miR-93-3p alleviates lipopolysaccharide-induced inflammation and apoptosis in H9c2 cardiomyocytes by inhibiting toll-like receptor 4

Pathology - Research and Practice xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

Pathology - Research and Practice journal homepage: www.elsevier.com/locate/prp

miR-93-3p alleviates lipopolysaccharide-induced inflammation and apoptosis in H9c2 cardiomyocytes by inhibiting toll-like receptor 4 Bi Tanga, Ling Xuana, Mingming Tanga, Hongju Wanga, Jing zhoua, Jinjun Liua, Shili Wua, ⁎ Miaonan Lia, Xiaojing Wangb, Heng Zhanga, a

Department of cardiovascular Medicine, The First Affiliated Hospital of Bengbu Medical College, Bengbu 233004, Anhui Province, PR China Clinical and Basic Provincial Laboratory of Respiratory System Diseases of Anhui Province, The First Affiliated Hospital of Bengbu Medical College, Bengbu 233004, Anhui Province, PR China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Cardiomyocytes Apoptosis LPS miR-93-3p TLR4

Background: miR-93 is recently recognized to perform anti-inflammatory action in the pathological process of cardiomyocytes dysfunction. However, it remains unclear whether miR-93-3p involves in lipopolysaccharide (LPS)-induced inflammation and apoptosis in H9c2 cells. The present study aimed to investigate the functions of miR-93-3p and its target, toll-like receptor 4 (TLR4), in LPS-stimulated cardiomyocytes. Material and methods: Cell viability was analyzed by CCK-8 assay. AnnexinV-FITC/PI staining and lactate dehydrogenase (LDH) assay were used to evaluate the cell death. The mRNA and protein levels were assayed by RT-qPCR and western blotting, respectively. The targeted gene was predicted by a bioinformatics algorithm and confirmed by a dual luciferase reporter assay. Results: LDH stimulation resulted in the suppression of cell viability and the increase in apoptosis rate, inflammatory cytokines and LDH levels, while inhibition of TLR4 with TAK-242 or overexpression of miR-93-3p dramatically blocked LPS-induced inflammation and apoptosis in cardiomyocytes. Intriguingly, bioinformatics analysis and experimental data suggested that TLR4 was a direct target of miR-93-3p, which could inhibit TLR4 expression by transfected with miR-93-3p mimics or elevate the expression of TLR4 by transfected with miR-933p inhibitors. Overexpression of TLR4 carried out an opposite effect to miR-93-3p and positively regulated LPSinduced inflammation and apoptosis in cardiomyocytes. Conclusion: miR-93-3p showed the protective effects against LPS-induced inflammation and apoptosis in cardiomyocytes by inhibiting TLR4 expression.

1. Introduction Cardiovascular diseases (CVDs), such as dilated cardiomyopathy, myocardial infarction, cardiac hypertrophy and heart failure, are now recognized that they may be associated with over-activation of inflammatory response and apoptosis in cardiomyocytes [4,5,21]. Previous studies have found that many detrimental stimuli, including palmitic acid, high glucose, oxidized low-density lipoprotein and lipopolysaccharide (LPS), can trigger inflammation and apoptosis in cardiomyocytes, which contribute to the pathogenesis of CVDs [6,17,22,23,31]. MicroRNA (miRNA) as a class of noncoding RNA is characterized by short and single-stranded RNA (18–25 nucleotides) and have recently emerged as a novel class of post-transcriptional regulators in a variety

of biological processes by binding to the 3′-untranslated regions (3′UTRs) of its target gene, thus inhibiting gene expression [9,10]. Numerous miRNAs have been reported in physiological and pathological processes of the heart [3,28]. In the process of LPS-induced cardiomyocyte apoptosis and inflammatory response, miR-29b and miR-155 are up-regulated [29,37], and miR-99a, miR-145, miR-181b and miR499 are down-regulated [7,14,15,26]. However, the possible mechanism of miR-93-3p in LPS-induced cardiomyocyte apoptosis and inflammation is unknown. miRNAs are believed to regulate multiple target messenger RNAs (mRNAs) in different pathologic conditions, however, one gene may be regulated by more than one miRNA [14]. Previous studies have shown that miR-93-3p regulates apoptosis and inflammation through multigene targeting, including interleukin-1 receptor-associated kinase 4

⁎ Corresponding author at: Department of cardiovascular Medicine, The First Affiliated Hospital of Bengbu Medical College, No. 287, Changhuai Road, Bengbu 233004, Anhui Province, PR China. E-mail address: [email protected] (H. Zhang).

https://doi.org/10.1016/j.prp.2018.08.024 Received 30 June 2018; Received in revised form 14 August 2018; Accepted 23 August 2018 0344-0338/ © 2018 Published by Elsevier GmbH.

Please cite this article as: Tang, B., Pathology - Research and Practice, https://doi.org/10.1016/j.prp.2018.08.024

Pathology - Research and Practice xxx (xxxx) xxx–xxx

B. Tang et al.

responsive 3′-UTR. An empty plasmid was served as a negative control. Overexpressed TLR4 plasmid (vector-TLR4) and control (vector-Con) were transfected into H9c2 cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientifc, Inc., Waltham, MA, USA), according to the manufacturer’s protocol.

(IRAK4), signal transducer and activator of transcription 3 (STAT3), Bcell lymphoma-2 (Bcl-2) and cyclin E1 [25,34,35]. TLR4 as a direct target of miR-93 contributes to myocardial infarction-associated transcript (MIAT)-regulated cardiac hypertrophy [18]. TLR4 is one receptor for LPS and plays a crucial role in the initiation of inflammatory response by leading to the activation of nuclear factor-kappa B (NF-κB), which accelerates the production of proinflammatory cytokines, such as tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and IL-6 [24,27]. These inflammatory mediators contribute to the dysfunction, apoptosis and necrosis of cardiomyocytes [13]. However, the association between miR-93-3p and TLR4 in LPS-induced inflammation and apoptosis in H9C2 cardiomyocytes has not been completely clarified. In the present study, a post-transcriptional regulatory mechanism by miR93-3p was found to regulate LPS-induced inflammation and apoptosis in cardiomyocytes by targeting inhibition of TLR4. miR-93-3p/TLR4 axis highlights a novel mechanism that contributes to the cardioprotective effects in inflammation or apoptosis-induced myocardial damage.

2.6. Luciferase reporter assay The sequence of miR-93-3p was obtained using online predict software and synthesized by RiboBio (Guangzhou, China). The wildtype (WT) or mutant-type (MUT) 3′-UTR of TLR4 was inserted into the multiple cloning sites of the luciferase expressing pMIR-REPORT vector (Ambion; Thermo Fisher Scientific, Inc.). For the luciferase assay, H9c2 cells (1 × 105) were seeded into 24-wells and co-transfected with luciferase reporter vectors containing the WT or MUT 3′-UTR of TLR4 (0.5 μg) combined with miR-Con or miR-93-3p mimics (100 nM) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer’s protocol. The luciferase activity was measured using a luciferase reporter assay kit (Promega, Madison, WI, USA) according to the manufacturer's protocol.

2. Material and methods 2.1. Cell culture

2.7. Reverse transcription-quantitative polymerase chain reaction (RTqPCR)

H9c2 cells were purchased from the American Type Culture Collection (ATCC, Bethesda, MD, USA) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), containing 10% fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.), 10% L-glutamine, 0.5% penicillin/streptomycin, with 5% CO2 atmosphere at 37 ℃. TAK-242 (an inhibitor of TLR4) and LPS were obtained from Invivogen (San Diego, CA, USA) and SigmaAldrich (St. Louis, MO, USA), respectively.

Total RNA was extracted using TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. miR-93 was detected using TaqMan® MicroRNA assay (Applied Biosystems, Foster City, USA) followed by manufacturer’s instructions. U6 snRNA was used as an endogenous control. cDNA was synthesized by reverse transcription reactions with 2 μg total RNA using moloney murine leukemia virus reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.). RT-qPCR was performed by Applied Biosystems 7300 Real-Time PCR System (Thermo Fisher Scientific, Inc.) with the TaqMan Universal PCR Master Mix (Thermo Fisher Scientific, Inc.). The relative expression levels of mRNA were calculated using the 2−ΔΔCt method [20] and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primers were used as follows: TLR4: forward 5′-AAGTTATTGTGGTGGTGTCTAG-3′ and reverse 5′-GAGGTA GGTGTTTCTGCTAAG-3′; TNF-α: 5′-AAGCCCGTAGCCCACGTCGTA-3′ and reverse 5′-GCCCGCAATCCAGGCCACTAC-3′; IL-1β: forward 5′-GACCTGGGCTGTCCTGATGA-3′ and reverse 5′-GTGCTGCTGCGAGA TTTGAA-3′; IL-6: forward 5′-TTCCATCCAGTTGCCTTCTTG-3′ and reverse 5′-GAAGGCCGTGGTTGTCACC-3′; GAPDH: forward 5′-GCACCGT CAAGCTGAGAAC-3′ and reverse 5′-TGGTGAAGACGCCAGTGGA-3′.

2.2. LDH assay H9c2 cells were seeded in 96-well plates (1 × 104 cells) and exposed to LPS (10 μg/mL) for 24 h. The plates were centrifuged at 1000g for 4 min. The LDH released from the damaged cells and total LDH was subjected to LDH kit (Beyotime Biotechnology, Haimen, China). 2.3. Inflammatory cytokine Cellular supernatant levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 were detected using the bioactive ELISA assay (Elabscience Biotechnology Co., Ltd., Wuhan, China) with a SpectraMax M5 ELISA plate reader (Molecular Devices, LLC, Sunnyvale, CA, USA), according to the manufacturer’s protocol.

2.8. Western blotting

2.4. Apoptosis assay

Proteins were extracted with radio immunoprecipitation assay (RIPA) buffer (Cat.No: P0013B; Beyotime Institute of Biotechnology, Haimen, China) and blotted as previously described [31]. The primary antibodies of TLR4 (dilution: 1:1000; Abcam, Cambridge, UK) and NFκB/p65 (dilution: 1: 500; Cell Signaling Technology, Inc., USA) were used to incubate the membranes. Subsequently, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (cat.no: sc-516102; dilution: 1:10,000; Santa Cruz Biotechnology) at room temperature for 2 h and visualized by chemiluminescence (Thermo Fisher Scientific, Inc.). Signals were analyzed with Quantity One® software version 4.5 (Bio Rad Laboratories, Inc., Hercules, CA, USA). β-actin (cat. no. sc-130065; 1: 2000; Santa Cruz Biotechnology) was used to as the control antibody.

H9c2 cells were incubated with different conditions for 24 h. AnnexinV-FITC/PI kit (Becton, Dickinson and Company, New Jersey, USA) was used to stain cells for 15 min, and then cell apoptosis assay was performed by flow cytometry assay (FACScan, BD Biosciences, San Jose, CA, USA) and analyzed by CELL Quest 3.0 software (BD Biosciences). 2.5. Cell transfection and plasmid constructs miR-Con, miR-93-3p mimics, scramble and miR-93-3p inhibitors were synthesized by RiboBio (Guangzhou, China) and transfected into H9c2 cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientifc, Inc., Waltham, MA, USA), according to the manufacturer’s protocol. A mammalian expression plasmid (pReceiver-M02-ERBB3; GeneCopoeia, Germantown, MD, USA) designed to express the fulllength open reading frame (ORF) of rat TLR4 without miR-93-3p

2.9. Statistical analysis Data were presented as mean ± SD. Statistical analysis was performed using IBM SPSS Statistics Version 19.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism Version 7.0 (GraphPad Software, Inc., La 2

Pathology - Research and Practice xxx (xxxx) xxx–xxx

B. Tang et al.

Fig. 1. LPS induces apoptosis and inflammation in H9c2 cardiomyocytes. H9c2 cells are exposed to LPS (10 μg/mL) for 0–48 h, cell viability is measured by CCK-8 assay (A). After treated with LPS (10 μg/mL) for 24 h, LDH is detected to reflect cell death (B), AnnexinV-FITC/PI staining is used to evaluate the cell apoptosis (C and D), the levels of TNF-α, IL-1β and IL-6 are analyzed by ELISA kit in cellular supernatant (E), and the mRNA levels of TNF-α, IL-1β and IL-6 are analyzed by RT-qPCR assay (F). * P < 0.05, ** P < 0.01, *** P < 0.001. n = 3 in each group.

3. Results

were significantly increased in LPS-exposed H9c2 cell compared with the control group. In addition, we also found that the levels of inflammatory cytokines, TNF-α, IL-1β and IL-6, were dramatically upregulated in LPS-treated H9c2 cell as compared to the control group by an ELISA assay (Fig. 1E) and RT-qPCR (Fig. 1F). These findings suggested that LPS-indiced inflammatory response might be involved in H9c2 cell apoptosis.

3.1. LPS induces apoptosis and inflammation in H9c2 cells

3.2. TLR4 is activated in LPS-stimulated H9c2 cells

Previous studies have shown that LPS is a potent stimulus for the inflammatory response and apoptosis in cardiomyocytes [14,24,29,34]. Consistent with these results, our study showed that H9c2 cells viability was inhibited by LPS (10 μg/mL) with a time-dependent manner (Fig. 1A), which might be associated with LPS-induced cell apoptosis. To demonstrate the potential mechanism of LPS-induced H9c2 cell growth inhibition, the levels of released lactate dehydrogenase (LDH), which is a marker of cell death [2], and apoptosis rate were measured in LPS-stimulated H9c2 cell. The results indicated that the rate of released LDH to total LDH (Fig. 1B) and apoptosis rate (Fig. 1C and D)

To further investigate the underlying molecular mechanisms of LPSinduced inflammation and apoptosis in H9c2 cells, the expression of TLR4 was detected in LPS-stimulated H9c2 cells with or without resatorvid (TAK-242), which is a novel selective TLR4 signal transduction inhibitor and can inhibit various kinds of inflammatory mediators, such as TNF-α, IL-1β and IL-6 [38]. The mRNA (Fig. 2A) and protein (Fig. 2B) levels of TLR4 were markedly increased in LPS-stimulated H9c2 cells, while the alterations in the expression of TLR4 were restrained by TAK-242 pretreatment for 12 h. In addition, LPS-induced apoptosis (Fig. 2C) and inflammatory response (Fig. 2D) were reversed

Jolla, CA, USA). Student’s t-test was used to analyze two-group differences. Inter-group differences were analyzed by one-way analysis of variance, followed by a post hoc Tukey test for multiple comparisons. Pvalue less than 0.05 was considered as a statistically significant difference.

3

Pathology - Research and Practice xxx (xxxx) xxx–xxx

B. Tang et al.

Fig. 2. Inhibition of TLR4 attenuates LPS-induced apoptosis and inflammation in H9c2 cardiomyocytes. H9c2 cells exposure to LPS (10 μg/mL), TAK-242 (20 μM) or LPS combined with TAK-242, the mRNA (A) and protein (B) expression of TLR4 are assayed by RT-qPCR and western blotting, respectively. AnnexinV-FITC/PI staining is used to evaluate the cell apoptosis (C), and the mRNA levels of TNF-α, IL-1β and IL-6 are analyzed by RT-qPCR assay (D). * P < 0.05 compared with control group; # P < 0.05 compared with LPS group. n = 3 in each group.

inflammatory response, over-activation of NF-κB is associated with cytoplasmic degradation of its inhibitor IκBα, p65, as a subunit of NFκB, into the nucleus binds to DNA and enhances the expression of inflammatory cytokines [8,31]. These results suggested that miR-93-3p could protect against LPS-induced inflammation and apoptosis in H9c2 cells.

by TAK-242 pretreatment. These results indicated that increased TLR4 expression in cardiomyocytes mediated LPS-induced inflammation and apoptosis. 3.3. miR-93-3p is suppressed in LPS-stimulated H9c2 cells miR-93 has shown to play the protective effects in several animal or cell model of myocardial damage [16,33]. In the present study, we found that the expression of miR-93-3p was suppressed by LPS with a dose- and time-dependent manner (Fig. 3A and B). To investigate the role of miR-93-3p in LPS-induced inflammation and apoptosis in H9c2 cells, the miR-93-3p mimics was transfected into H9c2 cells, and the expression of miR-93-3p was dramatically up-regulated after transfected with miR-93-3p mimics, suggesting that miR-93-3p mimics can efficiently be expressed in H9c2 cells (Fig. 3C). Interestingly, we found that overexpression of miR-93-3p blocked LPS-triggered apoptosis (Fig. 3D) and inflammation (Fig. 3E) in H9c2 cells. Furthermore, to investigate the possible mechanism of miR-93-3p reduced inflammation in H9c2 cells, the protein expression of NF-κB/p65 in nucleus was measured in LPS-treated H9c2 cells after transfected with miR-Con or miR-93-3p mimics. The results demonstrated that an increased NF-κB/ p65 level in the nucleus was detected in LPS-treated H9c2 cells, while miR-93-3p mimics had the capacity to reduction of nucleic NF-κB/p65 (Fig. 3F). NF-κB as a key transcription factor has been implicated in

3.4. TLR4 is a direct target of miR-93-3p Based on above results, we found that miR-93-3p performed an opposite effect to TLR4 in LPS-induced inflammation and apoptosis in H9c2 cells. However, the direct correlation between miR-93-3p and TLR4 is still unclear. Using online predict softwares, we found that there was a binding site for miR-93-3p in the 3′-UTR of TLR4 as shown in Fig. 4A. To further investigate whether miR-93-3p directly regulated the expression of TLR4, a luciferase reporter assay was performed after transfected with wild-type (WT) or mutant-type (MUT) 3′-UTR sequence of TLR4 combined with miR-Con or miR-93-3p mimics in H9c2 cells. The results demonstrated that transfection with miR-93-3p mimics significantly decreased the luciferase enzyme activity co-transfected with WT 3′-UTR of TLR4, but no change on luciferase enzyme activity in MUT 3′-UTR of TLR4 (Fig. 4B). We also found that the mRNA and protein expression of TLR4 were significantly suppressed by miR93-3p mimics and increased by miR-93-3p inhibitors in cardiomyocytes 4

Pathology - Research and Practice xxx (xxxx) xxx–xxx

B. Tang et al.

Fig. 3. Overexpression of miR-93-3p attenuates LPS-induced apoptosis and inflammation in H9c2 cardiomyocytes. The expression of miR-93-3p is analyzed by RTqPCR assay after treated with LPS for different concentrations (0–10 μg/mL; A) and treated with LPS (10 μg/mL) for different time (0–48 h; B). After transfected with miR-93-3p mimics, miR-93-3p is analyzed by RT-qPCR assay (C). H9c2 cells exposure to LPS (10 μg/mL), miR-93-3p mimics (100 nM) or LPS combined with miR-933p mimics for 24 h, AnnexinV-FITC/PI staining is used to evaluate the cell apoptosis (D), and the mRNA levels of TNF-α, IL-1β and IL-6 are analyzed by RT-qPCR assay (E). The protein expression of nucleic NF-κB/p65 was measured by western blotting (F). * P < 0.05 compared with control group; # P < 0.05 compared with LPS group. n = 3 in each group.

(Fig. 4C and D). Taken together, these findings suggested that TLR4 was a direct target of miR-93-3p.

93-3p was implicated in LPS-induced inflammation and apoptosis in H9c2 cardiomyocytes by inhibiting TLR4 expression.

3.5. TLR4 possesses an antagonistic effect to miR-93-3p in LPS-induced inflammation and apoptosis in H9c2 cells

4. Discussion Inflammation and apoptosis can be triggered by a variety of stimuli, such as high glucose, fatty acid and LPS, in cardiomyocytes [17,22,32]. Importantly, cardiomyocytes inflammation and apoptosis are two of the major pathogenic factors in cardiac diseases [4,5,14,21]. Therefore, it is crucial important to elucidate the underlining mechanisms in the progression of cardiomyocytes inflammation and apoptosis. In the present study, we found that up-regulation of TLR4 and down-regulation of miR-93-3p were simultaneously observed in LPS-stimulated H9c2

To further explain the interaction between miR-93-3p and TLR4, overexpression of miR-93-3p and TLR4 were co-transfected into H9c2 cells in the presence of LPS. The results validated that LPS-triggered apoptosis and inflammation could be magnified by overexpressed TLR4 (Fig. 5A and B). Moreover, the inhibitory actions of miR-93-3p mimics in LPS-induced inflammation and apoptosis were eliminated by overexpressed TLR4 (Fig. 5A and B). These observations revealed that miR5

Pathology - Research and Practice xxx (xxxx) xxx–xxx

B. Tang et al.

Fig. 4. TLR4 is a direct target of miR-93-3p. The putative miR-93-3p binding sites in the 3′-UTR of TLR4 are predicted by on-line softwares (A). The luciferase activity assay was performed (B). After transfected with miR-93-3p mimics or inhibitors, the mRNA (C) and protein (D) expression of TLR4 are assayed by RT-qPCR and western blotting, respectively. * P < 0.05 compared with miR-Con group; # P < 0.05 compared with scramble group. n = 3 in each group.

These results and our findings suggest that TLR4 acts as pro-inflammatory and pro-apoptotic roles in LPS-stimulated cardiomyocyte and can serve as a potential therapeutic target for cardiac dysfunction. To investigate how TLR4 expression was regulated in LPS-stimulated cardiomyocytes, we detected the expression of miRNAs that functioned as the post-transcriptional modulators suppression of target genes expression by binding to its 3′-UTR [3]. Therefore, we hypothesized that miRNAs by inhibiting TLR4 expression attenuated LPS-induced cardiomyocyte dysfunction. Some miRNAs, including miR-21-3p, miR-25, miR-99a, miR-146a, miR-375 and miR-499, have been confirmed to participate in LPS-induced cardiomyocyte dysfunction [11,12,14,15,30,36]. Among these miRNAs, up-regulation of miR-25, miR-99a, miR-146a and miR-499 and down-regulation of miR-21-3p and miR-375 reduce inflammatory response and decrease cardiomyocyte death in the presence of LPS dysfunction [11,12,14,15,30,36]. Therefore, miRNAs are closely correlated with the pathological process of cardiomyocyte.

cardiomyocytes. We also validated that miR-93-3p targeted to the 3′UTR of TLR4 and inhibited its expression. Overexpression of miR-93-3p carried out an opposite effect to TLR4 and negatively regulated LPSinduced inflammation and apoptosis in cardiomyocytes. Emerging evidence indicates that robust binding of TLR4 with LPS, a pathogen-associated molecular pattern ligand for TLR4, exacerbates the development of heart failure [19]. Usually, TLR4-mediated the over-activation of downstream signaling of transforming growth factorβ-activated kinase-1 (TAK-1) phosphorylation and NF-κB signaling associates with LPS-induced cardiac dysfunction [8]. Silencing TLR4 with small interfering RNA (siRNA) in cardiomyocyte reduces injury and decreases the damage markers LDH and TNF-α following hypoxia [1]. In our study, LPS was found to induce the expression of TLR4 in cardiomyocytes, while TLR4 inhibitor, TAK-242, significantly attenuated LPS-induced apoptosis and over-activation of inflammatory response. TAK-242 has also been reported to alleviate ox-LDL-induced apoptosis in primary myocardial cells by inhibiting TLR4/NF-κB signaling [31]. 6

Pathology - Research and Practice xxx (xxxx) xxx–xxx

B. Tang et al.

Fig. 5. TLR4 possesses an antagonistic effect to miR-93-3p in LPS-induced inflammation and apoptosis in H9c2 cells. After miR-93-3p mimics and TLR4 plasmids are co-transfected into H9c2 cells in the presence of LPS, AnnexinV-FITC/PI staining is used to evaluate the cell apoptosis (A), and the mRNA levels of TNF-α, IL-1β and IL-6 are analyzed by RT-qPCR assay (B). * P < 0.05 compared with PA group; # P < 0.05 compared with PA + miR-93-3p mimics group. n = 3 in each group.

Acknowledgements

In the present study, LPS stimulation down-regulated the expression of miR-93-3p, which resulted in elevation of the apoptosis rate and the mRNA expression of TNF-α, IL-1β and IL-6 in cardiomyocyte, while overexpressed miR-93-3p had beneficial effect on LPS-induced cardiomyocyte dysfunction by repressing inflammation and apoptosis. Moreover, bioinformatics analysis and experimental data indicated that TLR4 was a direct target gene of miR-93-3p, which could inhibit or promote TLR4 expression by transfection of miR-93-3p mimics or inhibitors, respectively. Furthermore, we also found that overexpressed TLR4 neutralized the beneficial effects of miR-93-3p mimics in LPSstimulated cardiomyocyte. Thus, miR-93-3p has protective effects against LPS-induced cardiomyocyte injuries by inhibiting TLR4 expression. In vivo and in vitro myocardial infarction models, elevation of miR-93 is suggested to improve cardiac function and inhibit cardiac remodeling and apoptosis [16]. miR-93 is also decreased in LPS-stimulated macrophages and inhibits NF-κB activation and pro-inflammatory cytokines by targeting IRAK4 expression [34]. These results indicate that miR-93 may target to multiple genes to regulate LPS-induced inflammation in different cell-types.

This research was supported by the National Natural Science Foundation of China (No. 81170046), Key Project of Top-Notch Talent of Discipline (specialty) of the Higher Education Institute of Anhui Province 2016 (No. gxbjZD2016072) and Major projects of Natural Science Research in Colleges and Universities of Anhui Province (No. KJ2018ZD023). References [1] O. Avlas, S. Srara, A. Shainberg, D. Aravot, E. Hochhauser, Silencing cardiomyocyte TLR4 reduces injury following hypoxia, Exp. Cell Res. 348 (2016) 115–122. [2] I. Barba, L. Chavarria, M. Ruiz-Meana, M. Mirabet, E. Agullo, D. Garcia-Dorado, Effect of intracellular lipid droplets on cytosolic Ca2+ and cell death during ischaemia-reperfusion injury in cardiomyocytes, J. Physiol. 587 (2009) 1331–1341. [3] R.A. Boon, K. Iekushi, S. Lechner, T. Seeger, A. Fischer, S. Heydt, D. Kaluza, K. Treguer, G. Carmona, A. Bonauer, A.J. Horrevoets, N. Didier, Z. Girmatsion, P. Biliczki, J.R. Ehrlich, H.A. Katus, O.J. Muller, M. Potente, A.M. Zeiher, H. Hermeking, S. Dimmeler, MicroRNA-34a regulates cardiac ageing and function, Nature 495 (2013) 107–110. [4] G. Bouras, G. Giannopoulos, G. Hatzis, D. Alexopoulos, G. Leventopoulos, S. Deftereos, Inflammation and chronic heart failure: from biomarkers to novel antiinflammatory therapeutic strategies, Med. Chem. (Shariqah (United Arab Emirates)) 10 (2014) 682–699. [5] E. Braunwald, Heart failure, JACC Heart Fail. 1 (2013) 1–20. [6] L. Chen, P. Liu, X. Feng, C. Ma, Salidroside suppressing LPS-induced myocardial injury by inhibiting ROS-mediated PI3K/Akt/mTOR pathway in vitro and in vivo, J. Cell. Mol. Med. 21 (2017) 3178–3189. [7] T.S. Chen, S. Battsengel, C.H. Kuo, L.F. Pan, Y.M. Lin, C.H. Yao, Y.S. Chen, F.H. Lin, W.W. Kuo, C.Y. Huang, Stemcells rescue cardiomyopathy induced by P. gingivalisLPS via miR-181b, J. Cell. Physiol. 233 (2018) 5869–5876. [8] W.P. Chen, H.J. Tzeng, H.C. Ku, Y.J. Ho, S.S. Lee, M.J. Su, Thaliporphine ameliorates cardiac depression in endotoxemic rats through attenuating TLR4 signaling in the downstream of TAK-1 phosphorylation and NF-kappaB signaling, Naunyn Schmiedeberg’s Arch. Pharmacol. 382 (2010) 441–453. [9] C. Emanueli, T. Thum, miRNAGE-34 induces cardiac damAGE, Cell Res. 23 (2013) 866–867. [10] A. Eulalio, M. Mano, M. Dal Ferro, L. Zentilin, G. Sinagra, S. Zacchigna, M. Giacca, Functional screening identifies miRNAs inducing cardiac regeneration, Nature 492

5. Conclusion Taken together, our findings collectively show that miR-93-3p as a post-transcriptional regulator for TLR4 inhibits LPS-induced inflammation and apoptosis in cardiomyocyte, All of these results provide a therapeutic strategy that elevation of miR-93-3p can play a protective role in cardiomyocyte dysfunction.

Competing interests The authors declare they have no competing interests. 7

Pathology - Research and Practice xxx (xxxx) xxx–xxx

B. Tang et al.

(2012) 376–381. [11] M. Gao, X. Wang, X. Zhang, T. Ha, H. Ma, L. Liu, J.H. Kalbfleisch, X. Gao, R.L. Kao, D.L. Williams, C. Li, Attenuation of cardiac dysfunction in polymicrobial sepsis by MicroRNA-146a is mediated via targeting of IRAK1 and TRAF6 expression, J. Immunol. 195 (2015) 672–682. [12] V.N.S. Garikipati, S.K. Verma, D. Jolardarashi, Z. Cheng, J. Ibetti, M. Cimini, Y. Tang, M. Khan, Y. Yue, C. Benedict, E. Nickoloff, M.M. Truongcao, E. Gao, P. Krishnamurthy, D.A. Goukassian, W.J. Koch, R. Kishore, Therapeutic inhibition of miR-375 attenuates post-myocardial infarction inflammatory response and left ventricular dysfunction via PDK-1-AKT signalling axis, Cardiovasc. Res. 113 (2017) 938–949. [13] A. Jenke, S. Wilk, W. Poller, U. Eriksson, A. Valaperti, B.H. Rauch, A. Stroux, P. Liu, H.P. Schultheiss, C. Scheibenbogen, C. Skurk, Adiponectin protects against toll-like receptor 4-mediated cardiac inflammation and injury, Cardiovasc. Res. 99 (2013) 422–431. [14] Z. Jia, J. Wang, Q. Shi, S. Liu, W. Wang, Y. Tian, Q. Lu, P. Chen, K. Ma, C. Zhou, SOX6 and PDCD4 enhance cardiomyocyte apoptosis through LPS-induced miR-499 inhibition, Apoptosis: Int. J. Program. Cell Death 21 (2016) 174–183. [15] R. Jing, Z. Zhou, F. Kuang, L. Huang, C. Li, microRNA-99a reduces lipopolysaccharide-induced oxidative injury by activating notch pathway in H9c2 cells, Int. Heart J. 58 (2017) 422–427. [16] K. Li, T. Lin, L. Chen, N. Wang, MicroRNA-93 elevation after myocardial infarction is cardiac protective, Med. Hypotheses 106 (2017) 23–25. [17] W. Li, Q. Fang, P. Zhong, L. Chen, L. Wang, Y. Zhang, J. Wang, X. Li, Y. Wang, J. Wang, G. Liang, EGFR inhibition blocks palmitic acid-induced inflammation in cardiomyocytes and prevents hyperlipidemia-induced cardiac injury in mice, Sci. Rep. 6 (2016) 24580. [18] Y. Li, J. Wang, L. Sun, S. Zhu, LncRNA myocardial infarction-associated transcript (MIAT) contributed to cardiac hypertrophy by regulating TLR4 via miR-93, Eur. J. Pharmacol. 818 (2018) 508–517. [19] L. Liu, Y. Wang, Z.Y. Cao, M.M. Wang, X.M. Liu, T. Gao, Q.K. Hu, W.J. Yuan, L. Lin, Up-regulated TLR4 in cardiomyocytes exacerbates heart failure after long-term myocardial infarction, J. Cell. Mol. Med. 19 (2015) 2728–2740. [20] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using realtime quantitative PCR and the 2(-delta delta C(T)) method, Methods (San Diego, Calif.) 25 (2001) 402–408. [21] S. Mangini, L. Higuchi Mde, J.T. Kawakami, M.M. Reis, R.N. Ikegami, S.A. Palomino, P.M. Pomerantzeff, A.I. Fiorelli, F.G. Marcondes-Braga, F. Bacal, S.M. Ferreira, V.S. Issa, G.E. Souza, P.R. Chizzola, E.A. Bocchi, Infectious agents and inflammation in donated hearts and dilated cardiomyopathies related to cardiovascular diseases, Chagas’ heart disease, primary and secondary dilated cardiomyopathies, Int. J. Cardiol. 178 (2015) 55–62. [22] Y. Pan, Y. Wang, Y. Zhao, K. Peng, W. Li, Y. Wang, J. Zhang, S. Zhou, Q. Liu, X. Li, L. Cai, G. Liang, Inhibition of JNK phosphorylation by a novel curcumin analog prevents high glucose-induced inflammation and apoptosis in cardiomyocytes and the development of diabetic cardiomyopathy, Diabetes 63 (2014) 3497–3511. [23] L. Perdomo, N. Beneit, Y.F. Otero, O. Escribano, S. Diaz-Castroverde, A. Gomez-

[24]

[25]

[26] [27] [28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36] [37]

[38]

8

Hernandez, M. Benito, Protective role of oleic acid against cardiovascular insulin resistance and in the early and late cellular atherosclerotic process, Cardiovasc. Diabetol. 14 (2015) 75. A. Plociennikowska, A. Hromada-Judycka, K. Borzecka, K. Kwiatkowska, Co-operation of TLR4 and raft proteins in LPS-induced pro-inflammatory signaling, Cell. Mol. Life Sci. 72 (2015) 557–581. M. Rouse, R. Rao, M. Nagarkatti, P.S. Nagarkatti, 3,3’-Diindolylmethane ameliorates experimental autoimmune encephalomyelitis by promoting cell cycle arrest and apoptosis in activated T cells through microRNA signaling pathways, J. Pharmacol. Exp. Ther. 350 (2014) 341–352. Q. Su, J. Yao, C. Sheng, Geniposide attenuates LPS-induced injury via up-regulation of miR-145 in H9c2 cells, Inflammation 41 (4) (2018) 1229–1237. L.A. Velloso, F. Folli, M.J. Saad, TLR4 at the crossroads of nutrients, gut microbiota, and metabolic inflammation, Endocr. Rev. 36 (2015) 245–271. C. Wahlquist, D. Jeong, A. Rojas-Munoz, C. Kho, A. Lee, S. Mitsuyama, A. van Mil, W.J. Park, J.P. Sluijter, P.A. Doevendans, R.J. Hajjar, M. Mercola, Inhibition of miR25 improves cardiac contractility in the failing heart, Nature 508 (2014) 531–535. H. Wang, Y. Bei, P. Huang, Q. Zhou, J. Shi, Q. Sun, J. Zhong, X. Li, X. Kong, J. Xiao, Inhibition of miR-155 protects against LPS-induced cardiac dysfunction and apoptosis in mice, Mol. Ther. Nucleic acids 5 (2016) e374. H. Wang, Y. Bei, S. Shen, P. Huang, J. Shi, J. Zhang, Q. Sun, Y. Chen, Y. Yang, T. Xu, X. Kong, J. Xiao, miR-21-3p controls sepsis-associated cardiac dysfunction via regulating SORBS2, J. Mol. Cell. Cardiol. 94 (2016) 43–53. X. Wang, Y. Sun, H. Yang, Y. Lu, L. Li, Oxidized low-density lipoprotein induces apoptosis in cultured neonatal rat cardiomyocytes by modulating the TLR4/NFkappaB pathway, Sci. Rep. 6 (2016) 27866. Y. Wang, Y. Wang, D. Yang, X. Yu, H. Li, X. Lv, D. Lu, H. Wang, Beta(1)-adrenoceptor stimulation promotes LPS-induced cardiomyocyte apoptosis through activating PKA and enhancing CaMKII and IkappaBalpha phosphorylation, Crit. Care (London, England) 19 (2015) 76. Y. Wo, J. Guo, P. Li, H. Yang, J. Wo, Long non-coding RNA CHRF facilitates cardiac hypertrophy through regulating Akt3 via miR-93, Cardiovasc. Pathol. 35 (2018) 29–36. Y. Xu, H. Jin, X. Yang, L. Wang, L. Su, K. Liu, Q. Gu, X. Xu, MicroRNA-93 inhibits inflammatory cytokine production in LPS-stimulated murine macrophages by targeting IRAK4, FEBS Lett. 588 (2014) 1692–1698. X.T. Yan, L.J. Ji, Z. Wang, X. Wu, Q. Wang, S. Sun, J.M. Lu, Y. Zhang, MicroRNA-93 alleviates neuropathic pain through targeting signal transducer and activator of transcription 3, Int. Immunopharmacol. 46 (2017) 156–162. Y. Yao, F. Sun, M. Lei, miR-25 inhibits sepsis-induced cardiomyocyte apoptosis by targetting PTEN, Biosci. Rep. 38 (2018). H. Zhang, H. Li, A. Ge, E. Guo, S. Liu, L. Zhang, Long non-coding RNA TUG1 inhibits apoptosis and inflammatory response in LPS-treated H9c2 cells by down-regulation of miR-29b, Biomed. Pharmacother. 101 (2018) 663–669. Y. Zhang, W. Peng, X. Ao, H. Dai, L. Yuan, X. Huang, Q. Zhou, TAK-242, a toll-like receptor 4 antagonist, protects against aldosterone-induced cardiac and renal injury, PLoS One 10 (2015) e0142456.