by directly repressing Ppara in MCD-fed mice. Thus, our study strongly suggested that MIR20B regulates the pathogenesis of NAFLD, but might also be relevant in the development of severe stages of liver fibrosis and even in HCC.

Fibrates are FDA-approved medications for the treatment of patients with dyslipidemia (hypercholesterolemia, mixed dyslipidemia, hypertriglyceridemia) (Katsiki et al., 2013). Fibrates are well-known PPARα agonists, and PPARα is a widely accepted promising target for the treatment of NAFLD and NASH. In animal studies, fenofibrate improved hypertriglyceridemia and hepatic steatosis. In a small pilot trail, fenofibrate improved ALT and AST levels and hepatocellular ballooning degeneration, but did not show significant improvements in steatosis, lobular inflammation, fibrosis, or NAS score (Fernández-Miranda et al., 2008). Clofibrate, another PPARα agonist, did not show clinical benefit in the treatment of NASH (Laurin et al., 1996). Recently, the effect of omega-3 poly-unsaturated FAs (n-3 PUFA), which are ligands for PPARα, on patients with NAFLD was evaluated. Omacor, which consists of purified docosahexaenoic and eicosapentaenoic acids as ethyl esters, showed a linear decrease in liver fat percentage without improvements in fibrosis score (Scorletti et al., 2014). Omacor has undergone a phase three clinical trial but the results have not been disclosed (NCT01277237). These results show that the effect of PPARα agonists on NAFLD is limited. Our results suggest one of the possible reasons for this limited effect. Here, we show that PPARA expression is suppressed by increased MIR20B expression, which may weaken the efficacy of fibrates. Indeed, our results showed that the effects of fenofibrate were limited in AAV-Mir20b-treated mice, and that the combination of fenofibrate and AAV-anti-Mir20b further improved the NAFLD phenotype in MCD-fed mice. Nowadays, it is generally accepted that treatment with PPARα agonists alone is limited and that a combination strategy is necessary for NAFLD. The inhibition of MIR20B presents an opportunity for the development of novel therapeutics for NAFLD.

Sequencing data have been deposited in GEO under accession codes GSE168484. Other data generated or analysed during this study are included in the manuscript. Source data files have been provided.

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Author details

1. Yo Han Lee

   Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Writing – original draft, Writing – review and editing

   Contributed equally with

   Hyun-Jun Jang and Sounkou Kim

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3422-0306

2. Hyun-Jun Jang

   Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Writing – original draft, Writing – review and editing

   Contributed equally with

   Yo Han Lee and Sounkou Kim

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2261-0067

3. Sounkou Kim

   Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea

   Contribution

   Data curation, Formal analysis, Methodology, Software, Visualization, Writing – original draft

   Contributed equally with

   Yo Han Lee and Hyun-Jun Jang

   Competing interests

   No competing interests declared

4. Sun Sil Choi

   Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea

   Contribution

   Conceptualization, Data curation

   Competing interests

   No competing interests declared

5. Keon Woo Khim

   Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea

   Contribution

   Investigation, Validation

   Competing interests

   No competing interests declared

6. Hye-jin Eom

   Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea

   Contribution

   Investigation, Validation

   Competing interests

   No competing interests declared

7. Jimin Hyun

   Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea

   Contribution

   Investigation, Validation

   Competing interests

   No competing interests declared

8. Kyeong Jin Shin

   Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea

   Contribution

   Investigation, Validation

   Competing interests

   No competing interests declared

9. Young Chan Chae

   Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea

   Contribution

   Supervision, Writing – review and editing

   Competing interests

   No competing interests declared

10. Hongtae Kim

    Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea

    Contribution

    Supervision, Writing – review and editing

    Competing interests

    No competing interests declared

11. Jiyoung Park

    Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea

    Contribution

    Supervision, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-3705-4769

12. Neung Hwa Park

    Department of Internal Medicine, Ulsan University Hospital, Ulsan, Republic of Korea

    Contribution

    Resources, Validation, Writing – review and editing

    Competing interests

    No competing interests declared

13. Chang-Yun Woo

    Department of Internal Medicine, Asan Medical Center, Seoul, Republic of Korea

    Contribution

    Resources, Validation

    Competing interests

    No competing interests declared

14. Chung Hwan Hong

    Department of Medical Science, Asan Medical Center, Seoul, Republic of Korea

    Contribution

    Resources, Validation

    Competing interests

    No competing interests declared

15. Eun Hee Koh

    Department of Internal Medicine, Asan Medical Center, Seoul, Republic of Korea

    Contribution

    Resources, Validation

    Competing interests

    No competing interests declared

16. Dougu Nam

    Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea

    Contribution

    Data curation, Software, Supervision, Validation, Writing – review and editing

    For correspondence

    dougnam@unist.ac.kr

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-0239-2899

17. Jang Hyun Choi

    Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea

    Contribution

    Conceptualization, Data curation, Funding acquisition, Project administration, Supervision, Writing – original draft, Writing – review and editing

    For correspondence

    janghchoi@unist.ac.kr

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-0526-9028

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