Down-regulation of miR-144 elicits proinflammatory cytokine production by targeting toll-like receptor 2 in nonalcoholic steatohepatitis of high-fat-diet-induced metabolic syndrome E3 rats

Molecular and Cellular Endocrinology 402 (2015) 1–12

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Molecular and Cellular Endocrinology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m c e

Down-regulation of miR-144 elicits proinflammatory cytokine production by targeting toll-like receptor 2 in nonalcoholic steatohepatitis of high-fat-diet-induced metabolic syndrome E3 rats Dongmin Li a,b,1, Xuan Wang a,b,1, Xi Lan a,b, Yue Li a,b, Li Liu a,b, Jing Yi a,b, Jing Li a,b, Qingzhu Sun a,b, Yili Wang c, Hongmin Li d, Nannan Zhong e, Rikard Holmdahl f, Shemin Lu a,b,* a Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi 710061, China b Key Laboratory of Environment and Genes Related to Diseases (Xi’an Jiaotong University), Ministry of Education of China, Beijing, China c Research Institute of Cancer, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, China d School of Life Sciences, Northwest University, Xi’an, Shaanxi 710061, China e Xi’an Health School, Xi’an, Shaanxi 710054, China f Division of Medical Inflammation Research, Department of Biochemistry and Biophysics, Karolinska Institute, SE-17177 Stockholm, Sweden

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Article history: Received 20 April 2014 Received in revised form 6 December 2014 Accepted 9 December 2014 Available online 19 December 2014 Keywords: miR-144 TLR2 Proinflammatory cytokines Nonalcoholic steatohepatitis (NASH) Kupffer cells High-fat-diet induced metabolic syndrome (HFD-MetS)

A B S T R A C T

Objective: To analyze regulatory microRNA(s) leading to increased TLR2 expression in livers of high-fatdiet induced metabolic syndrome (HFD-MetS) in rats with non-alcoholic steatohepatitis (NASH). Methods: TLRs, inflammatory cytokines, candidate miRNAs targeting key TLR and its cellular localization were determined in liver. The miR-144 targeting TLR2 and regulating TLR2 signaling were further determined by dual luciferase reporter assay and miR-144 mimics or inhibitor. Results: Expression of miR-144 was negatively correlated with TLR2 expression in Kupffer cells. The miR144 bound to 3′UTR of rat TLR2 mRNA. In addition, compared to control group, TLR2, TNF-α, IFN-γ and activation of NF-κB decreased after miR-144 mimic challenge in NR8383 cells and BMM from E3 rats, which could be compensated by Pam3CSK4; while opposite effects on their expressions were observed after miR-144 inhibitor administration, augmented by Pam3CSK4. Conclusion: Decreased miR-144 could enhance TNF-α and IFN-γ production by targeting TLR2 in vitro, and might contribute to TLR2 up-regulation and the progression of NASH in HFD-MetS E3 rats. This might offer a novel and potential target for NASH therapy. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Nonalcoholic steatohepatitis (NASH) is the progressive stage of non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of metabolic syndrome (Boppidi and Daram, 2008; Kim and

Abbreviations: BMC, bone marrow cells; BMM, bone marrow-derived macrophages; DMEM, Dulbecco’s modification of Eagle’s medium; FBS, fetal bovine serum; HFD, high fat diet; HFD-MetS, high-fat-diet induced metabolic syndrome; miRNA, microRNA; miR-144, microRNA 144; NAFLD, non-alcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; RT-qPCR, real-time quantitative PCR; TBST, Trisbuffer saline with 0.1% Tween; TLR2, Toll like receptor 2; ELISA, enzyme-linked immunosorbent assay. * Corresponding author. Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi 710061, China. Tel.: +86 29 82657764; fax: +86 29 82657764. E-mail address: [email protected] (S. Lu). 1 These two authors contributed equally to this work. http://dx.doi.org/10.1016/j.mce.2014.12.007 0303-7207/© 2014 Elsevier Ireland Ltd. All rights reserved.

Younossi, 2008; Medina-Santillan et al., 2013). It is well known that NASH not only promotes the development of hepatic diseases such as cirrhosis, hepatic decompensation, and hepatocellular carcinoma (Rahimi and Landaverde, 2013), but also acts as a driving force for several extra-hepatic diseases such as cardiovascular diseases and type 2 diabetes mellitus (Armstrong et al., 2014). Although NAFLD pathogenesis remains incompletely understood (Dowman et al., 2011; Petta et al., 2009), hepatic inflammation is necessary for progression from simple steatosis to NASH (Farrell et al., 2012). Mobilization of cytokines, particularly TNF-α, IL-6, IFN-γ and other Th1 type cytokines, is one of many processes implicated in hepatic inflammatory cell recruitment of NASH and the key step in initiation and perpetuation of liver injury (Farrell et al., 2012; Mari et al., 2008). The activation of hepatic inflammatory signaling pathways is believed to play a crucial role in the development of NASH. Accumulating lines of evidence have demonstrated that Tolllike receptors (TLRs) play important roles in the progression of NASH (Roh and Seki, 2013). TLRs are sensors that recognize molecular

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patterns presenting on a broad range of pathogens and altered or specialized host molecules, trigger inflammatory and antimicrobial responses and mediate the production of proinflammatory cytokines (Farrell et al., 2012). Among them, TLR2, TLR4 and TLR9 were closely associated with NASH. TLR4 is triggered by saturated fatty acid (palmitic acid) and endotoxin from intestinal microbiota (Sawada et al., 2014), and mediates the progression of obesityinduced NASH through activation of X-box binding protein-1 in mice (Ye et al., 2012) and fructose-induced NASH through TNFα signaling in Kupffer cells (Spruss et al., 2009). Both mutation and deficiency of TLR4 lead to decreased NASH (Rivera et al., 2010; Spruss et al., 2009). TLR2 and TLR9 have been reported to promote NASH induced by choline-deficient–amino acid-deficient diet (CDAA). These effects operate through activation of inflammasome (Furusho et al., 2013; Miura et al., 2013) and induction of interleukin-1beta (Miura et al., 2010). MicroRNAs (miRNA) are a class of ~22 nucleotide, noncoding RNA, and play a critical role in regulating gene expression either by translational repression or mRNA degradation, depending on incomplete or complete complementarity between miRNA seed sequence and target mRNA sequence, respectively (Huntzinger and Izaurralde, 2011; Mack, 2007; Rota et al., 2011; Shukla et al., 2011). miRNAs widely participate in the activities of cells, such as cellular growth, differentiation (He et al., 2011; Sun and Lai, 2013), metabolism (Davalos et al., 2011), endoplasmic reticulum stress, oxidative stress, inflammation (Dudda et al., 2013; Nahid et al., 2013) and apoptosis. Also, accumulating evidence has supported that miRNAs are implicated in the pathogenesis and progression of NASH (Ferreira et al., 2014). The hepatocyte-specific miR122 accounts for about 70% of total hepatic miRNAs and plays a critical role in hepatic lipid metabolism, and loss of function of miR-122 potentially contributes to the pathogenesis and development of NASH (Hsu et al., 2012; Hu et al., 2012; Jopling, 2012; Tsai et al., 2012). Interestingly, miR-370, regulating the expression of miR-122 and carnitine palmitoyl transferase to affect lipid metabolism, is also involved in the pathogenesis of NASH (Iliopoulos et al., 2010). In addition, such other miRNAs as miR-10b, miR-33a/b, miR-34a, miR-216 and miR-302a can be up-regulated or down-regulated during the development of NASH (Castro et al., 2013; Hoekstra et al., 2012; Rayner et al., 2011; Zheng et al., 2010). Recently, how miRNAs regulate TLR-signalling pathways has been highlighted, which provides new insights into understanding molecular mechanisms of inflammation (He et al., 2014). miRNAs modulate the expression of TLRs, such as TLR2 (Benakanakere et al., 2009; Philippe et al., 2012) and TLR4 (Androulidaki et al., 2009; Chen et al., 2007; Yang et al., 2011), and key signal molecules of the TLR pathways, such as MyD88 (Huang et al., 2010; Tang et al., 2010) and NFκB1 (Bazzoni et al., 2009; Qi et al., 2012). In previous study, we found that the mRNA expression of TLR2 was significantly increased in nonalcohol steatohepatitis of HFD-MetS E3 rats. Therefore, we hypothesize that certain miRNAs may be involved in upregulation of TLR2 and its downstream molecules and affect the development of NASH. In present study, we mainly investigated the expression of predicted candidate miRNAs targeting TLR2 mRNA in this model. We found that decreased miR-144 was negatively related to up-regulated TLR2 and activated the downstream pathway. The results from miR-144 mimics and inhibitor and dual luciferase reporter assay further verified the targeting–regulation relationship between miR-144 and TLR2. 2. Materials and methods 2.1. Animal model of nonalcoholic steatohepatitis Thirty-two E3 rats (8–12 weeks, age–gender matched) were randomly divided into control group fed with control diet and highfat-diet (HFD) group fed with HFD for 24 weeks to induce metabolic

syndrome (HFD-MetS) with nonalcoholic steatohepatitis. The ingredients of control diet and HFD and evaluation methods of HFDMetS with nonalcoholic steatohepatitis were the same as we described previously (Li et al., 2011). After HFD-MetS with nonalcoholic steatohepatitis in E3 rats was evaluated, portions of liver tissues were collected and stored at −80 °C for total RNA and protein extraction, and other portions of liver tissues were preserved in 4% polyoxymethylene, embedded in paraffin, sectioned 5 μm thick and stained for immunohistochemistry. The experiments were approved by the Institutional Animal Ethics Committee of the Xi’an Jiaotong University Health Science Center (NO.XJ20120117). 2.2. Bioinformatics Candidate miRNAs targeting TLR2 were selected from the unanimous predictive outcomes by two widely advocated yet distinct algorithms of EBL: TargetScan 6.0 (http://www.targetscan.org) and miRanda (http://www.microRNA.org). The candidate miRNAs targeting TLR2 included rno-miR-101a, miR-101b, miR-132, miR144, miR-212, miR-31, miR-320, miR-410 and miR-9. 2.3. Cell culture and BMM differentiation Rat macrophage cell line, NR8383, and HEK-293T cells were respectively cultured in F-12K medium (Sigma-Aldrich) containing 20% fetal bovine serum (FBS) (Hyclone) and low-glucose Dulbecco’s modification of Eagle’s medium (DMEM, 1 g/l glucose) medium (Hyclone, USA) containing 10% FBS (Hyclone), as well as 100 units/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich). Cells were incubated at 37 °C in a humidified chamber supplemented with 5% CO2. Bone marrow-derived macrophages (BMM) from E3 rats were obtained according to the previous protocol with slight modification (Weischenfeldt and Porse, 2008). Briefly, the intact femurs of E3 rats (male, 4 weeks old) were aseptically dislocated from the hind legs. The bone marrow cells (BMC) were flushed from the femurs with cold lymphocyte medium (RPMI-1640 containing 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin) using a 5-ml syringe and a 25-gauge needle, and then pipetted cells up and down into single-cell suspension. The cells were filtered, centrifuged and resuspended in BMM medium (lymphocyte medium with 30% (Boltz-Nitulescu et al., 1987) L929-conditioned medium which was prepared as described (Weischenfeldt and Porse, 2008)). Lastly, 8 × 106 cells per well were seeded into 6-well plates, washed twice with PBS every other day, and incubated in a humidified incubator with 5% CO2 at 37 °C for 7-day differentiation (Weischenfeldt and Porse, 2008). Differentiated BMM in 6-well plates was used for subsequent experiments. 2.4. Real-time quantitative PCR Total RNA was isolated with TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized using 5 μg total RNA with RevertAidTM First Strand cDNA Synthesis Kit (Thermo scientific, USA). Real-time quantitative PCR (RT-qPCR) was performed with SYBR® Premix Ex Taq™ II (TaKaRa, China) by iQ5 system (Bio-Rad, USA) and normalized by β-actin. The RT-qPCR parameters were as follows: 3 min at 95 °C, followed by 40 cycles of 30 s at 95 °C and 30 s at annealing temperatures. The information of genes, primer sequences and annealing temperatures is depicted in Supplementary Table S1. For analyses of miRNA expression, 2 μg total RNA was used to generate miRNA cDNA by reverse transcription with PrimeScript® miRNA cDNA Synthesis kit (Takara, Dalian, China). The candidate miRNAs were quantitated with SYBR® Premix Ex Taq™ II (TaKaRa) using miRNA cDNA as the template by miRNA-specific RT-qPCR running in the iQ5 system (Bio-Rad). The expression of candidate

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miRNAs was normalized by U6 snRNA. The RT-qPCR parameters for miRNA quantification were as follows: 2 min at 95 °C, then 40 cycles of 15 s at 95 °C and 45 s at 60 °C. The sequence and annealingtemperature information of forward primers (synthesized by BGI Company, Shenzhen, China) for microRNA RT-qPCR analysis are depicted in Supplementary Table S2. Reverse primer is the Uni-miR RT-qPCR primer provided by PrimeScript® miRNA cDNA Synthesis kit. 2.5. Western blotting Proteins (100 μg) from the hepatic tissues, NR8383 cells and BMM were prepared with ice-cold cell lysis buffer (Beyotime Co., Shanghai, China), separated with 10% SDS–polyacrylamide gel (SDS– PAGE), and then transferred onto PVDF membranes (Bio-Rad Laboratories). The PVDF membrane was then blocked with 5% nonfat milk in Tris-buffer saline with 0.1% Tween (TBST) and incubated overnight at 4 °C with primary antibodies against TLR1 (1:100, 195001-AP, Proteintech, USA),TLR2 (1:200, SC-10739, Santa Cruz, USA), TLR4 (1:100, Santa Cruz, USA), TLR7 (1:100, 17232-1-AP, Proteintech, USA), TLR9 (1:100, 17230-1-AP, Proteintech, USA) and β-actin (1:500, SC-47778, Santa Cruz), respectively. The membranes were washed and then incubated for 1 h with secondary antibodies of HRPconjugated goat anti-mouse IgG or goat anti-rabbit IgG (Pierce, USA). Immunoreactive protein bands were detected by the kit from Supersignal® West Pico (Thermo Scientific). To determine activation of NF-κB signaling, a Nuclear and Cytoplasmic Protein Extraction Kit (P0027, Beyotime, China) was used. Cytosolic and nuclear protein extracts of the cells were subjected to Western blotting analysis with anti-IκBα antibody (1:1000, #4814, Cell Signaling Technology®, USA) and anti-NF-κB p65 antibody (1:1000, #3037, Cell Signaling Technology®, USA), respectively. TBP (1:5000, 66166-1-AP, Proteintech, USA) and α-tublulin (1:5000, 66031-1-AP, Proteintech, USA) were used as housekeeping genes of nuclear and cytoplasmic protein, respectively. 2.6. Enzyme-linked immuno sorbent assay Rat serum and culture supernatants were collected, and TNF-α and IFN-γ contents were determined using the Enzyme-Linked Immuno Sorbent Assay (ELISA) Development Kit (Peprotech, USA). Briefly, 100 μl serum or supernatant was respectively added into the TNF-α or IFN-γ antibody-coated plate and then incubated at 25 °C for 2 h. Streptavidin-HRP was added and 3,3′-5,5′ tetramethylbenzidin (TMB) was used for development after respectively adding the biotinconjugated detecting antibody of TNF-α or IFN-γ and incubating for 2 h. The optical density (OD) value was obtained at the wave of 450 nm by Multiskan Spectrum (Thermo, USA). Complete medium was used as blank control, and TNF-α or IFN-γ concentrations were respectively calculated from the standard curves, which were obtained using the series dilution of recombinant rat TNF-α (from 1500 pg/ml to zero) or recombinant rat IFN-γ (from 1000 pg/ml to zero). 2.7. Immunohistochemical and immunofluorescent double labeled staining The hepatic parraffin sections (5 μm) were used for immunohistochemical staining according to the conventional procedure of immunohistochemistry. Rabbit polyclonal antibody against rat TLR2 (sc-10739, Santa Cruz, USA) was diluted in 1:50. The goat biotinconjugated antibody against rabbit IgG and horseradish peroxidase (HRP)-conjugated streptavidin were purchased from Boster (China). Hematoxylin was used for counterstaining. In order to verify TLR2-producing cells, we run a double immunofluorescent staining in hepatic sections. A mixture of primary

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antibodies included mouse monoclonal antibody against rat ED1 (Abcam ab31630, 1:200) also called CD168 and rabbit polyclonal antibody to rat TLR2, and the secondary antibodies were FITClabeled goat anti-rabbit IgG (H + L) (Beyotime A0562, 1:200) and Cy3-labeled goat anti-mouse IgG (H + L) (Beyotime A0521,1:200). Double-labeled immunofluorescent staining was carried out following the described procedure with slight modification (Lin et al., 2008). Lastly, the immunofluorescent images were taken under a fluorescence microscope (Olympus, Japan) and merged by using Photoshop 7.0 software. 2.8. Transfection of miR-144 mimics and inhibitor NR8383 cells (5 × 105/well) were seeded into 6-well plates and cultured with F-12K medium containing 20% FBS. As the confluence of NR8383 cells reached 80–90%, 100 nM of mimics, inhibitor and negative control (NC) of miR-144 were transfected, respectively, via Lipofectamine™ 2000 (Invitrogen) under a serum-free condition. After 5 h transfection, serum-free F-12K medium was replaced by F-12K medium with 20% FBS. All the cells were incubated at 37 °C in a CO2 incubator for 24 h, and then treated with 1 μg/ml Pam3CSK4 for another 24 h before harvested with TRIzol® reagent or ice-cold cell lysis buffer (Beyotime Co., Shanghai, China). Similarly, differentiated BMM in 6-well plates was transiently transfected with the mimic, inhibitor and negative control (NC) of miR-144, respectively, following the same procedures. 2.9. Co-transfections and dual luciferase activity assay The pmir-TLR2-3′UTR vector and pmir-mutant-TLR2-3′UTR vector were constructed, respectively, by inserting TLR2 3′UTR with the putative binding site of miR-144 and mutant TLR2 3′UTR with mutant binding site of miR-144 at the downstream of the firefly luciferase gene into pmir vector (Promega). Plasmids were prepared with E.Z.N.A.TM Endo-free Plasmid Maxi Kit (Omega Bio-tek). HEK-293T cells (5 × 104 cells per well) were seeded in a 48-well culture plate and cultured with DMEM medium with 10% FBS. As the confluence of HEK-293T cells reached 80–90%, the pmir vector (100 ng), pmir-TLR2-3′UTR vector (100 ng) and pmir-mutant-TLR2-3′UTR vector (100 ng) were transfected into HEK-293T cells, respectively, simultaneously with 100 nM miRNA small molecules via Lipofectamine 2000TM (Invitrogen). Twenty-four hours later, the luciferase activity was detected using Dual-Luciferase® Reporter 1000 Assay System (Promega) by a plate-reading luminometer (VICTOR™ 2030 Multilabel Plate Reader, PE, USA), and the relative luciferase activity value showed as FL/RL that was achieved from the firefly luciferase activity by the Renilla luciferase as control per sample. The normalized relative luciferase activity of different groups was used for statistical analysis. 2.10. Statistics Quantitative data were expressed as means ± SEM. The statistical analysis was performed by ANOVA Post Hoc Tests (Bonferroni) using StatView Software (SAS Institute Inc, Cary, NC, USA). P-value less than 0.05 was considered significant. 3. Results 3.1. Increase of TLR2 expression in Kupffer cells of HFD-MetS E3 rats with nonalcoholic steatohepatitis In livers of HFD-MetS E3 rats with nonalcoholic steatohepatitis, we firstly detected the mRNA expression of TLRs and some cytokines using RT-qPCR. The data indicated that the mRNA expression of TLR2, TLR9, TNF-α, IFN-γ and TGF-β significantly increased in livers of HFD

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Fig. 1. The expressions of TLRs and cytokines in livers and the concentration of cytokines in serum of those E3 rats. A. The mRNA expressions of TLRs (upper panel) and cytokines (lower panel) were measured in livers of four-group E3 rats by RT-qPCR. B and C. The protein level of TLRs with differential mRNA expressions were determined by western blotting (B) and normalized by β-actin (C). D. Concentrations of TNF-α and IFN-γ in serum of four-group E3 rats were detected by ELISA. The above-mentioned data were expressed as means ± SEM (n = 8 for each group). * and ** represent p < 0.05 and 0.01, respectively. HFD-MetS, high-fat-diet induced metabolic syndrome; RTqPCR, real-time quantitative PCR; TLR2, Toll-like receptor 2; TNF-α, tumor necrosis factor α; IFN-γ, interferon-γ; ELISA, enzyme linked immunosorbent assay.

group E3 rats. The mRNA expression of TLR1, TLR4 and TLR7 decreased in livers of HFD group E3 female rats, while they did not change in livers of HFD group male E3 rats, comparing with their corresponding control groups. However, the mRNA expression of

TLR3, TLR5, TLR6 and TLR8 did not show any differences in fourgroup livers of E3 rats (Fig. 1A). Secondly, the protein expressions of the differential expressed TLRs and several cytokines were further determined using Western blotting (TLR1, TLR2, TLR4, TLR7 and TLR9)

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Fig. 2. The cellular localization of TLR2 in livers of HFD-MetS E3 rats with steatohepatitis. A. The representative images of immunohistochemistry analysis of TLR2 were showed here from livers of four-group E3 rats. B. Cellular localization of TLR2 was determined in livers of HFD-MetS E3 rats with steatohepatitis by double-labeled immunofluorescent staining.TLR2 expression was observed green by staining with the FIFC-conjugated antibody, while ED1 expression was observed red by staining with the Cy3-conjugated antibody. HFD-MetS, high-fat-diet induced metabolic syndrome; RT-qPCR, quantitative real-time PCR; TLR2, Toll like receptor 2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

(Fig. 1B and 1C) and ELISA (the serum concentration of TNF-α and IFN-γ) (Fig. 1D). The data indicated that the changes of their protein expression were in accordance with those of their mRNA expression. Notably, TLR2 and its downstream signaling molecules, TNF-α and IFN-γ, were up-regulated in both male and female of HFD groups (Fig. 1). Further analysis by immunohistochemistry (Fig. 2A) showed that not only the protein expression of TLR2 was increased in livers of the HFD groups as compared to the control groups, but also more positive cells (brown cells) expressing TLR2 could be found in HFD groups, especially in female HFD livers. Furthermore, TLR2 was mainly located in mesenchymal cells rather than hepatic cells. In order to identify the cell type with increased TLR2 expression, double-label immunofluorescent staining was performed, in which TLR2 was green-labeled by FIFC while ED1 was red-marked with Cy3. The most of stained cells were presented yellow color in the merged graph (Fig. 2B), suggesting that elevated TLR2 was mainly located in Kupffer cells in livers of HFD group E3 rats. 3.2. Negative correlation between miR-144 and TLR2 in livers of HFD-MetS E3 rats with nonalcoholic steatohepatitis We presumed that some miRNAs might be involved in TLR2 posttranscriptional regulation. To verify the hypothesis, we selected 9 candidate miRNAs targeting the 3′UTR of rat TLR2 mRNA depending on the unanimous predictive outcomes from TargetScan and miRanda, two well known miRNA databases. We used RT-qPCR to determine the miRNA expressions, and the results showed that miR144 expression was significantly decreased in the HFD-group liver of both male and female E3 rats compared with those of control groups (Fig. 3A), while the expressions of miR-132 and 9 were increased only in the HFD-group liver of male rats (Fig. 3A). The expressions of miR-101a, miR-101b, miR-212, miR-31, miR-320 and miR-410 showed no significant change among these groups (Fig. 3A). Among the 9 detected miRNAs, only elevated miR-144 was negatively correlated with TLR2 expression (Fig. 3B). 3.3. miR-144 directly targeting 3′UTR of rat TLR2 mRNA To validate whether miR-144 hinders TLR2 expression, pmirTLR2-3′UTR vector and pmir-mutant-TLR2-3′UTR vector were

constructed, and DNA sequencing confirmed the integrity of both recombinant vectors that were inserted wild TLR2 3′UTR and mutant TLR2 3′UTR, respectively (Fig. 4A and 4B). After transiently cotransfecting HEK 293 T cells with 100 nM miR-144 mimics or mNC and 100 ng three kind of vectors for 24 h respectively, the result showed that in the presence of miR-144 mimics, the normalized relative luciferase activity in HEK 293 T cells transfected with pmirTLR2-3′UTR vector decreased significantly, comparing with that in HEK 293 T cells transfected with pmir-vector or pmir-mutant-TLR23′UTR vector, while the normalized relative luciferase activity among 3 groups showed no difference in the mNC group (Fig. 4C). However, with 100 nM miR-144 inhibitor, the normalized relative luciferase activity of HEK 293 T cells, transfected by 100 ng pmir-TLR23′UTR vector, increased significantly, comparing with that of HEK 293 T cells transfected with 100 ng pmir-vector or 100 ng pmirmutant-TLR2-3′UTR vector, while the normalized relative luciferase activity among 3 groups showed no difference in the presence of iNC (Fig. 4D). These results indicated that miR-144 was directly bound to the 3′UTR of rat TLR2 mRNA. In other words, the rat TLR2 is a target gene of miR-144. 3.4. Negative regulation of TLR2 signaling by miR-144 in macrophage cell line To explore the potential effect of miR-144 on TLR2 in macrophage cells, miR-144 mimics and inhibitor were used to challenge NR8383 cells, a rat macrophage cell line, at the concentrations of 100 nM. After 24 h transient transfection with miR-144 mimics, the expressions of miR-144, TLR2, TNF-α, IFN-γ and activation of NFκB in NR8383 cells were detected, respectively. After treated by miR144 mimics, the expression of miR-144 was augmented about 600fold, while subsequent Pam3CSK4 treatment hardly affected the change (Supplementary Fig. S1A). Compared with mNC groups, TLR2 decreased in miR-144 mimics group in both the presence and absence of Pam3CSK4, and its protein expression rescued by Pam3CSK4 was much lower in miR-144 mimics group than that in mNC group (Fig. 5A). As compared to mNC groups, the mRNA expression of TNF-α and IFN-γ showed the same results as TLR2 in miR-144 mimics group (Fig. 5B). Compared with mNC groups, the concentrations of TNF-α and IFN-γ in cell culture supernatants

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Fig. 3. The correlation between TLR2 and candidate microRNAs in livers of HFD-MetS E3 rats with steatohepatitis. A. The relative expressions of 9 candidate microRNAs, which were predicted by using two online softwares (http://www.microrna.org and http://www.targetscan.org), in livers of HFD-MetS E3 rats with steatohepatitis were determined by miRNA RT-qPCR. U6 snRNA was used as endogenous control for data normalization. B. The correlation between candidate microRNAs and TLR2 mRNA levels. The data were expressed as means ± SEM (n = 8 for each group). * represents p < 0.05, between HFD and control group. HFD-MetS, high-fat-diet induced metabolic syndrome; RT-qPCR, real-time quantitative PCR; TLR2, Toll-like receptor 2.

decreased in miR-144 mimics group in both the presence and absence of Pam3CSK4, and the production of TNF-α and IFN-γ rescued by Pam3CSK4 in miR-144 mimics group was far below than that in mNC group (Fig. 5C). In addition, analyses from the fraction of nuclear and cytoplasmic protein by Western blotting showed that miR-144 mimic could decrease nuclear translocation of p65 and degradation of IκBα, which could be compensated by Pam3CSK4 treatment (Supplementary Fig. S2A). With the same procedure, the expressions of miR-144, TLR2, TNF-α, IFN-γ and activation of NFκB in NR8383 cells treated with miR-144 inhibitor were also detected. Similar results could be found in miR-144 inhibitor effect on the decrease of miR-144, no significant difference could be found after Pam3CSK4 treatment in both iNC and inhibitor groups (Supplementary Fig. S1B). In contrast, TLR2 increased in miR-144 inhibitor group with or without Pam3CSK4, compared with iNC groups, while the protein expression of TLR2 activated by Pam3CSK4 was much higher in miR-144 inhibitor group than that in iNC group (Fig. 5D). Downstream signaling molecules, TNF-α and IFN-γ, showed the same change just like TLR2 in those groups (Fig. 5E). Compared with iNC groups, the concentrations of TNF-α and IFN-γ in culture

supernatants increased in miR-144 inhibitor group in both the presence and absence of Pam3CSK4, and the production of TNF-α and IFN-γ significantly promoted by Pam3CSK4 in miR-144 mimics group was much higher than that in mNC group (Fig. 5F). In addition, analyses of nuclear and cytoplasmic fraction by Western blotting showed that inhibitor of miR-144 could promote NF-κB p65 nuclear translocation, and increase the degradation of IκBα, and the effect could be augmented by Pam3CSK4 treatment (Supplementary Fig. S2B). These data suggested that miR-144 negatively regulated TLR2 expression, activation of NF-κB and subsequent production of proinflammatory cytokine in the downstream of TLR2 signal pathway in NR8383 cells. 3.5. Negative regulation of TLR2 signaling by miR-144 in primary macrophages Besides macrophage cell line, we also explore the potential impact of miR-144 on TLR2 in primary macrophages. miR-144 mimics and inhibitor were used to challenge differentiated BMM cells according to the same procedures as described earlier. The expressions of

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Fig. 4. Identification of targeted relationship between miR-144 and 3′UTR of rat TLR2 mRNA A. The seed sequence of miR-144, the binding site of miR-144 in wild 3′ UTR and 3 mutant bases as indicated in mutant 3′ UTR of rat TLR2 mRNA. B. Three luciferase reporter constructs: pmir-vector (empty vector as control), pmir-TLR2-3′ UTR vector with wild 3′ UTR of TLR2 mRNA, and pmir-mutant TLR2-3′ UTR vector with mutant 3′ UTR of TLR2 mRNA. C and D. HEK293 T cells were transiently cotransfected with luciferase reporter constructs and miR-144 mimics (C) or inhibitors (D). Luciferase activities were detected after transient transfection for 24 h and normalized to the luciferase activities from the control pmir-vector. The data were expressed as means ± SEM from 3 independent experiments. * and ** represent p < 0.05 and 0.01, respectively, compared to the control pmir-vector.

miR-144, TLR2, TNF-α, IFN-γ and activation of NF-κB in BMM cells after 24 h transient transfection with miR-144 mimics were detected respectively. After being treated by miR-144 mimics or inhibitor, the corresponding changes similar to NR8383 cells were observed in BMM cells (Supplementary Fig. S3A and S3B). Compared with mNC groups, TLR2 decreased on protein level in miR144 mimics group in the presence or absence of Pam3CSK4, while TLR2, rescued by Pam3CSK4, was much lower in miR-144 mimics group than that in mNC group (Fig. 6A). Compared with mNC groups, the same challenges brought the same corresponding change in TNF-α and IFN-γ following TLR2 (Fig. 6B and 6C). Analyses from nuclear and cytoplasmic fraction further showed that miR-144 mimics could decrease nuclear translocation of p65 and degradation of IκBα, which could be compensated by Pam3CSK4 treatment in BMM cells (Supplementary Fig. S4A). In the same way, the expressions of miR-144, TLR2, TNF-α, IFN-γ and activation of NF-κB in BMM cells after 24 h transient transfection with miR-144 inhibitor were also detected. In contrast, TLR2 increased in miR-144 inhibitor group with or without Pam3CSK4 as compared to iNC groups, while its protein expressions activated by Pam3CSK4 in miR144 inhibitor group were higher than that in iNC group (Fig. 6D). The same change occurred to TLR2 downstream molecules TNF-α and IFN-γ among these groups (Fig. 6E and 6F). Analyses from nuclear and cytoplasmic fraction further showed that inhibitor of miR144 could also promote NF-κB p65 nuclear translocation and degradation of IκBα, and the effect could be augmented by Pam3CSK4 treatment in BMM cells (Supplementary Fig. S4B). These data suggested that miR-144 negatively regulated TLR2 expression, activation of NF-κB and subsequent production of proinflammatory cytokines in the downstream of TLR2 signal pathway in macrophages.

4. Discussion Nonalcohol steatohepatitis (NASH) has become a major health concern along with the global obesity epidemic (Kim and Younossi, 2008; Medina-Santillan et al., 2013). The activation of hepatic TLR signaling pathways and the production of proinflammatory cytokines are believed to play a crucial role in the development of NASH (Miura et al., 2010; Petrasek et al., 2013; Wagnerberger et al., 2012). In a fructose-induced 8-week NAFLD, TLRs 1–9 and proinflammatory cytokine including TNF-α were all up-regulated (Wagnerberger et al., 2012). In a choline-deficient amino acid-defined (CDAA)-diet induced NASH for 22 weeks (Miura et al., 2013), TLR2 mediates the development of NASH through inflammasome activation in the presence of palmitic acid. In present NASH model of E3 rats for 24 weeks, TLR2 and its downstream molecules (TNF-α and IFN-γ) and inflammasome component NLRP3 (data not show) were up-regulated in livers of both male and female HFD groups. We concluded that the upregulation/activation of TLR2 and the proinflammatory cytokine production play a key role in the development of NASH. On the contrary, in another CDAA-diet induced NASH for 8 weeks, TLR2 deficiency could enhance NASH (Rivera et al., 2010). The reason for the discrepant expression and function of TLR2 in NASH seems to be related with the inducers and the induced periods. Increasing experimental and clinical data indicate significant genderrelated differences regarding liver injury in patients with nonalcoholic steatohepatitis (Caballero et al., 2012; Morais et al., 2011; Oda and Kawai, 2009). In morbidly-obese patients, males displayed more severe inflammation and liver derangement (Caballero et al., 2012; Morais et al., 2011). In present NASH model, E3 male rats also displayed more severe hepatic steatosis and inflammation than female rats after feeding with HFD for 24 weeks (data not shown), which

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Fig. 5. Effect of miR-144 mimics and inhibitor on TLR2 and its signaling in NR8383 cells. A, B and C. After 24 hour transient transfection with 100 nM mNC or miR-144 mimics and subsequent 24 hour treatment with Pam3CSK4Pam3CSK4 (1 μg/ml), the expressions of TLR2 protein (A) and cytokines mRNA (B) were respectively detected by Western blotting and RT-qPCR in NR8383 cells, while the concentrations of cytokines in culture supernatant of NR8383 cells (C) were determined by ELISA . D, E and F. After 24 hour transient transfection with 100 nM iNC or miR-144 inhibitor and subsequent another 24 hour treatment with Pam3CSK4 (1 μg /ml), the expressions of TLR2 protein (D) and cytokine mRNA (E) were respectively detected by Western blotting and RT-qPCR in NR8383 cells, while ELISA was operated to detect the concentration of cytokines in culture supernatant of NR8383 cells (F). The data were expressed as means ± SEM. * and ** represent p < 0.05 and 0.01 respectively at indicated groups (3 independent experiments). mNC, negative control of mimics; iNC, negative control of inhibitor; RT-qPCR, real-time quantitative PCR; TLR2, Toll-like receptor 2; ELISA, enzyme linked immunosorbent assay.

is consistent with previous reports. Furthermore, Imahara et al. reported that less severe innate immune responses in women were observed and possibly associated with TLR4 genomic variation (Imahara et al., 2005). Thus, the decreased expression of TLR1 4 and 7 in livers of female HFD groups is probably linked with the weakened inflammatory signalings, and might contribute to the genderrelated different reactions to high fat diet. Recent studies have shown that miRNAs are implicated in the pathogenesis and development of NASH (Cheung et al., 2008; Hsu et al., 2012). Especially, miR-105, miR-19a/b and miR-143 modulate TLR2 expression in human oral keratinocytes (Benakanakere et al., 2009),

fibroblast-like synoviocytes (FLS) of rheumatoid arthritis patients (Philippe et al., 2012) and human colorectal carcinoma cells (Guo et al., 2013) respectively. Thus, we hypothesized that some miRNAs might be responsible for TLR2 up-regulation in nonalcoholic steatohepatitis of HFD-MetS E3 rats. Although human miR-105, miR-19a/b and miR143 have been validated that they suppress TLR2 protein expression in three kinds of human cells (Benakanakere et al., 2009; Guo et al., 2013; Philippe et al., 2012), there is no binding site of rat miR-105, miR19a/b and miR-143 in 3′UTR of rat TLR2 mRNA. Therefore, 9 predicted candidate miRNAs which have putative binding sites in 3′UTR of rat TLR2 mRNA were checked using miRNA RT-qPCR.

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9

Fig. 6. Effect of miR-144 mimics and inhibitor on TLR2 and its signaling in E3 BMM cells. A, B and C. After 24 hour transient transfection with 100 nM mNC or miR-144 mimics and subsequent 24 hour treatment with Pam3CSK4 (1 μg/ml), the expressions of TLR2 protein (A) and cytokines mRNA (B) were respectively detected by Western blotting and RT-qPCR in BMM cells, while the concentrations of cytokines in culture supernatant of BMM cells (C) were determined by ELISA. D, E and F. After 24 hour transient transfection with 100 nM iNC or miR-144 inhibitor and subsequent another 24 hour treatment with Pam3CSK4 (1 μg/ml), the expressions of TLR2 protein (D) and cytokine mRNA (E) were respectively detected by Western blotting and RT-qPCR in BMM cells, while ELISA was operated to detect the concentration of cytokines in culture supernatant of BMM cells (F). The data were expressed as means ± SEM. * and ** represent p < 0.05 and 0.01 respectively at indicated groups (3 independent experiments). BMM, bone marrow-derived macrophages; mNC, negative control of mimics; iNC, negative control of inhibitor; RT-qPCR, real-time quantitative PCR; TLR2, Toll-like receptor 2; ELISA, enzyme linked immunosorbent assay.

Except increased miR-132 and miR-9 in HFD-group livers of male E3 rats, only miR-144 significantly decreased in livers of both male and female HFD-groups. TLR2 agonist, TLR4 agonist and TNF-α could induce miR-132 and miR-9 in human monocytes (Bazzoni et al., 2009; Taganov et al., 2006). So, up-regulated miR-132 and miR-9 might contribute to NASH by activated TLR2 and/or increased TNFα. miR-144 has been characterized as “common miRNA signature” of a number of different tumors in humans (Wang et al., 2011) and found to promote erythropoiesis (Rasmussen et al., 2010). In a high fat diet (HFD, consisting of 39% fat, 40% carbohydrate and 21% protein) and low dose of STZ (40 mg/kg) induced type 2 diabetes

mellitus (T2D), circulating level and tissue expression of miR-144 in pancreas, fat and liver are all increased. Additionally, increased circulating miR-144 could impair insulin signaling via inhibiting the expression of insulin receptor substrate 1 (Karolina et al., 2011). However, in our present study, miR-144 expression significantly decreased in livers of HFD-MetS E3 rats with steatohepatitis, and was negatively related with TLR2 expression. This opposite expression of miR-144 may be related to different disease models and induced methods. Currently, it is generally accepted that down-regulation of some key microRNAs is a well-characterized phenomenon in various malignant transformation and tumor progression, which may

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result from the loss of genomic copy number and epigenetic silencing such as DNA hypermethylation of CpG islands in microRNA genes and the transcriptional and epigenetic repression through suppressive transcriptional factors or recruiting a corepressor complex (Baer et al., 2013; Zhang et al., 2012). In addition, it has been also reported that some microRNAs are downregulated under certain conditions such as high glucose (Tang et al., 2009), high-cholesterol diet (Cirera et al., 2010), sodium arsenite (Marsit et al., 2006), folate deficiency (Marsit et al., 2006), hypoxia (Donker et al., 2007), and oxidative stress or ER stress (Muratsu-Ikeda et al., 2012). In view of the present cell and animal model and no-CpG island in a 3-kb upstream region of the 5′ end of pre-miR-144 gene which predicted by three online softwares (http://www.uscnorris.com /cpgislands2/cpg.aspx, http://www.ebi.ac.uk/Tools/seqstats/emboss _newcpgreport/, and http://rulai.cshl.org/tools/FirstEF/), the downregulation of miR-144 in the E3 rat model might be related to number factors such as high glucose, high-cholesterol, the transcriptional and epigenetic repression, oxidative stress or ER stress in NASH, while the exact mechanism remains unclear. However, a series of dual luciferase reporter assay validated that miR-144 was directly bound to the 3′UTR of rat TLR2 mRNA. In other words, miR144 can directly target TLR2 and regulate its expression in vitro. Also it has been reported that when inflammatory injury occurs in liver, Kupffer cells are activated by both damage-associated molecular patterns and pathogen-associated molecular patterns through TLRs (Jaeschke, 2011; Schwabe et al., 2006). Double-labeled immunofluorescent staining displayed that up-regulated TLR2 mainly expressed in hepatic Kupffer cells of HFD-MetS E3 rats suggested that TLR2 in Kupffer cells might be responsible for triggering hepatic inflammation and proinflammatory cytokine production, leading to the onset of NASH in this model. TNF-α (Tomita et al., 2006; Tosello-Trampont et al., 2012) and IFN-γ (Luo et al., 2013), the target molecules of TLR2 signaling, are believed to play a pivot role in the pathophysiology of NASH by recruiting hepatic inflammatory cells

(Tomita et al., 2006), impairing insulin receptor signaling (Tosello-Trampont et al., 2012) and inducing hepatocyte apoptosis (Kudo et al., 2009). In addition, activated neutrophils and monocytes are recruited into the inflammatory liver (Jaeschke, 2011), BMM as primary macrophages and rat macrophage cell line NR8383 cells were used to further investigate the effect of miR-144 on TLR2 expression, activation of NF-κB and the production of its downstream proinflammatory cytokines. Pam3CSK4, a synthetic TLR2 ligand, induces the activation of proinflammatory transcription factor NF-κB (Aliprantis et al., 1999; Ozinsky et al., 2000) and leads to the expression and secretion of IFN-γ and TNF-α (Paludan et al., 2001). In NR8383 cells as well as in BMM, after transient transfection with miR-144 mimics, besides TLR2 expression, IFN-γ and TNF-α production significantly decreased even after subsequent Pam3CSK4 stimulation for 24 h, while the opposite result was obtained after miR-144 inhibitor administration. This implied that up-regulated miR-144 suppressed the activation of TLR2 signal pathway to Pam3CSK4, whereas downregulated miR-144 enhanced the activation, and further confirmed that miR-144 negatively regulated TLR2 expression, activation of NFκB and the production of its downstream proinflammatory cytokines. Taken together, down-regulated miR-144 untied its targeting at TLR2 and enhanced the production of proinflammatory cytokines (TNF-α and IFN-γ) in vitro, this might be involved in the development and progression of NASH in HFD-MetS E3 rats. The finding provides the first insight into the regulation of TLR2 by miR-144, and may offer a novel and potential target for NASH intervention (Fig. 7). Acknowledgement The project was supported by the National Natural Science Foundation of China (No. 81370952), the Research Project of Shaanxi Provincial Key Laboratory of Biotechnology (No. 14JS088), Key Science

Fig. 7. The schematic diagram of miR-144 roles in pathogenesis of steatohepatitis in HFD-MetS E3 rats. In hepatic Kupffer cells of HFD-MetS E3 rats with steatohepatitis, the downregulation of miR-144 de-suppressed TLR2 protein expression, led to activation of TLR2 signaling and triggered the onset of inflammation. As a consequence, the inflammation cell infiltration increased. Our data indicate that miR-144 may play a crucial role in the pathogenesis of steatohepatitis in HFD-MetS by targeting TLR2.

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