miR‐212 downregulation contributes to the protective effect of exercise against non‐alcoholic fatty liver via targeting FGF‐21

Abstract Non‐alcoholic fatty liver disease (NAFLD) is associated with obesity and lifestyle, while exercise is beneficial for NAFLD. Dysregulated microRNAs (miRs) control the pathogenesis of NAFLD. However, whether exercise could prevent NAFLD via targeting microRNA is unknown. In this study, normal or high‐fat diet (HF) mice were either subjected to a 16‐week running program or kept sedentary. Exercise attenuated liver steatosis in HF mice. MicroRNA array and qRT‐PCR demonstrated that miR‐212 was overexpressed in HF liver, while reduced by exercise. Next, we investigated the role of miR‐212 in lipogenesis using HepG2 cells with/without long‐chain fatty acid treatment (±FFA). FFA increased miR‐212 in HepG2 cells. Moreover, miR‐212 promoted lipogenesis in HepG2 cells (±FFA). Fibroblast growth factor (FGF)‐21, a key regulator for lipid metabolism, was negatively regulated by miR‐212 at protein level in HepG2 cells. Meanwhile, FFA downregulated FGF‐21 both at mRNA and protein levels in HepG2 cells. Also, FGF‐21 protein level was reduced in HF liver, while reversed by exercise in vivo. Furthermore, siRNA‐FGF‐21 abolished the lipogenesis‐reducing effect of miR‐212 inhibitor in HepG2 cells (±FFA), validating FGF‐21 as a target gene of miR‐212. These data link the benefit of exercise and miR‐212 downregulation in preventing NAFLD via targeting FGF‐21.


Introduction
Non-alcoholic fatty liver disease (NAFLD) is an important public health problem closely associated with genetic factors, obesity, type 2 diabetes and lifestyle act [1]. Non-alcoholic fatty liver disease comprises a spectrum of hepatic disorders, usually beginning with simple steatosis, characterized by accumulation of triglyceride (TG) within lipid droplets in hepatocytes, which can possibly progress to nonalcoholic steatohepatitis, liver cirrhosis, and even worse, hepatocellular carcinoma [2]. Although the dysregulated metabolism of TG and insulin resistance have been known to be critical for the development of NAFLD, the pathogenesis of NAFLD is incompletely understood and current therapeutic strategies are far from satisfactory [3].
Population-based studies indicate that NAFLD is associated with sedentary life style [4]. Physical exercise has been known to be beneficial for NAFLD prevention and treatment [5][6][7][8]. It has been reported that physical exercise at least once a week was effective to attenuate hepatic steatosis in NAFLD patients [9]. Physical exercise is supposed to reduce intrahepatic TG accumulation, improve insulin sensitivity and attenuate oxidative stress [10]. To those who have cardiorespiratory limitations, resistance exercise also displays beneficial effects on NAFLD through improving insulin sensitivity, reducing intrahepatic lipids and inducing hepatic fat oxidation [11][12][13]. A better understanding of the molecular mechanisms mediating the protective effect of exercise against NAFLD is of great importance.
In the present study, we demonstrate that hepatic miR-212 is upregulated in high-fat (HF)-diet fed mice, while exercise protects the liver from HF-diet induced hepatic steatosis with blunted miR-212 expression. Our data further show that miR-212 participates in the lipogenesis in HepG2 cells in vitro, through targeting fibroblast growth factor (FGF)-21 which is a central regulator for lipid metabolism. This work provides strong evidence suggesting that miR-212 might be a novel therapeutic target mimicking the benefit of exercise in the treatment of NAFLD.

Animal experimentation
Male C57BL/6 mice aged 8-10 weeks were purchased from SLAC Laboratory Animal Center, Shanghai. Mice were housed in a temperaturecontrolled room on a 12 hrs light/dark cycle and fed ad libitum with a HF or control diet for 16 weeks. Mice were randomized into four groups: (i) control group (n = 10), mice fed with standard chow (n = 10); (ii) exercise group (n = 10), mice fed with standard chow and subjected to exercise; (iii) HF diet group (n = 10), mice fed with HF diet (20.26% carbohydrate, 19.74% protein, and 60% fat) and (iv) HF diet & exercise group (n = 10). Exercised-mice were put on a running machine at the speed 10 m/min. for 60 min./day for a period of 16 weeks. Bodyweight was recorded once a week during the study. After 16 weeks, animals were weighted and killed. Lee's index was calculated as bodyweight (g)^(1/3) 91000/naso-anal length (cm) to evaluate obesity degree. The study protocol was reviewed and approved by the ethics committee of Shanghai University and all animal experiments were conducted under the guidelines on humane use and care of laboratory animals for biomedical research published by National Institutes of Health (No. 85-23, revised 1996).

Histopathological analysis
Liver segments (three selected specimens from different regions of the liver) were removed from each mouse, fixed in 4% buffered formalin and processed for embedding in paraffin. The 5 lm-thick liver sections were stained with haematoxylin and eosin and Oil Red O staining for evaluation of hepatic steatosis.

Serum analysis
Serum alanine transaminase (ALT) and aspartate transaminase (AST) activities (U/l), as well as total cholesterol (TCH) and TG levels (mmol/l) were measured using routine clinical chemical assays (Nanjing Jiancheng, China).

Microarray analysis
Total RNA were isolated from liver tissues and quantified by the Nano-Drop ND-2100 (Thermo Scientific, Hudson, NH, USA) and the RNA integrity was assessed using Agilent 2100 (Agilent Technologies, Palo Alto, CA, USA). The sample labelling, microarray hybridization and washing were performed based on the manufacturer's standard protocols. Briefly, total RNA were tailed with Poly A and then labelled with Biotin. After, the labelled RNA were hybridized for 16 hrs at 48°C on Affemetrix miRNA 3.0 Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450. The arrays were scanned by the Affymetrix Scanner 3000 (Affymetrix, Cleveland, OH, USA). Affymetrix Gene-Chip Command Console software (version 4.0; Affymetrix) was used to analyse array images to get raw data and then offered Robust Multi-Array Analysis (RMA) normalization. Next, Genespring software (version 12.5; Agilent Technologies) was used to proceed the data analysis. Probes that at least 75 percent of samples in any 1 condition out of 2 conditions have flags in 'Present' were chosen for further data analysis. Differentially expressed miRNAs were then identified through fold change as well as P-value calculated using t-test. The threshold set for  up-and down-regulated genes was a fold change ≥2.0 and a P ≤ 0.05. The MIAME-compliant data have been submitted to Gene Expression Omnibus (platform ID: GSE65978).

Cell culture and FFA treatment
Human hepatocellular carcinoma HepG2 cells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in high glucose-DMEM (Hyclone, Logan, UT, USA) containing 10% foetal bovine serum (Hyclone) and 1% penicillin/streptomycin in a humidified atmosphere of 5% CO 2 at 37°C. HepG2 cells were treated with 1 mM long-chain fatty acid (FFA) (oleate: palmitate at a molar ration of 2:1; Sigma-Aldrich, St. Louis, MO, USA) in 1% bovine serum albumin for 24 hrs to induce lipogenesis in vitro.

Nile Red staining for intracellular lipid droplets analysis
HepG2 cells were seeded at 1.5 9 10 5 cells/well in 24-well plates. After treated with 1 mM FFA for 24 hrs, cells were washed twice with PBS and fixed with 4% paraformaldehyde for 15 min. Intracellular lipids were stained with Nile Red dye (0.1 lmol/ml) for 15 min. Cell nuclei were stained with Hoescht 33258 dye for 20 min. The staining process was conducted at room temperature avoiding direct light. Images were acquired with a fluorescence microscope with magnification of 1009 and 2009 (Leica, Wetzlar, Germany).

Flow cytofluorometry for intracellular lipid content assessment
Intracellular lipid content was determined by flow cytofluorometry based on Nile Red staining. Cells were washed twice with PBS and detached by trypsinization. After centrifugation at 166g for 5 min., the cell pellet was resuspended in 1 ml PBS and incubated with 0.75 lg/ml Nile Red dye for 15 min. at room temperature. Nile Red intracellular fluorescence was determined by flow cytofluorometry using MoFlo XDP Cell Sorter (Beckman Coulter, Miami, FL, USA) on the FL3 emission channel through a 585 AE 21 nm band pass filter, following excitation with an argon ion laser source at 488 nm [33]. A total of 15,000 cells were collected and data were analysed using FlowJo software (Treestar Inc., Ashland, OR, USA). Fluorescence intensity was expressed indirectly as the percentage of events above the median value of fluorescence.

Real time quantitative PCR
Isolation of total RNA was performed using Trizol reagent (Invitrogen, Paisley, UK). For mRNA analysis, a total of 400 ng RNA was used for cDNA synthesis using Bio-Rad iScripTM cDNA Synthesis Kit (Bio-Rad, Richmond, CA, USA). PCR amplification was carried out with Takara SYBR Premix Ex Taq TM (Tli RNaseH Plus, Takara, Kusatsu, Japan) in CFX96TM Real-Time PCR Detection System (Bio-Rad). b-actin was used as internal control. For miRNA analysis, the Bulge-LoopTM miRNA qPCR Primer Set (RiboBio) was used to detect miR-212 expression by qRT-PCR with Takara SYBR Premix Ex Taq TM in CFX96TM Real-Time PCR Detection System. U6 and 5s were used as internal controls for in vivo and in vitro experiments, respectively. The utilized primers were listed in Table 1.

Western blot analysis
Liver tissues or HepG2 cells were lysed using RIPA lysis buffer (Beyotime Institute of Biotechnology, China) containing 1% phenylmethanesulfonyl fluoride. A total of 30 lg protein was subjected to electrophoreses on 12% SDS-Page gels, transferred to Polyvinylidene

Statistical analysis
The relative expression levels of mRNA and miRNA were calculated using the 2 ÀDDCt method. Data are expressed as mean AE S.E.M. Oneway ANOVA test was performed to analyse difference between groups, followed by Bonferroni's post-hoc test for multiple comparisons. All analysis were performed using (SPSS software, International Business Machines Corporation, Armonk, NY, USA) (version 19.0). P-value less than 0.05 was considered significant.

Exercise reduces HF-diet induced liver steatosis and hepatic injury
After a 16-week HF-diet, mice became markedly obese compared to controls, while HF-diet induced increase in bodyweight and Lee's index was reduced by exercise ( Fig. 1A and B). The livers of HF group were macroscopically enlarged and pale in colour (Fig. 1C). Haematoxylin and eosin staining and Oil Red O staining for liver tissues showed increased accumulation of lipid droplets in hepatocytes (Fig. 1D). Meanwhile, HF mice displayed elevated serum levels of ALT, AST, TCH and TG (Fig. 1E). All of these changes were significantly attenuated by exercise ( Fig. 1C-E), indicative of protective effect of exercise against liver steatosis and hepatic injury in HF mice.

miR-212 is induced in FFA-treated HepG2 cells in vitro
HepG2 cells with or without FFA treatment were used to examine the role of miR-212 in lipogenesis in vitro. FFA-treated HepG2 cells demonstrated increased intracellular lipid content as represented by Nile Red staining (Fig. 3A) and Nile Red intracellular fluorescence determined by flow cytofluorometry (Fig. 3B). Moreover, FFA-treated HepG2 cells displayed upregulation of miR-212 level (Fig. 3C), indicating that miR-212 could also be induced in an in vitro cell model mimicking NAFLD.

miR-212 participates in lipogenesis in HepG2 cells regardless of FFA treatment
To determine whether miR-212 could regulate lipogenesis in vitro, HepG2 cells were transfected with miR-212 mimics, inhibitor or their NC for 48 hrs. As measured by qRT-PCR, miR-212 mimics upregulated, while miR-212 inhibitor downregulated miR-212 level in HepG2 cells ( Fig. 4A and B), confirming that the inhibitors and mimics used in this study took effects. Next, we demonstrated that miR-212 mimics increased, while miR-212 inhibitor reduced intracellular lipid content in HepG2 cells regardless of FFA treatment using Nile Red staining and flow cytofluorometry ( Fig. 4C and D). These data provide important evidence showing that miR-212 might be a crucial regulator for lipogenesis.

FGF-21 is a target gene of miR-212 involved in lipogenesis
As shown in miRWalk database, FGF-21 is a validated target gene of miR-212. Knowing that FGF-21 is a central regulator contributing to lipid metabolism, we continued to examine whether FGF-21 could be a target gene of miR-212 involved in lipogenesis. We found that miR-212 mimics repressed, while miR-212 inhibitor induced the expression of FGF-21 at protein but not mRNA level in HepG2 cells regardless of FFA treatment (Fig. 5A-D). Meanwhile, FFA-treated HepG2 cells also displayed a reduction in FGF-21 expression at both mRNA and protein level ( Fig. 5E and F). Furthermore, we tested FGF-21 expression in mice liver samples showing that the protein level but not mRNA level of FGF-21 was reduced by HF-diet while reversed with exercise in HF  Fig. 5G and H). These data reveal that FGF-21 could be negatively regulated by miR-212 and/or under conditions of lipogenesis. Next, FGF-21 expression was silenced using si-FGF-21 which aimed to clarify if FGF-21 was responsible for the effects of miR-212 in lipogenesis. FGF-21 mRNA level was efficiently suppressed by transfection of HepG2 cells with si-FGF-21-102 and si-FGF-21-103 (Fig. 6A). As examined by Nile Red staining and flow cytofluorometry, silencing FGF-21 (by either si-FGF-21-102 or si-FGF-21-103) caused notable increase in lipid content in HepG2 cells regardless of FFA treatment ( Fig. 6B and C). Furthermore, silencing FGF-21 significantly blunted the inhibitory effect of miR-212 inhibitor on lipogenesis in HepG2 cells ( Fig. 6B and C). These data clearly indicate that FGF-21 is a target gene of miR-212 involved in lipogenesis.

Discussion
Exercise is known to be beneficial for NAFLD treatment. Recent studies have shown the critical involvement of miRNA in NAFLD. However, it is unclear whether exercise could prevent NAFLD via miRNA targeting. Our data demonstrate that: (i) HF-diet induced hepatic steatosis is attenuated by exercise; (ii) Hepatic miR-212 is upregulated in HF mice, while restored by exercise; (iii) miR-212 is induced during lipogenesis in FFA-treated HepG2 cells; (iv) miR-212 upregulation induces, while miR-212 downregulation reduces lipogenesis in HepG2 cells regardless of FFA treatment and (v) miR-212 contributes to lipogenesis via targeting FGF-21.
Our results demonstrating that exercise is beneficial to reduce liver steatosis and hepatic injury in HF-diet fed mice are in line with other publications. Exercise has previously been shown to reduce hepatic lipid content via downregulation of lipid metabolism-associated genes, such as FAS, SREBP-1c, ACC and SIRT-1 [34,35], which can be attributable to AMP-activated protein kinase (AMPK) activation [36]. Also, the effect of exercise on hepatic lipid accumulation is linked to reduced oxidative stress and improved insulin sensitivity [37,38]. Moreover, the benefit of exercise has been found in NAFLD patients engaged in exercise regardless of aerobic and resistant training [9,11]. The current study shows that 16-week running training reduces bodyweight, attenuates liver   steatosis, and restores ALT, AST, TCH and TG serum levels in HF mice, although it has previously been indicated that the beneficial effect of exercise in NAFLD patients could be independent of bodyweight loss [11,39,40]. Thus, other mechanisms should relate exercise and reduced steatosis in NAFLD.
It has increasingly been reported that miRNA dysregulation is responsible for the development of NAFLD [14,17]. Hereby using microarray analysis, we detected a spectrum of upregulated miRNA in livers of HF mice, including miR-34a, -149, -155, -205, -212 and -1224, among which miR-34a and miR-155 have already been reported to be dysregulated in NAFLD [25,[41][42][43]. Furthermore, we found that HF-diet induced upregulation of these miRNA was notably reversed by exercise. As miR-212 was one of the most upregulated miRNA in fatty liver in the present study and FGF-21, a central regulator for lipid metabolism, is a previously validated target gene of miR-212, the following work was focused on the role of miR-212 in liver steatosis.
The HF-diet induced miR-212 overexpression and exercise-associated reverse of miR-212 level were confirmed by qRT-PCR. Then we used HepG2 cell model with or without FFA treatment which aimed to further clarify the functional and molecular mechanisms of miR-212 in lipogenesis. Our data show that FFA-treated HepG2 cells have increased intracellular lipid content with an elevated level of miR-212. Furthermore, miR-212 mimics increases, while miR-212 inhibitor reduces lipid synthesis in HepG2 cells regardless of FFA treatment. Our findings suggest that miR-212 upregulation is not only an accompanying phenomenon during lipogenesis, but also could be an essential molecular factor initiating lipid synthesis. In fact, miR-212 dysregulation has previously been reported in cancers [44][45][46][47][48][49], angiogenic responses [50], and cardiac hypertrophy [51]. To our knowledge, we are the first to reveal the potential role of miR-212 in hepatic lipogenesis which could be used as a therapeutic tool for NAFLD.
Fibroblast growth factor-21, a potential metabolic regulator, has been shown to be protective against obesity, type 2 diabetes and hepatic steatosis in animal studies [52][53][54][55]. These beneficial effects of FGF-21 are mediated by its pleiotropic metabolic actions, including increasing energy expenditure, lowering serum and hepatic lipids (e.g. TG and cholesterol), and increasing insulin resistance [56][57][58][59][60][61]. As FGF-21 is a validated target gene of miR-212 as referred to miR-Walk database, we wished to determine whether miR-212 exerts its effect on lipogenesis through targeting FGF-21. We have found that miR-212 negatively regulates FGF-21 at protein but not mRNA level in HepG2 cells regardless of FFA treatment. Meanwhile, FFA treatment reduces FGF-21 expression at both mRNA and protein levels. Furthermore, interfering FGF-21 could abolish the lipogenesis-reducing effect of miR-212 inhibitor in HepG2 cells. These results clearly indicate that FGF-21 is a target gene of miR-212 involved in lipogenesis.
High level of circulating FGF-21 as well as elevated hepatic FGF-21 mRNA expression have been reported in obesity, type 2 diabetes and NAFLD in both animal models and human subjects [52][53][54][55][62][63][64][65]. However, it has been reported that lower level of FGF-21 may occur when hepatic steatosis progresses to liver inflammation and/or fibrosis [65]. Thus, our data showing lower FGF-21 protein level in HF-diet fed mice might be probably associated with more hepatic injury. In contrast, reversed FGF-21 protein level was detected in exercised HF mice in comparison to sedentary HF mice. In fact, increased serum and liver FGF-21 level has been reported with exercise training [66,67]. Based on current understanding of favourable metabolic effect of FGF-21, exercise-induced FGF-21 might contribute to protect against hepatic steatosis and hepatic injury. Last but not least, to fully prove miR-212 downregulation contributes to the protective effect of exercise against non-alcoholic fatty liver via targeting FGF-21, further study is required to subject miR-212 transgenic mice to exercise training with HF diet. Taken together, this study shows that miR-212 downregulation contributes to the protective effect of exercise against NAFLD via targeting FGF-21, linking the benefit of exercise and the downregulation of miR-212 in preventing NAFLD. Pharmacological inhibition of miR-212 using antagomiRs, chemically engineered oligonucleotides which used to silence endogenous miRNAs, might be a novel therapeutic strategy mimicking the benefit of exercise in the treatment of NAFLD.