MicroRNA-194 inhibition improves dietary-induced non-alcoholic fatty liver disease in mice through targeting on FXR

Accepted Manuscript MicroRNA-194 inhibition improves dietary-induced nonalcoholic fatty liver disease in mice through targeting on FXR

Hezhongrong Nie, Chunli Song, Daming Wang, Shengjin Cui, Tingyu Ren, Zhaopeng Cao, Qing Liu, Zeyan Chen, Xiaoyong Chen, Yiwen Zhou PII: DOI: Reference:

S0925-4439(17)30335-6 doi:10.1016/j.bbadis.2017.09.020 BBADIS 64903

To appear in: Received date: Revised date: Accepted date:

21 March 2017 25 July 2017 21 September 2017

Please cite this article as: Hezhongrong Nie, Chunli Song, Daming Wang, Shengjin Cui, Tingyu Ren, Zhaopeng Cao, Qing Liu, Zeyan Chen, Xiaoyong Chen, Yiwen Zhou , MicroRNA-194 inhibition improves dietary-induced non-alcoholic fatty liver disease in mice through targeting on FXR. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Bbadis(2017), doi:10.1016/ j.bbadis.2017.09.020

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ACCEPTED MANUSCRIPT MicroRNA-194 inhibition improves dietary-induced non-alcoholic fatty liver disease in mice through targeting on FXR

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Hezhongrong Nie1*, Chunli Song1, Daming Wang1, Shengjin Cui1, Tingyu Ren1, Zhaopeng Cao1,

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Qing Liu1, Zeyan Chen1, Xiaoyong Chen1, Yiwen Zhou1*

1 Center of Clinical Laboratory, Shenzhen Hospital of Southern Medical University, Shenzhen,

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China

* Corresponding author: Center of Clinical Laboratory, Shenzhen Hospital of Southern Medical

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University, No.1333 Xinhu Road, Baoan District, Shenzhen, Guangdong province 518100, P.R.

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China.

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Email address: [email protected] (Yiwen Zhou); [email protected] (Hezhongrong

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Nie). Tel: 86-755-2332-9999.

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ACCEPTED MANUSCRIPT Abstract Non-alcoholic fatty liver disease (NAFLD) affects obesity-associated metabolic syndrome, which exhibits hepatic steatosis, insulin insensitivity and glucose intolerance. Previous studies indicated that hepatic microRNAs (miRs) play critical roles in the development of NAFLD. In this study,

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we aim to explore the pathphysiological role of miR-194 in obesity-mediated metabolic

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dysfunction. Our findings show that the high fat diet or palmitic acid treatment significantly

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increase hepatic miR-194 levels in vivo and in vitro. Silence of miR-194 protects palmitic acid-

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induced inflammatory response in cultured hepatocytes, and attenuates structural disorders, lipid deposits and inflammatory response in fatty liver. MiR-194 inhibitor also improves glucose and

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insulin intolerance in obese mice. Through dual luciferase assay, we demonstrate that miR-194

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directly binds to FXR/Nr1h4 3’-UTR, and inhibits gene expression of FXR/Nr1h4. Furthermore, overexpression of miR-194 downregulates FXR/Nr1h4 in cultured hepatocytes, but miR-194

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inhibitor reversely increases FXR/Nr1h4 expression in obese mouse liver tissues. On the contrast,

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silence of FXR/Nr1h4 abolishes the hepatic benefits in obese mice treated with miR-194 inhibitor. Present study provides a novel finding that suppression of miR-194 attenuates dietary-induced

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NAFLD via upregulation of FXR/Nr1h4. The findings suggest miR-194/FXR are potential

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diagnostic markers and therapeutic targets for NAFLD. Abbreviation: NAFLD: non-alcoholic fatty liver disease miR: microRNA Nuclear FXR/Nr1h4: farnesoid X receptor HFD: high-fat diet STC: standard chow NF-κB: nuclear factor-κB Keywords: non-alcoholic fatty liver disease; miR-194; inflammation; FXR/Nr1h4

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ACCEPTED MANUSCRIPT 1. Introduction Nonalcoholic fatty liver disease (NAFLD), the hepatic manifestation of metabolic syndrome, is the most common liver disease in clinical practice, affecting most obese individuals [1, 2]. Disorders related to metabolic syndromes, such as obesity, type 2 diabetes mellitus and

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dyslipidemia are identified as the main risk factors for the development of NAFLD [1, 2].

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NAFLD exhibits amounts of hepatic pathology, including inflammatory disorder, structural

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damages and lipid deposits [3-5]. NAFLD in rodents is typically induced by high-fat diet (HFD),

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which stimulates excess accumulation of triglyceride in liver and insulin resistance [5]. Although a widely accepted two-hit hypothesis may partially explain the progressive liver damage by non-

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alcoholic steatosis and steatohepatitis [3-5], much of the pathogenesis of NAFLD remains

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undiscovered and requires further study.

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MicroRNAs (miRs) are short noncoding RNA molecules of 18-25 nucleotides in length that repress specific target mRNAs by degradation or translational repression. Recently, several

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miRNAs have been implicated in fatty liver diseases [6-8]. The expression of miR-194 in the

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liver has been known for a long time, but its function has not been clearly characterized. One study suggested that miR-194 was highly expressed in hepatocytes, stellate cells and Kupffer

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cells [9, 10]. A second study suggested that hepatic miR-194 is induced in ob/ob mouse model [11]. These two reports provided the first evidence that hepatic miR-194 is upregulated during fatty liver development. However, whether miR-194 has physiological roles in the process of liver injuries is still unknown. Mechanistically, explore evidences for endogenous targets of miR194 in vivo is also critical for understanding its biological effects. Nuclear farnesoid X receptor (FXR, Nr1h4) is a ligand activated transcription factor, playing a critical role in maintaining lipid and glucose homeostasis by regulating its downstream genes in 3

ACCEPTED MANUSCRIPT the liver [12-14]. It is mainly expressed in the liver, intestines, kidney, and adrenal glands [12]. FXR controls several key genes involved in human bile acid synthesis and metabolism, including cholesterol 7α-hydroxylase 1 (Cyp7a1), bile salt export pump (Bsep), small heterodimer partner (Shp) and intestinal bile acid-binding protein (Ibabp) [12]. Studies on FXR knockout mice have

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demonstrated that FXR exerts hepatoprotective effects, which exhibit downregulation of plasma

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cholesterol and triglyceride levels and suppression of hepatic lipid accumulation [12, 13].

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Diminished FXR expression has also been linked to an increase of inflammatory responses in obese mice [15]. In contrast, strongly enhanced FXR expression effectively improves obesity-

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induced metabolic dysfunction [15]. Preclinical and clinical evidences have suggested that FXR

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agonist has beneficial effect in the treatment of NAFLD by decreasing hepatic inflammatory damages, steatosis, and insulin resistance in patients [16, 17]. Therefore, targeting of FXR is a

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potential therapeutic approach to combat NAFLD progress.

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Here, through cultured hepatocytes and dietary-induced mouse model, we demonstrated that

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hepatic miR-194 was an important factor in mediating the obesity-induced hepatic injuries. Our results further indicated that hepatic miR-194 inhibition attenuated liver damages and metabolic

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dysfunction through upregulation of FXR/Nr1h4 signalling.

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2. Materials and methods 2.1 Reagents

Palmitic acid, bovine serum albumin, D-glucose, recombinant insulin and miR-194 mimic were purchased from Sigma chemicals (Sigma, St. Louis, USA). Triglyceride assay kit was purchased from Cayman Chemical (Ann Arbor, MI). For western blot experiments, anti-phosp-IκB, antiIκB and anti-Tubulin antibodies were from Cell Signaling (Danvers, MA). Anti-FXR antibody

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ACCEPTED MANUSCRIPT was purchased from Santa Cruz Biotech (Santa Cruz, CA). For immunohistological staining, anti-F4/80 was from R&D systems. 2.2 Cell culture The human derived HepG2 cell was offered from the Institute of Biochemistry and Cell Biology

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at the Chinese Academy of Sciences (Shanghai, China), and maintained in EMEM medium

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containing 10% FBS and 1% Penicillin/Streptomycin antibiotics in humidified chamber (37°C, 21% O2 and 5% CO2). For stimuli treatment, cells were stimulated with palmitic acid for

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indicated time.

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2.3 Animal experiments

Male C57BL/6J mice, aged 8-week, were obtained from Animal Centre of Southern Medical

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University (Guangzhou, China). All animals were housed at a constant room temperature with a

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12:12 hour light-dark cycle. Mice were fed with high fat diet (SLAC Animal Laboratories,

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Shanghai, China), providing 59% of calories from fat, 25% from carbohydrates and 16% from protein for 8 weeks, then randomly divided into 2 groups with 8 mice in each group: 1) high fat

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diet-treated mice that were injected with control lentivirus (HFD+Ctrl group); 2) hfd-induced mice that were injected with miR-194 anti-sense lentivirus (HFD+miR-194 inhibitor group).

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Another animal experiment was divided into 3 groups, including HFD+Ctrl group, HFD+miR194 inhibitor group and HFD+miR-194 inhibitor plus siNr1h4 group. These mice were fed HFD for another 4 weeks. Mice treated with standard diet (12% fat, 59% total carbohydrate and 29% protein) were used as standard chow group (STC group. Monitor body weight every two weeks, and collect liver tissues after sacrificing the mice. Procedures involving the animal experiments were approved by the Southern Medical University Animal Policy and Welfare Committee.

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ACCEPTED MANUSCRIPT 2.4 Liver histopathology Liver tissues were fixed in 4% paraformaldehyde for 24-h and embedded in paraffin. 7 µm paraffin sections were prepared and stained with hematoxylin and eosin. To investigate the histological damage, the slides were observed under a light microscope (100×, Olympus, Japan).

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2.5 Immunohistochemistry

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After deparaffinization and rehydration, liver sections (7 µm) were deprived endogenous enzymes with 3% H2O2 for 10 min and blocked with 10% BSA in PBS for 30 min. Slides were

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incubated overnight at 4 ℃ with anti-F4/80 antibodies (1:100), then treated with secondary

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antibody (Cell Signaling, Danvers, MA; 1:200) for 1 hour at room temperature. PBST washed slides for 3 times, and DAB (Sigma, Louis, MO) developed for 3 minutes. Then slides were

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microscope (200×, Olympus, Japan).

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stained the nuclear with hematoxylin for 10 seconds, and the images were imaged by a light

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2.6 DNA constructs and virus generation

The miR-194 anti-sense (miR-194 inhibitor) or nonsense control lentiviral plasmid was

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constructed by using synthetic oligonucleotide, which contains either miR-194 binding sites or

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nonsense sequence. MiR-194 inhibitor or control lentivirus was produced by co-transfecting HEK-293T cells with the corresponding lentiviral constructs and lentiviral mix (Genomeditech) according to the manufacturer’s instruction. 72 hours after transfection, the medium was collected and concentrated using Polyethylene glycol 6000 (PEG 6000, Sigma) precipitation approach and the titer was determined by the frequency of GFP-positive HEK-293T cells. For in vivo lentivirus treatment, fifty microliter of 1 x 1010 IU/ml lentivirus encoding miR-194 inhibitor or control sense was administrated into mice after HFD feeding for 8 weeks. Liver was 6

ACCEPTED MANUSCRIPT collected 4 weeks after injection and the efficiency of infection was validated by measuring the miR-194 levels. 2.7 Luciferase reporter gene constructs and luciferase assay The construct psicheck-3’-UTR of Nr1h4 was achieved by digesting double restriction enzyme

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(XhoI and NotI) in psicheck2 vector (T7 promoter, Addgene), followed by ligation of sequence

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encoding the corresponding 3’UTR to miR-194 binding sites. The 3’-UTR of Nr1h4 was confirmed by sequencing and named psicheck-Nr1h4. Site-directed mutagenesis of the Nr1h4 3’-

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UTR was performed using a QuikChange site-directed mutagenesis kit (Agilent, Santa Clara, CA) and conduct the psicheck-Nr1h4-Mut plasmid. HEK-293T cells were transfected with one of the

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above plasmids using Lipofectamine™ 3000 (Invitrogen). Constitutively active firefly luciferase

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plasmid (pGL3-Promoter Vector, Promega) was simultaneously delivered to cells for

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normalization. Luciferase activity was measured by the dual-luciferase assay system (Promega, Madison, WI) 48 h post transfection.

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2.8 Real-time quantitative PCR

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30-50 mg liver tissues were homogenized in TRIZOL (Invitrogen, Carlsbad, CA) for extraction of RNA according to the manufacturer’s protocol. Both reverse transcription and quantitative

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PCR were carried out with the Invitrogen kits (Invitrogen, Shanghai, China). The ABI Prism 7900 Sequence Detection System (Applied Biosystems, Alameda, CA) was used for real timeqPCR analysis. The primers of genes including TNF-α, IL-6, MCP-1, Nr1h4 and 18s were synthesized from Invitrogen (Invitrogen, Shanghai, China). The primer sequences used were listed in Table S1. For miR-194 expression analysis, Taqman-based miR RT for miR-194 and sno202 analysis.

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ACCEPTED MANUSCRIPT 2.9 Western immunoblot 50 µg of the lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to a nitrocellulose membrane. Each membrane was preincubated for 1 h at room temperature in Tris-buffered saline, pH 7.6, containing 0.05% tween 20

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and 5% non-fat milk, and incubated with specific antibodies. Immunoreactive bands were then

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detected by incubating the membrane with secondary antibodies conjugated to horseradish

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peroxidase, and visualized using enhanced chemiluminescence reagents (Bio-Rad, Hercules, CA).

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The amounts of the proteins were analysed using Image J analysis software version 1.38e and normalized with their respective control.

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2.10 Statistical analysis

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The results were shown as means ± SEM. The Students’ t-test or ANOVA analysis were used for

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analysing the statistical significance of difference among groups, respectively. P<0.05 was

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considered as statistical significance. 3. Results

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3.1 High fat diet or palmitic acid induces miR-194 product in vivo and in vitro

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MiR-194 is highly expressed in liver tissues, especially in hepatocytes, and positively associated with hepatic fibrogenesis [9-11]. Previous one study implied that ob/ob mice exhibited significant higher level of miR-194 in liver [11], so we firstly determined whether high fat diet (HFD) also stimulated hepatic miR-194 levels. C57BL/6J mice were fed with HFD for 1, 4, 8 and 12-week. As compared with standard chow (STC)-fed mice, HFD time-dependently increased hepatic miR194 levels (Fig. 1A). Furthermore, palmitic acid (PA), as a toxic lipid, also dose-dependently stimulated miR-194 expression in cultured HepG2 cells (Fig.1B). 8

ACCEPTED MANUSCRIPT 3.2 Inhibition of miR-194 attenuates PA-induced inflammatory response in HepG2 cells Therefore, we generated lentivirus encoding miR-194 anti-sense to silence hepatic miR-194 levels. As Fig. 2A shown, miR-194 inhibitor effectively suppressed miR-194 level in HepG2 cells (p < 0.001). With lipid overload in hepatocytes, there are obvious inflammatory response,

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including secretion of inflammatory cytokines [18]. As Fig. 2B shown, PA significantly

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stimulated the mRNA levels of TNF-α, IL-6 and MCP-1 (p < 0.001), but miR-194 suppression

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could effectively decrease their upregulation. NF-κB signalling plays a critical role in mediating

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inflammatory response in hepatocytes [18, 19]. With inflammatory stimulation, IκB is phosphorylated and degraded at early response.[19] We found HepG2 cells exhibited higher

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phosphorylated-IκB (p-IκB) and degradation of IκB in PA-treated group (p < 0.001), but miR-

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194 inhibition attenuated NF-κB activation (p < 0.01) (Fig. 2C-D).

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3.3 Suppression of miR-194 improves HFD-induced fatty liver disease Next we aim to investigate the physiological role of miR-194 in HFD-induced fatty liver injuries.

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C57BL/6J mice were fed HFD for 8 weeks, and tail veil injection with lentivirus encoding miR-

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194 anti-sense or nonsense for another 4 weeks. As Fig. S1 shown, miR-194 inhibitor significantly reduced hepatic miR-194 level in HFD-fed mice (p < 0.001). MiR-194 inhibitor

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decreased HFD-stimulated liver weight, as compared with HFD+Ctrl group (Fig. 3A, p < 0.05). In histological analysis, we found treatment with miR-194 inhibitor could effectively improve histological feature of liver steatosis, mainly exhibited accumulation of lipid droplets and immune cell infiltration (Fig. 3B). Previous studies have demonstrated that the accumulation of lipid deposit in HFD-fed hepatic tissues [5]. As compared with HFD+Ctrl group, the hepatic triglyceride content was significantly decreased in miR-194 inhibitor-treated HFD-fed mice (Fig. 3C, p < 0.01). The increasing infiltrated macrophage is a key characteristic in fatty liver disease 9

ACCEPTED MANUSCRIPT [3]. As Fig. 3D-E shown, HFD stimulated macrophage percentage in liver, but miR-194 inhibitor could block the recruitment of macrophages (p < 0.05). Furthermore, suppression of miR-194 also decreased mRNA expression of inflammatory cytokines, including TNF-α, IL-6 and MCP-1 (Fig. 3F, p < 0.05). Besides, miR-194 silence decreased p-IκB, but increased IκB protein, to

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inactivate NF-κB signalling pathway (Fig. 3G-H).

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3.4 MiR-194 silence improves HFD-induced body weight gain, glucose intolerance and insulin

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resistance, but no effect on fasting blood glucose

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To further identify the benefits of miR-194 inhibition, we measured the changes of metabolic function. As Fig. 4A shown, HFD significantly increased body weight, but miR-194 inhibitor

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could reduce the body weight gain after lentivirus injection for 2 and 4 weeks (p < 0.05 and p <

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0.01 respectively). However, there was no difference between HFD+Ctrl and HFD+miR-194

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inhibition groups in fasting blood glucose levels (Fig. 4B). Glucose intolerance and insulin resistance are key parameters in determining metabolic homeostasis [20]. As Fig. 4C shown,

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mice exhibited higher glucose levels in HFD+Ctrl group, but miR-194 silence effectively

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decreased glucose levels in glucose tolerance test. Furthermore, HFD+miR-194 inhibitor-treated mice had stronger sensitive response to insulin, as compared with HFD+Ctrl group (Fig. 4D).

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3.5 MiR-194 directly inhibits Nr1h4 expression in HepG2 cells and hepatic tissues in obese mice To screen the possible genes involved in miR-194-mediated hepatic damages, we utilized mirSVR score for predicting miR-194 target site, and PhastCons score for the conservation in different species. As Fig. 5A shown, Nr1h4 3’-UTR contained the binding sequence of miR-194. Therefore, we constructed plasmids encoding Nr1h4 wild-type and mutant 3’-UTR sequence, and co-transfected with plasmid encoding miR-194 into HEK-293T cells. Through luciferase reporter

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ACCEPTED MANUSCRIPT assay, we found that miR-194 could directly bind to Nr1h4 3’-UTR, but not to mutant Nr1h4 3’UTR (Fig. 5B). This result demonstrated Nr1h4 was a potential target of miR-194. Next we analysed the inhibition of miR-194 on Nr1h4 expression in PA-treated HepG2 cells and hepatic tissues in obese mice. On the contrast to increment of miR-194 expression in liver, HFD

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time-dependently decreased the mRNA expression of Nr1h4 (Fig. 5C). Similarity, PA treatment

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also dose-dependently down-regulated Nr1h4 expression in HepG2 cells (Fig. 5D). To further

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delineate their relationship, we treated HepG2 cells with miR-194 RNA mimic. As Fig. 5E

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shown, miR-194 mimic significantly increased miR-194 levels in HepG2 cells (p < 0.001). These induction of miR-194 decreased Nr1h4 mRNA (Fig. 5F) and protein levels (Fig. 5G) in HepG2

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cells, as compared with control group. In obese mice, miR-194 inhibitor obviously increased

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FXR/Nr1h4 protein (Fig. 5H-I, p < 0.05) and mRNA levels (Fig. 5J, p < 0.05), as compared with

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HFD group.

To further confirm the critical role of miR-194/FXR signalling in obesity-induced liver injuries,

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we next utilized FXR/Nr1h4 siRNA to investigate whether blocking FXR/Nr1h4 could reverse

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the benefits of miR-194 inhibition. As Fig. S2 shown, treatment with Nr1h4 siRNA decreased expression of Nr1h4 mRNA (Supple Fig. 2A) and FXR protein (Supple Fig. 2B) levels in miR-

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194 inhibitor-injected obese mice. We further confirmed the suppression of miR-194 on FXR by measuring its downstream targets. As Figure S3A shown, HFD increased the gene expression of Cyp7α1, but decreased Bsep, which were reversed by the treatment of miR-194 inhibitor. Therefore, the obesity-induced upregulation of serum bile acid was inhibited by miR-194 inhibitor, but silence of FXR lowered these suppression (Figure S3B). Although intestine is a critical tissue for bile acid metabolism, silence of hepatic miR-194 or FXR by tail-vein injection of viruses had less effect on FXR downstream genes, including Shp and Ibabp (Figure S3C). 11

ACCEPTED MANUSCRIPT Then we examined whether the hepatic benefits of miR-194 suppression depended on FXR signalling. As compared with the benefits of miR-194 inhibitor-treated obese mice, siNr1h4 reversely increased liver weight (Fig. 6A, p < 0.05), accumulated cellular bubble and immune cell infiltration (Fig. 6B) and stimulated triglyceride deposit (Fig. 6C). Furthermore, siNr1h4-

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treated mice had severe hepatic macrophage infiltration (Fig. 6D) and increased mRNA

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expression of TNF-α, IL-6 and MCP-1 (Fig. 6E). Mechanistically, Nr1h4 silence activated NF-κB

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signalling pathway via increasing p-IκB/IκB protein level (Fig. 6F-G). These results demonstrated that the hepatic protective effect of miR-194 inhibition relied on the upregulation of

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FXR/Nr1h4.

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4. Discussion

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In present study, we firstly provided evidences that silence of hepatic miR-194 attenuated high fat

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diet-induced fatty liver disease and metabolic dysfunction. Mechanistically, the benefits of hepatic miR-194 inhibition relied on the direct upregulation of the transcriptional factor

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FXR/Nr1h4.

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The role of miRNA-mediated gene regulation in liver function and diseases has gained increasing attention. Many characteristics of NAFLD pathogenesis, including hepatic inflammatory

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response, structural damages and lipid deposits, are associated with one or more species of miRNA [6-8]. Some miRNA, such as miR-21a, miR-34a and miR-122, are overexpressed in fatty liver tissues, while the levels of miR-99a, miR-134, and miR-296 are decreased [6-8]. Previous studies reported that miR-194 was induced in ob/ob mouse liver and highly expressed in hepatocytes [11]. Hepatic miR-194 participated in liver fibrogenesis and epithelial cell differentiation, which determined liver function [9, 10]. However, there is no evidence to unveil the physiological role of miR-194 in dietary-induced NAFLD. Current study firstly found the 12

ACCEPTED MANUSCRIPT incremental miR-194 level during obesity in high fat diet-fed mice, and overload of palmitic acid stimulated miR-194 expression in hepatocytes (Fig. 1). Although the regulation of miR-194 production is unknown, the induction of miR-194 may represent a pathphysiological response. Excess energy uptake leads ectopic lipid deposition, which is mainly attributed to deposition of

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lipids outside of the adipose tissues, including liver or skeletal muscle [3-5]. Toxic lipids trigger

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pro-inflammatory signalling by bind to TLR4/MD2 complex, then activate transcriptional factors,

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such as NF-κB and AP-1 [19, 21]. And tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6)

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and monocyte chemokine protein-1 (MCP-1) are considered to be the major inflammatory mediators in NAFLD [18]. Furthermore, the infiltrated macrophages amplifies inflammatory

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response by secreting additional cytokines. All these chronic reaction will damages hepatic

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function and structure [3-5]. Present study demonstrated hepatic miR-194 inhibition attenuated expression of inflammatory cytokines and signalling activation, and blocked macrophage

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infiltration (Fig. 2-3).

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Previous studies indicate that bile acids (BAs) promote regeneration and repair after fatty liver

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injuries [12, 22, 23]. Mechanistically, BAs play important roles not only in the absorption of dietary fat as detergent, but also in the regulation of cholesterol homeostasis via cholesterol

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degradation [22]. However, the effects of BAs on promoting NAFDL is absent in FXR-KO mice, suggesting FXR is the mediator of BAs signalling in liver diseases [12, 13]. Obesity reduces hepatic FXR expression, and weakens the metabolic benefits mediating by BA/FXR signalling [24]. Therefore, no feedback inhibition of BA synthesis by FXR occurred, which further stimulates the deposit of Bas in obese conditions. Similarity, present study also found HFD decreased hepatic FXR expression, and further exacerbated triglyceride accumulation and inflammatory response (Fig. 3), but increased circulating BA levels (Fig. S3B). The reduction of 13

ACCEPTED MANUSCRIPT FXR was closely associated with increasing miR-194 levels in liver, suggesting miR-194 might supress FXR expression. Through dual-luciferase assay, present study firstly demonstrated miR194 directly targeted on FXR 3’-UTR and inhibited its gene expression (Fig. 5). Although we could not exclude the contribution of other miRs to FXR regulation, gain-of-function and loss-of-

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function of mR-194 in vivo and in vitro indicated that FXR was a possible mediator of miR-194’s

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effect on NAFLD. Our further study determined that silencing FXR/Nr1h4 could abolish the

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benefits in miR-194 inhibitor-treated obese mice (Fig. 6). All these results demonstrated that

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miR-194/FXR signalling played a critical role in obesity-induced mouse fatty liver diseases. It should be noted that hepatic miR-194 inhibition improved metabolic parameters, including

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body weight reduction, improvement of glucose disposal and insulin response (Fig. 4). These

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findings supported hepatic miR-194 could mediate dietary-induced metabolic disorders, suggesting pharmaceutical or genetic modification of miR-194 was a promising approach to

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combat obesity and related complications.

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In conclusion, our results suggested that miR-194 was a potential mediator in NAFLD

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development, and miR-194/FXR affected hepatic inflammatory response and metabolic dysfunction. Therefore, miR-194/FXR might be an attractive therapeutic agent against obesity-

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related NAFLD.

Acknowledgements

This work was supported by grants from Science and Technology Planning Project of Shenzhen (JCYJ20140415151845365 to YW Zhou), Nature Science Foundation of Guangdong Province (2015A030313825 to DM Wang) and Scientific Research Fund of Southern Medical University (C1033337 to CL Song).

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ACCEPTED MANUSCRIPT Author contributions HZR Nie, CL Song, DM Wang, SJ Cui and ZP Cao performed the animal and cell research; HZR Nie, TY Ren and Q Liu contributed to generate virus and luciferase plasmids; HZR Nie, ZY Chen and XY Chen analysed the data; HZR Nie and YW Zhou designed the research study; HZR

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Nie wrote and edited the manuscript.

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All the authors declare no competing financial interest.

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Conflicts of interest

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ACCEPTED MANUSCRIPT Figure Legends Figure 1. Expression of miR-194 in liver is associated with obesity. (A) Abundance of miR194 in hepatic tissues of C57BL/6J mice fed with standard chow (STC) or high fat diet (HFD) for 1, 4, 8, 12 weeks (n = 5-6). (B) Expression of miR-194 in HepG2 cells treated with BSA or 125,

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Figure 2. Inhibition of miR-194 attenuates PA-induced inflammatory response in HepG2

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cells. (A) HepG2 cells were transfected with lentivirus encoding miR-194 anti-sense (miR-194 inhibitor) or relative control sense (Ctrl) for 48 hours, and RT-qPCR analyzed the expression

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level of miR-194. (B-D) HepG2 cells were pre-transfected with miR-194 inhibitor or Ctrl for 48

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hours, then incubated with 500 µM PA. (B) After 24-hour treatment, RT-qPCR measured inflammatory mRNA levels of TNF-α, IL-6 and MCP-1. (C-D) After 1-hour treatment, western

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blot detected protein expression of phosp-IκB, total IκB and Tubulin (C). Quantitative analysis of

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p-IκB/IκB density (D). Data represent Mean ± SEM. Differences between groups were determined by Students’ t-test or ANOVA; n = 4 independent experiments, *P < 0.05, **P < 0.01,

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***P < 0.001. Gene levels were normalized to 18s level. Figure 3. Suppression of miR-194 improves high fat diet-induced hepatic injuries. Male C57BL/6J mice fed with HFD for 8 weeks were transfected with lentivirus encoding miR-194 anti-sense or Ctrl by tail vein injection. Sacrifice mice after 4 weeks of lentivirus injection, and collect liver for further analysis. The lean mice fed with chow diet for 12 weeks as STC group. (A-C) Liver weight (A), representative images of H&E staining (B, 100× amplification) and

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ACCEPTED MANUSCRIPT hepatic triglyceride levels (C). (D-E) Immunohistological staining of F4/80+ macrophages in liver (D, brown color, 200× amplification) and quantitative analysis of F4/80+ cell percentage (D). (F) RT-qPCR measured mRNA levels of TNF-α, IL-6 and MCP-1. (G-H) Western blot analyzed protein expression of phosp-IκB, total IκB and Tubulin (G), and quantitative analysis of

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Figure 4. MiR-194 inhibition confers mice protection against high fat diet-induced body weight gain, glucose intolerance and insulin resistance. (A-B) Monitor mouse body weight (A)

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Figure 5. MiR-194 inhibits Nr1h4 expression in HepG2 cells and hepatic tissues in obese mice. (A) The conserved consequential site of target region on Nr1h4 mRNA 3’-UTR for miR194, and mutation at Nr1h4 3’-UTR sequence. (B) Luciferase assay with wild-type Nr1h4 3’UTR or mutated 3’-UTR, transfected with miR-194 over-expression or control plasmid in HEK293T cells. (C) Abundance of Nr1h4 mRNA in hepatic tissues of C57BL/6J mice fed with STC or HFD for 1, 4, 8, 12 weeks (n = 5-6). (D) Expression of Nr1h4 mRNA in HepG2 cells treated with BSA or 125, 250, 500 µM PA for 24 hours (n = 4 independent experiments). (E-G) HepG2 18

ACCEPTED MANUSCRIPT cells were transfected with lentivirus encoding miR-194 mimic or control for 48 hours, then RTqPCR analyzed expression levels of miR-194 (E) and Nr1h4 (F), and western blot measured FXR expression (G) (n = 4 independent experiments). (H-J) Western blot analyzed protein expression of FXR and Tubulin (H), and quantitative analysis of FXR/Tubulin protein density (I) and

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mRNA levels in hepatic tissues (J) (n = 6-8 mice). Data represent Mean ± SEM. Differences

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Figure 6. Silence Nr1h4 abolishes the benefits of miR-194 inhibition in hepatic tissue. (A-C) Liver weight (A), representative images of H&E staining (B, 100× amplification) and hepatic

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triglyceride levels (C). (D-E) Immunohistological staining of F4/80+ macrophages in liver (D,

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200× amplification) and quantitative analysis of F4/80+ cell percentage (D). (F) RT-qPCR measured mRNA levels of TNF-α, IL-6 and MCP-1. (G-H) Western blot analyzed protein

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expression of phosp-IκB, total IκB and Tubulin (G), and quantitative analysis of p-IκB/IκB

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density (H). Data represent Mean ± SEM. Differences between groups were determined by

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to 18s level.

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ACCEPTED MANUSCRIPT Highlights 1. Lipid overload induced hepatic miR-194 levels in vivo and in vitro. 2. MiR-194 inhibition improved obesity-induced NAFLD and metabolic disorders. 3. MiR-194 directly suppressed FXR/Nr1h4 gene expression in mice and cultured hepatocytes.

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4. Silence of FXR/Nr1h4 abolished the benefits of miR-194 inhibition in NAFLD.

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