MiR-103-3p promotes hepatic steatosis to aggravate nonalcoholic fatty liver disease by targeting of ACOX1

Background Nonalcoholic fatty liver disease (NAFLD) is a major risk factor for hepatocellular carcinoma, and alterations in miRNA expression are related to the development of NAFLD. However, the role of miRNAs in regulating the development of NAFLD is still poorly understood. Methods We used qRT-PCR to detect the level of miR-103-3p in both cell and mouse models of NAFLD. Biochemical assays, DCF-DA assays, Oil red O staining and HE staining were used to detect the role of miR-103-3p in NAFLD development. Target genes of miR-103-3p were predicted using the TargetScan database and verified by qRT-PCR, western blot and dual-luciferase assays. Results The expression of miR-103-3p increased in both NAFLD model cells and liver tissues from the NAFLD mouse model. Inhibition of miR-103-3p significantly alleviated the accumulation of lipid droplets in free fatty acid-treated L02 cells and liver tissues from mice with NAFLD. Inhibition of miR-103-3p reduced the contents of H2O2, TG, ALT, and AST and ROS production while increasing the ATP content. Moreover, the miR-103-3p antagomir alleviated liver tissue lesions in mice with NAFLD. Further studies identified ACOX1, a key enzyme for the oxidation and decomposition of fatty acids, as a direct target of miR-103-3p. Conclusions These findings identified a negative regulatory mechanism between ACOX1 and miR-103-3p that promotes the pathogenesis of NAFLD and suggested that inhibition of miR-103-3p may be a potential treatment strategy for NAFLD.


Introduction
Nonalcoholic fatty liver disease (NAFLD) develops into progressive nonalcoholic steatohepatitis (NASH), liver fibrosis, and cirrhosis and eventually causes liver cancer, which results in an increasing global public health burden that affects both adults and children [1,2]. Studies on the pathogenesis of NAFLD in recent decades have revealed that NAFLD is initiated by aberrant lipid metabolism in the liver [3]. Aberrant lipid metabolism of the liver is caused by mitochondrial dysfunction, endoplasmic reticulum stress (ERS), oxidative stress, and activation of inflammation [4]. In addition to the classic factors, epigenetic mechanisms such as noncoding RNA are involved in the progression of NAFLD [5,6]. Therefore, studying the mechanism of noncoding RNA in NAFLD may provide a promising strategy for NAFLD.
Noncoding RNA refers to RNAs such as rRNA, tRNA, snRNA, snoRNA, lncRNA, circRNA and miRNA that do not encode protein [7]. To date, 28,645 miRNAs that regulate one-third of human genes have been found in animals, plants and viruses [8]. MicroRNA is a type of 21-23 nt single-stranded small RNA that targets the 3′UTR of mRNAs and silences or degrades mRNA, which regulates the progression of NAFLD [9,10]. Patients with NAFLD displayed dramatic upregulation of miR-132 expression, and suppression of miR-132 expression decreased lipid accumulation to improve NAFLD in vivo [11]. Intriguingly, miR-26a was decreased in NAFLD patients' livers, and overexpression 1 3 of miR-26a inhibited high-fat diet (HFD)-induced oxidative stress, lipid accumulation, activation of inflammation and hepatic damage [12]. Anti-miR-214-3p increased ULK1induced autophagic activity to mitigate hepatic steatosis [13].
MiR-103-3p is an important member of the miRNA family and is related to the regulation of pathological processes such as osteoporosis, diabetes, and obesity [14,15]. Recent research has shown that miR-103-3p is a noninvasive prospective biomarker for NAFLD diagnosis [16]. The increase in miR-103-3p is related to liver steatosis, a type of NAFLD [17]. However, the regulatory mechanism of miR-103-3p in NAFLD has not yet been studied.
In this research, we used HFD-fed mice and FFA-treated L02 cells to research the possible regulatory effect of miR-103-3p on NAFLD. MiR-103-3p expression in hepatic and hepatocellular tissues increased. Moreover, suppression of miR-103-3p alleviated NAFLD damage both in vivo and in vitro by regulating hepatic lipid metabolism. Finally, we found that acyl-CoA oxidase 1 (ACOX1) was the target gene of miR-103-3p.

Cell culture and cellular steatosis model construction
Human normal liver L02 cells were purchased from ATCC and cultured in RPMI 1640 medium (Gibco™, A4192301) with 10% FBS (Gibco™, 12483020) and 1% penicillin/streptomycin in 5% CO 2 in a 37 °C humidified atmosphere. We used 1 mM FFA [FFA; oleate acid and palmitate acid (2:1)] to induce L02 cells to establish a NAFLD cell model. L02 cells were cultured in RPMI 1640 medium with FBS until the cells reached 60-80% confluence and then treated with 1 mM FFA for 48 hours (48 h).

Animals and treatment
Six-week-old male C57BL/6 mice (Changsheng Biotechnology) were fed for 1 week and then randomly divided into four groups: (1) the control group; (2) the HFD group; (3) the HFD + 15 mg/kg Antagomir-NC; and (4) the HFD + 15 mg/ kg Antagomir-103-3p. All animal studies were approved by the Animal Care and Use Committee of Zhejiang University in accordance with the Chinese guidelines for the care and use of laboratory animals.

qRT-PCR assays
Total RNA was extracted by TRIzol reagent (Invitrogen™, A33251), and a TaqMan™ MicroRNA Reverse Transcription Kit (Applied Biosystems™, 4366596) or First Strand cDNA Synthesis Kit (Thermo Scientific™, K1632) was used to reverse transcribe complementary DNA (cDNA) for miRNAs or mRNA, respectively. qRT-PCR experiments of miR-103-3p, FASN, ACOX1, and ACSL1 were performed using the StepOnePlus Real-Time PCR system with Maxima SYBR Green qPCR Master Mixes. The relative expression of genes was calculated by the 2 −△△Ct method. The qRT-PCR primer sequences were shown in Table 1.

Oil red O staining
Then, 10% paraformaldehyde was applied to fix the slides, and Oil red O staining solution (Sigma, SLBP5248V) was added to the slides for 15 min. After washing with 60% isopropanol, the slides were counterstained with haematoxylin and imaged by light microscopy.

Haematoxylin and eosin (HE) staining
Liver tissues were fixed with 4% paraformaldehyde, and 5-µm-thick tissue sections were stained with HE. After airdrying, the slides were mounted with neutral gum, and the morphological changes in the liver tissues were detected under a light microscope.

Statistical analysis
Data were analysed by SPSS 18.0 and are presented as the mean ± standard deviation (SD). The means between groups were evaluated using Student's t test or one-way ANOVA. P values < 0.05 were considered significant.

MiR-103-3p expression increased in NAFLD, and suppression of miR-103-3p improved the NAFLD cell phenotype
qRT-PCR assays showed that miR-103-3p expression was increased in the FFA-treated cells and liver tissues from mice with NAFLD (Fig. 1A). These results showed that miR-103-3p may regulate the development of NAFLD. To determine the role of miR-103-3p in the pathological process of NAFLD, we successfully constructed an Antagomir-103-3p that downregulated miR-103-3p expression in the FFA-treated L02 cells (Fig. 1B). Oil Red O staining assays showed that Antagomir-103-3p significantly alleviated the accumulation of lipid droplets in NAFLD group cells (Fig. 1C).

Suppression of miR-103-3p alleviated the damage to NAFLD group cells
Abnormal lipid metabolism results in hepatic steatosis in NAFLD [18]. The biochemical test results showed that compared with the Antagomir-NC-treated cells, the Antagomir-103-3p-treated NAFLD group cells had significantly  GGT TAC ACT GTG CTA GGT GTTG  TCC AGG CGC ATG AGG CTC AGC  Mus-ACOX1  CCT GAT TCA GCA AGG TAC GG  TCG CAG ACC CTG AAG AAA TC  Mus-ACSL1  TGG GGT GGA AAT CAT CAG CC  CAC AGC ATT ACA CAC TGT ACA ACG G  Human-ACSL1  AAC AGA CGG AAG CCC AAG C  TCG GTG AGT GAC CAT TGC  decreased TG, ALT, and AST contents ( Fig. 2A). Moreover, the suppression of miR-103-3p decreased the H 2 O 2 content and ROS production (Fig. 2B). The ATP content was significantly increased in the Antagomir-103-3p-treated NAFLD group cells (Fig. 2C). In addition, qRT-PCR and western blot results showed that NAFLD model cells treated with Antagomir-103-3p displayed inhibition of fatty acid synthase (FASN) and long-chain acyl coenzyme A synthase  (Fig. 2D). The above findings revealed that suppression of miR-103-3p significantly alleviates inflammation, abnormal lipid metabolism, oxidative stress and damage to cells in the NAFLD group.

Suppression of miR-103-3p alleviated the damage to mice with NAFLD
To research the correlation between miR-103-3p and NAFLD in vivo, we constructed a mouse model of NAFLD with an HFD for 8 weeks, and Antagomir-103-3p was used for tail vein injection to interfere with miR-103-3p expression. The qRT-PCR results showed that Antagomir-103-3p effectively downregulated miR-103-3p expression in liver tissue from the mice with NAFLD (Fig. 3A). Oil Red O staining revealed that the number of red-stained lipid droplets increased in the liver tissue of the mice with NAFLD. The number of red-stained lipid droplets in the liver tissue of the mice with NAFLD in the Antagomir-103-3p group was reduced. HE staining revealed that the liver tissues of the mice with NAFLD had increased steatosis hepatocytes, ballooning degenerated hepatocytes and visible focal necrosis, and Antagomir-103-3p alleviated liver tissue lesions in the mice with NAFLD (Fig. 3B). In summary, Antagomir-103-3p improves the pathological changes in fatty liver tissue, indicating that miR-103-3p is related to NAFLD development. The biochemical test results showed that the serum TG, ALT, and AST contents of the Antagomir-103-3p-treated mice with NAFLD were significantly reduced. Moreover, Antagomir-103-3p inhibited ROS production and H 2 O 2 in the liver tissues and serum of the mice with NAFLD. The ATP content was significantly increased in the liver tissues of the Antagomir-103-3p-treated mice with NAFLD (Fig. 3C). In addition, Antagomir-103-3p inhibited FASN and ACSL1 mRNA levels and FASN and ACSL1 expression in liver tissues from the mice with NAFLD while promoting ACOX1 mRNA levels and ACOX1 expression (Fig. 3D). The above results revealed that suppression of miR-103-3p significantly alleviates inflammation, abnormal lipid metabolism, oxidative stress and damage in the mice with NAFLD.

Discussion
NAFLD is characterized by abnormal lipid accumulation in hepatocytes in the absence of alcohol intake [19]. In particular, NAFLD can progress to advanced NASH, fibrosis and cirrhosis and even hepatic carcinoma, which is a threat to human health [20]. However, there is no effective drug for NAFLD therapy. MiRNAs as a noninvasive marker may show promise in the diagnosis of NAFLD by replacing liver biopsy and provide a new strategy for NAFLD [21,22]. The expression of miR-103-3p is related to steatosis activity, fibrosis score, stages, and prognostic markers of NAFLD [16]. We found that miR-103-3p was increased in the FFA-treated hepatocytes and liver tissues of the mice with NAFLD and may be a prospective biomarker for NAFLD diagnosis.
Abnormal lipid accumulation in hepatocytes results in TG deposition, upregulation of the levels of H 2 O 2 , ALT and AST and decreases in ATP [23][24][25]. Excessive TG accumulation is a feature of NAFLD, leading to hepatic lipid droplets [26]. We found that suppression of miR-103-3p alleviates lipid droplet accumulation in NAFLD group cells and liver tissues of the mice with NAFLD, accompanied by an increase in TG content. ALT and AST were increased in the NAFLD group compared to the control group [27].
We found that suppression of miR-103-3p downregulated ALT and AST levels in NAFLD cell supernatant and mouse serum. Mitochondrial oxidative function has a key role in the development of NAFLD, and fatty acid oxidation, ATP synthesis and ROS production influence lipogenesis and gluconeogenesis [28]. Consistent with our finding, ATP was significantly increased by suppression of miR-103-3p. Moreover, suppression of miR-103-3p decreased ROS production in NAFLD group cells and liver tissues from the mice with NAFLD. However, the specific mechanism by which miR-103-3p regulates NAFLD damage requires further study.
MiRNAs target the 3′UTR of mRNAs and silence or degrade mRNA, which regulates the progression of NAFLD [29]. MiR-17 targets Pknox1 to reduce hepatocyte steatosis by inhibiting intracellular TG and lipid accumulation [30]. MiR-130b-5p targets IGFBP2 to upregulate SCD1, ACC1 and FAS expression, and suppression of miR-130b-5p prevented lipid accumulation in hepatocytes [31]. Overexpression of miR-183-5p inhibited Btg1 to upregulate lipogenic gene expression [32]. We found that miR-103-3p regulates the mRNA levels and protein expression of the key enzymes for fatty acid synthesis (FASN and ACSL1) and the oxidation and decomposition of fatty acids (ACOX1) to alleviate liver tissue lesions in NAFLD. Moreover, miR-103-3p targets ACOX1 to modulate the development of NAFLD. ACOX1 regulates lipid homeostasis, oxidative stress, and hepatic inflammation, and the suppression of ACOX1 regulated the accumulation of TG in NAFLD [33,34]. The miR-31-5p-ACOX1 axis was shown to alter lipid metabolomes in oral squamous cell carcinoma [35]. MiR-103a-3p targets HMGB1 to alleviate LPS-induced inflammation [36]. Moreover, ACOX1 was verified to be the specific target of miR-103-3p by qRT-PCR, western blot and dual-luciferase assays. Our research showed that miR-103-3p may inhibit ALT and AST to improve inflammation, decrease ROS and H 2 O 2 levels to improve oxidative stress, and increase ATP levels to alleviate NAFLD damage by targeting ACOX1.

Conclusions
In summary, we demonstrated the possible regulation of miR-103-3p in NAFLD. MiR-103-3p expression was increased in the FFA-treated cells and liver tissues from the mice with NAFLD. Suppression of miR-103-3p alleviates abnormal lipid metabolism, oxidative stress and NAFLD damage by targeting ACOX1. Our research may provide a prospective biomarker for NAFLD diagnosis and a new strategy for NAFLD. Antagomir-103-3p alleviated the damage to mice with NAFLD. Mice with NAFLD were fed an HFD for 8 weeks, and Antagomir-NC or Antagomir-103-3p was used for tail vein injection once a week for 2 weeks. A MiR-103-3p expression in mouse liver tissues was examined by qRT-PCR. B Oil Red O staining detected lipid droplet accumulation in mouse liver tissues, and HE staining detected liver tissue lesions in mice. C The TG, ALT, AST and H 2 O 2 contents in mouse serum were examined, while ROS generation and ATP content were examined in mouse tissues. D The protein and mRNA levels of ACOX1, FASN and ACSL1 were examined by western blotting and qRT-PCR, respectively. *P < 0.05 compared with the control group; # P < 0.05 compared with the NAFLD+Antagomir-NC group ◂ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The binding of miR-103-3p to the ACOX1 3′UTR was verified by a dual luciferase reporter assay. C The ACOX1 mRNA level was deter-mined by qRT-PCR. D ACOX1 protein expression was determined by western blots. The data are expressed as the mean ± standard deviation, *P < 0.05 A model for miR-103-3p regulation of NAFLD by targeting ACOX1 (see text for details)