TNFα-induced downregulation of microRNA-186 contributes to apoptosis in rat primary cardiomyocytes

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TNF␣-induced downregulation of microRNA-186 contributes to apoptosis in rat primary cardiomyocytes Hua Xu ∗,1 , Jingyao Li 1 , Yue Zhao, Dayi Liu Department of Cardiology, Daqing Oil Field General Hospital, NO. 9 Saertu District, Daqing City, 163000, Heilongjiang Province, China

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Article history: Received 16 January 2017 Received in revised form 14 February 2017 Accepted 14 February 2017 Available online xxx Keywords: Cardiomyocytes TNF␣ AIF MicroRNA-186 Apoptosis Inflammation

a b s t r a c t Progressive loss of cardiac cardiomyocytes is involved in pathogenesis of heart failure. Inflammation is considered as a major risk factor that triggers cardiomyocytes apoptosis or induces cellular damage. Proinflammatory cytokines such as TNF␣ can directly activate cell apoptosis or promote oxidant production that damages cellular structure eventually. We investigated TNF␣ mediated apoptosis in cultured rat primary cardiomyocytes. Annexin V/PI staining and apoptosis biomarker expression were used to examine cardiomyocytes cell apoptosis response. We also identified key microRNA that plays a regulatory role in this pathway with genetic and biochemical approaches. Apoptosis Inducing Factor (AIF) expression was found to be upregulated with 10 ␮g/ml or 50 ␮g/ml TNF␣ stimulation for 24 h, which was associated with apoptotic index. Subsequently, miR-186 was identified as direct regulator of AIF in TNF␣ mediated cardiomyocytes apoptosis from microRNA expression profiling. miR-186 level was downregulated with TNF␣ treatment that was correlated with AIF induction. Last, in the rescue experiment, miR-186 mimic protected cardiomyocytes against TNF␣ mediated apoptosis. Collectively, the results suggest TNF␣-induced AIF upregulation contributes to apoptosis in rat primary cardiomyocytes through regulating miR-186 expression, which implies miR-186 could be a potential therapeutic target for preventing inflammation associated cardiac damage. © 2017 Elsevier GmbH. All rights reserved.

1. Introduction Cardiac failure is the major cause of human pathological death globally. The pathogenesis of heart failure involves the progressive loss of cardiac cardiomyocytes, which suggests apoptosis as an important mode leading to cell death during heart failure (Haunstetter and Izumo, 1998; Kang and Izumo, 2000; Narula et al., 1996). Inflammation is one of the major risk factors underlining cardiovascular disease. Inflammation causes over-production of proinflammatory cytokines such as TNF␣ that can directly activate cell apoptosis pathway or that leads to oxidant production to cause tissue damage (Finkel, 2003; Moe et al., 2004). Studies with TNF␣ have revealed its important role in apoptosis associated cardiac pathologies such as cardiac hypertrophy and cardiomyopathy (Al-Shudiefat et al., 2013; Lin et al., 2010; Shanmugam et al., 2016). Over-expression of TNF␣ in mouse model

Abbreviations: AIF, apoptosis inducing factor. ∗ Corresponding author. E-mail address: [email protected] (H. Xu). 1 Contributed equally.

reduced Bcl-2 (anti-apoptotic protein) expression and triggered the downstream caspase cascade, leading to intrinsic apoptotic pathway activation, that eventually contribute to adverse cardiac remodeling in the adult heart (Engel et al., 2004). Clinical study demonstrated that inhibition of TNF␣ pathway could be effective approach for anti-inflammatory associated with antioxidant benefits (Moe et al., 2004; Sugano et al., 2002). Therefore identifying the downstream effectors or other regulators for TNF␣ induced apoptosis will inspire new therapeutic strategy, in order to reduce cardiovascular incidents due to inflammation or oxidative stress induced apoptosis. AIF is apoptotic effector protein residing in mitochondria membrane. In response to apoptotic stimulus, AIF translocates from the mitochondria into the nucleus to induce nuclear condensation and DNA fragmentation that resemble key cellular features during cell apoptosis (Daugas et al., 2000). Recent study has revealed AIF translocation and activation are involved in aldosterone induced cardiomyocyte apoptosis that play a part in ALD induced cardiomyocyte injury (Xiao et al., 2013). MicroRNAs are sequence specific regulators for gene expression and were first discovered in worms in 1990s (Lee et al., 1993). They usually contain 21–25 nucleotides, and fine-tune the expres-

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Please cite this article in press as: Xu, H., et al., TNF␣-induced downregulation of microRNA-186 contributes to apoptosis in rat primary cardiomyocytes. Immunobiology (2017), http://dx.doi.org/10.1016/j.imbio.2017.02.005

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sion of thousands of target mRNAs, with each mRNA targeted by multiple microRNAs. Most microRNAs bind to the complementary sites in 3 -untranslated regions (UTRs) of target mRNAs to suppress gene expression, by either directing mRNAs degradation or protein translation repression (Pillai, 2005). During the last 20 years, microRNAs have been proposed as key regulators in various biological processes, including cell growth, differentiation and apoptosis (Bartel, 2004). Dysregulation of microRNAs in cells may lead to cancerous phenotype (Farazi et al., 2013) or be associated with inflammatory and autoimmune diseases (Dai and Ahmed, 2011). The role of microRNA regulation in cardiovascular diseases is still unclear. Therefore, it will provide more insight for managing cardiovascular diseases by characterizing microRNA candidates involved in cardiomyocytes apoptosis pathway. In this study, we established a system to study the TNF␣ mediated apoptosis in rat primary cardiomyocytes. With this approach we established AIF as an apoptotic effector induced by TNF␣ to cause cell death in cardiomyocytes. We further identified miR-186 as key regulator in this axis. Finally the in vitro rescue experiment supported the hypothesis that miR-186 was a potential therapeutic target to confer cardiomyocytes the protection against TNF␣ mediated apoptosis.

prepared in SDS sample buffer and separated on an 8–12% SDSpolyacrylamide gel and then transferred to a PVDF membrane. Membranes were washed in TBS-T and then blocked with 5% nonfat milk in TBS-T for 1 h. Membranes are then incubated with indicated primary antibody in 5% non-fat milk prepared for overnight at 4 ◦ C. After washed with TBS-T three times for 10 min each, the membranes were incubated with the corresponding secondary antibodies coupled to HRP (Horse Radish Peroxidase) for 1 h at room temperature. After washed with TBS-T three times for 10 min each, western signals were visualized using Western Pico Super ECL reagent (Pierce, WI, USA). The results shown here were the representatives of at least three times of independent experiments. 2.5. MicroRNA microarray

2. Methods

MicroRNA microarray was customized by Agilent Technologies. Briefly oligos probes for miR-17-5p, miR-20b-5p, miR-106b, miR-75B, miR-455, miR-186, miR-375, miR-20a, miR-93, miR-33, miR-291a-3p, miR-399-5p, miR-1281 and miR-370 were designed and synthesized on SurePrint custom MicroRNA microarray platform. About 100 ng RNAs from each sample were dephosphorylated, labeled with Cyanine 3-pCP and desalted before the hybrodozation (20 h, 55 ◦ C). The MicroRNA expression profile was acquired with Agilent SureScan microarray scanner.

2.1. Materials and cell culture

2.6. Luciferase assay

Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin and trypsin were supplied by Gibco (Thermo Fisher, Waltham, MA, USA). Fetal bovine serum (FBS) was supplied from Hyclone (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). Recombinant TNF␣ (cat no. PHC3016) for treatment was obtained from Thermo Fisher. Rabbit monoclonal Anti-AIF antibody [E20] was purchased from Abcam, Cambridge, MA, USA. Both miR-186 mimic and inhibitor were customized by Exiqon. Primary culture of neonatal rat cardiomyocytes was based on the method described previously (Franke et al., 2007).

Luciferase reporter gene vectors were transfected into rat primary cardiomyocytes in 6-well plate with TurboFect transfection reagent (Fermentas). Renilla luciferase gene was also co-transfected for all experiments as internal control. After 48 h, the cells were harvested and the luciferase activities were subsequently measured with Dual-Luciferase Reporter assay kit (Promega, Madison, WI, USA). Luciferase activities were normalized to the cell number.

2.2. NF˛ treatment Cardiomyocytes were seeded with 5,000 cells/cm2 on Day 1. On Day 3, cells were washed with PBS and cultured with serum free medium. After over-night incubation, the cells were stimulated with TNF␣ in respective concentration for 24 h and then harvested for different assay measurements. 2.3. RT-PCR analysis Total RNA was extracted from 1 × 106 cells using TRIzol solution (Invitrogen, Carlsbad, CA, USA). 1 ␮g of total RNA was reverse transcribed into cDNA for 1 h at 50 ◦ C using oligo dT primer and reverse transcriptase in the presence of RNAse inhibitor. Transcribed cDNA template (50 ng) was incubated with 200 nM AIF primers or GAPDH primers (normalization control) in a total volume of 20 ␮l using KAPA SYBR FAST qPCR kit. Primers used in the study were: AIF forward 5 -CTA TAG GGA GAT CCA GGC AAC TTG-3 , reverse 5 -TAT AGG GAG ACC TCT GCT CCA GCC-3 ; GAPDH forward 5 -CGT CTT CAC CAC CAT GGA GAA GGC-3 , reverse 5 -AAG GCC ATG CCA GTG AGC TTC CC-3 . 2.4. Western blot analysis After treatment, the cells were washed once in PBS and harvested in RIPA lysis buffer. The protein concentration was measure with protein BCA protein assay. 40 ␮g protein lysate was

2.7. Flow cytometry analysis Cardiomyocyte apoptosis was quantified by a flow cytometer. Annexin V/Propidium iodide (PI) Apoptosis Detection Kit for flow cytometry was used to detect the proportion of apoptotic cells. Only those counts showing both Annexin V and PI positive were considered dead cells. 2.8. Statistical analysis All experimental data were presented as mean ± standard deviation. Experiments were repeated three times. Statistical differences were analyzed by student t-test. A P value < 0.05 was considered significant. 3. Results 3.1. Dosage effect of TNF˛ on primary cardiomyocytes TNF␣ is a prominent proinflammatory factor that can induce cardiomyocytes apoptosis during cardiovascular inflammation (Bajaj and Sharma, 2006; Nian et al., 2004). In order to elucidate the molecular mechanism that regulates this process, we first sought to measure the dosage response of rat primary cardiomyocytes to TNF␣ in cell culture. Primary cardiomyocytes were treated with different concentrations of TNF␣ (2, 10 or 50 ng/ml) for 24 h. Their apoptotic responses were then measured by annexin V-FITC/PI staining. Cells stained with both annexin V-FITC and PI were considered apoptotic cells. As shown in Fig. 1 (A&B), 24 h treatment of TNF␣ at doses of 10 ng/ml and 50 ng/ml significantly induced

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Fig. 1. Effects of TNF-␣ treatment at different doses (2, 10 or 50 ng/ml) for 24 h on cell apoptosis in the cardiomyocytes. (A) Representative images of flow cytometry analysis. Annexin V/PI staining was used to detect cell apoptosis. (B) Quantitative results of the percentages of apoptotic cells in the experimental groups. Experiments were repeated in triplicate. Data was given as mean ± SD. **p < 0.01 versus control.

cell apoptosis, with 15.49% and 36.48% of total population showing double positives in flow cytometry respectively. However, 2 ng/ml of TNF␣ did not provoke any cell death in 24 h incubation time. These indicated the response threshold of cardiomyocytes to TNF␣ mediated apoptotic induction is about 10 ng/ml, which agrees with the observation in other study (Yu et al., 2014). 3.2. TNF˛ induces AIF expression in primary cardiomyocytes AIF is known as downstream effector to activate cell apoptosis pathway in response to different apoptotic insults (Cande et al., 2002). But few studies have characterized the activation of AIF by TNF␣. Therefore, we sought to understand the role of AIF in TNF␣ mediated apoptosis. We first proceed to measure AIF mRNA and protein expression in cardiomyocytes treated with different concentrations of TNF␣. In accordant with what we observed in flow cytometry experiment, TNF␣ treatment at 10 ng/ml and 50 ng/ml increased AIF mRNA expression (Fig. 2A). To confirm this, AIF protein level was also examined by western blot. As shown (Fig. 2B&C), AIF protein expression was upregulated in cells incubated with 10 ng/ml or 50 ng/ml TNF␣, with nearly two folds increase at higher dose (50 ng/ml) stimulation. As expected, we observed no change of AIF level in cardiomyocytes treated with 2 ng/ml TNF␣. Therefore, in order to induce an optimal apoptotic response in rat primary cardiomyocytes, 24 h culture incubation of 50 ng/ml TNF␣ was adopted as standard experimental condition to investigate the mechanistic process for TNF␣ mediated cellular apoptosis in this study. 3.3. miR-186 regulates AIF expression under TNF˛ pathway Next, we moved on to identify the potential microRNAs that regulate TNF␣ pathway through targeting AIF. Fourteen microRNAs that may target AIFM1 (gene encoding AIF) were predicted. The customized microarray targeting these microRNAs were employed

to study their expressional changes in Rat primary cardiomyocytes in response to TNF␣. As shown in Fig. 3A, seven of them (miR-17-5p, miR-20b-5p, miR-106b, miR-75b, miR-455, miR-186 and miR375) were downregulated by TNF␣ treatment. Further on, mature MicroRNA assay confirmed that TNF␣ leaded to a downregulation of miR-186 expression to nearly 80% (Fig. 3B). To confirm AIF level was regulated by miR-186, we measure AIF mRNA in cells transfected with anti-miR-186 (inhibitor of miR-186). As shown (Fig. 3C), inhibiting miR-186 induced AIF mRNA in nearly two folds. The increase in protein expression was also confirmed in western blot (Fig. 3D&E). MicroRNA targeting site is usually at the 3 UTR of their targeting genes. The predicted miR-186 targeting sequence on AIFM1 3 UTR was shown in Fig. 4A. Based on this, we constructed two luciferase reporter gene vectors: ATFM1-WT denoted the one with aforementioned AIFM1 3 UTR directly conjugated to 3’end of luciferase gene; while ATFM1-Mut referred to the one with mutated AIFM1 3 UTR sequence cloned downstream of luciferase gene (Fig. 4B). In theory the mutation on AIFM1 3 UTR sequence should abolish the sequence site binding to miR186. Next, primary cardiomyocytes were transfected with either of these two reporter constructs. The luciferase activities in the cells were then measured with or without TNF␣ treatment. In cells expressing AIFM1-WT, luciferase activity increased around two folds after TNF␣ stimulation, which was explained by TNF␣ mediated down-regulation of miR-186 that attenuated microRNA suppression effect on luciferase reporter gene AIFM1-WT. While in cells expressing AIFM1-Mut, luciferase activity showed no change after TNF␣ treatment (Fig. 4C). In another experiment, we treated these two reporter lines with miR-186 mimic. As expected, miR-186 mimic directly reduced the luciferase activity in cells expressing AIFM1-WT to an extent of nearly 80%, but had no impact on that of AIFM1-Mut (Fig. 4D). Taken together, TNF␣ pathway can negatively regulate miR-186 activity, which in turn leads to AIF upregulation in primary cardiomyocytes that may eventually trigger apoptosis.

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Fig. 2. Effects of TNF-␣ treatment at different doses (2, 10 or 50 ng/ml) for 24 h on AIF mRNA and protein expression in the cardiomyocytes. (A) Levels of AIF mRNA expression were analyzed by RT-PCR. GAPDH was employed as an internal control. (B, C) Levels of AIF protein expression in each group was examined by Western blot. ␤-actin was used as a loading control. AIF protein expressions were normalized to control. Experiments were repeated in triplicate. Data was given as mean ± SD. *p < 0.05 and **p < 0.01 versus control.

Fig. 3. 50 ng/ml TNF-␣ treatment for 24 h increases AIF mRNA and protein expressions in the cardiomyocytes, which may be due to down-regulation of miR-186 expression. (A) Microarray analysis of potential miRs which are predicted to target AIFM1. (B) TNF-␣ treatment causes down-regulation of miR-186, which was analyzed by mature miRNA assay. (C) Levels of AIF mRNA expressions in the cardiomyocytes were quantified by RT-PCR. GAPDH was employed as an internal control. Cells were treated with or without anti-miR-186. (D, E) Levels of AIF protein expressions in the cardiomyocytes were analyzed by Western blot. ␤-actin was used as a loading control. AIF protein expressions were normalized to control group. Experiments were repeated in triplicate. Data was given as mean ± SD. **p < 0.01 versus control.

3.4. miR-186 mimic rescues primary cardiomyocytes from TNF˛ mediated apoptosis

effect is probably mediated through the suppression of TNF␣ AIF1 activation.

Last, we proceeded to study the regulatory effect of miR-186 on TNF␣ induced apoptosis in rat primary cardiomyocytes. Cells treated with TNF␣ for 24 h showed 31.88% cell death by Annexin V/PI staining. In contrast, the presence of miR-186 mimic reduced the TNF␣ induced apoptosis index to 14.27% (Fig. 5). The rescuing

4. Discussion The factors leading to the progression of cardiovascular diseases such as cardiac failure, myocardial infarction or myocarditis are not fully understood. Progressive loss of cardiomyocytes medi-

Please cite this article in press as: Xu, H., et al., TNF␣-induced downregulation of microRNA-186 contributes to apoptosis in rat primary cardiomyocytes. Immunobiology (2017), http://dx.doi.org/10.1016/j.imbio.2017.02.005

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Fig. 4. Direct targeting of miR-186 with the 3 -UTR on the mRNA of AIFM1. (A) Sequences of the predicted miR-186 targeting sites on the 3 -UTR of AIFM1 mRNA, indicated by the vertical black lines. (B) Wild type (-WT) or mutated (-Mut) sequences from AIFM1 mRNA 3 -UTR were cloned downstream of luciferase reporter gene (LUF). (C, D) Luciferase activities of −WT and −Mut constructs for AIFM1 were determined in cardiomyocytes with TNF-␣ (50 ng/ml) or miR-186 mimic treatment. Experiments were repeated in triplicate. Data was given as mean ± SD. **p < 0.01 versus control.

Fig. 5. Effects of miR-186 mimic on the apoptosis in cardiomyocytes induced by TNF-␣ (50 ng/ml, 24 h) treatment. (A) Representative images of flow cytometry analysis. Annexin V/PI staining was used to detect cell apoptosis. (B) Quantitative results of the percentages of apoptotic cells in control, TNF-␣ group, TNF-␣ + miR-186 mimic group. Experiments were repeated in triplicate. Data was given as mean ± SD. **p < 0.01 versus control.

ated through apoptosis is considered as one of the pathological factors (Kang and Izumo, 2000; Kocher et al., 2001). Histological evidence supports the apoptosis mediated myocytes loss model in patients with end-stage cardiomyopathy (Narula et al., 1996). A cascade of caspases activation leads to cell apoptosis. Such event is usually mediated through either receptor or mitochondriondependent mechanisms. Receptor binding mediated apoptosis includes TNF␣ pathway in which cytokine TNF␣ directly activates TNFRI on cell surface and triggers cell apoptosis (Chen and

Goeddel, 2002). Serum elevation of TNF␣ is observed in many human cardiac related pathogenic conditions. Since TNF␣ can be produced by myocardium although the cell type was not clearly defined, local concentrations of TNF␣ in cardiac tissue in these pathogenic conditions are considered to be high (Kleinbongard et al., 2011). Previous studies have shown cardiomyocyte expressing functional TNFR1 can undergo apoptosis after stimulation with TNF␣ in vitro (Shanmugam et al., 2016). Our study confirms this finding and shows low dose TNF␣ (10 ng/ml, 24 h) is sufficient to trigger cultured primary rat cardiomyocyte apoptosis as measured in Annexin V/PI staining. Moreover, many studies have been exploring the strategies by targeting TNF␣ mediated apoptotic pathway to confer the protection on cardiomyocytes. For instance, Leptin, an adipose-derived hormone, is found to exert a protective effect on cardiomyocytes exposed to TNF␣. Authors further showed that apoptotic effect in cardiomyocytes is associated with TNF␣ induced p38 and NFkB activation which can be inhibited by Leptin (Yu et al., 2014). Another example is oleic acid, a major component of olive oil. In this study, TNF␣ treated rat cardiomyocytes showed significant increase of oxidative stress, apoptosis and the expression of apoptotic proteins Bax, Caspase 3 and PARP cleavage, whereas cotreatment of oleic acid attenuated these changes, suggesting the beneficial effects of oleic acid on TNF␣ induced oxidative stress, apoptosis and cell damage (Al-Shudiefat et al., 2013). Therefore to elucidate the molecular mechanism of TNF␣ mediated apoptosis in cardiomyocytes will provide more therapeutic targets or protection strategies to protect cardiac tissues from inflammation or oxidative stress induced damages. AIF was discovered in 1999 as an apoptosis effector (Susin et al., 1999). It is associated with the outer mitochondrial membrane and translocate into nucleus after an apoptotic insult, where it induces DNA fragmentation and chromatin condensation that resemble the key apoptotic phenotypes (Daugas et al., 2000). This effector pathway is triggered in many models of regulated cell death in a caspase cascade independent mechanism (Cande et al., 2002). In some pathological apoptosis models, AIF mediated apoptosis is particularly important. For instance, AIF activation has been observed

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in acute neuronal apoptosis induced by trauma, hypoglycemia or transient ischemia (Cande et al., 2002). AIF mediated apoptosis has also been reported being involved for death of cortical neurons induced by exposure to heat-inactivated Streptococcus pneumonia (Braun et al., 2001), hydrogen peroxide, peroxynitrite (Zhang et al., 2002), or the excitotoxin N-methyl-d-aspartate (NMDA) (Yu et al., 2002). However, the role of AIF in TNF␣ mediated apoptosis is less studied. For the first time in rat primary cardiomyocytes we show that AIF expression is regulated by TNF␣. It will be intriguing to further study the AIF activation, translocation and the therapeutic implication of inhibiting AIF in TNF␣ pathway. Cardiomyocytes loss is known to contribute to cardiac injury associated diseases. This suggests TNF␣ induced AIF mediated apoptosis may participate the pathogenesis of heart injury induced by inflammation. MiR-186 has been characterized in various tumor models and cancer studies. MicroRNA expression study in patients suffering from oral squamous cell carcinoma (OSCC) reveals a downregulation of miR-186 in whole blood samples, which suggests it as a potential cancer biomarker in OSCC (Ries et al., 2014). Expression of miR-186 is also found being suppressed in human non-small cell lung cancer. In vitro study shows over-expressing of miR-186 suppressed lung cancer cell proliferation, migration and invasion through targeting MAP3K2 (Huang et al., 2016). Epithelial-Mesenchymal Transition (EMT) has an established role in the acquisition of therapeutic resistance in tumor tissues. Such EMT phenotype was detected in cisplatin-resistant ovarian cancer tissues and cells with a correlation with decreased miR-186 expression. Over-expressing miR-186 in epithelial ovarian cancer cells reversed EMT phenotype, enhanced cell apoptosis and eventually rendered the cells more sensitive to cisplatin in vitro and in vivo (Yao et al., 2015; Zhu et al., 2016). This implicated miR186 as a potential candidate for overcoming chemoresistance in cancer therapy. Collectively, all these studies reveals miR-186 may function as a tumor suppressor in various malignancies. In our study, miR-186 is found being downregulated by TNF␣ in primary cardiomyocytes. The downregulation of miR-186 leads to the upregulation of AIF that subsequently promotes cell apoptosis. Further on, we validated AIF as a target of miR-186 with luciferase reporter assay incorporated with AIF 3 UTR and miR-186 mimic expression study. The rescue experiment also supported miR-186 expression protects cells from TNF␣ mediated apoptosis in cardiomyocytes. Therefore, our study suggests miR-186 may have a more complex role in cells, which is determined by its regulatory targets in pathways or specific cell types. In cancer models, miR186 acts as tumor suppressor and its expression suppresses cell proliferation and promotes apoptosis. The proposed target genes of miR186 in cancer model include MAP3K2 in non-small cell lung cancer (Huang et al., 2016), Twist1 in epithelial ovarian cancer cells (Yao et al., 2015) and NSBP1 in human bladder cancer tissue (Yao et al., 2015). In contrast, in non-cancerous cells, miR-186 regulates different target genes such as AIFM1 in cardiomyocytes. AIF is cell apoptosis inducer and decreases in response to TNF␣ stimulation in our model. The increased AIF in cardiomyocytes is associated the downregulation of miR-186 by TNF␣. As a result, miR-186 expression negatively regulates TNF␣ mediated cardiomyocytes apoptosis. More experiments are required to elucidate the fundamental mechanism that determines target specificity of miR-186 in different cell types. From the therapeutic aspect, miR-186 may represent a new candidate to rescue cell death caused by inflammation in cardiovascular system. To validate this hypothesis, it will be important to confirm the pro-apoptotic role of miR-186 in inflammation-mediated cardiac injury mouse model. And it is also important to examine the miR-186 expression in patients with heart diseases such as myocardial infarction to confirm therapeutic value of miR-186 in protecting or treating cardiac failure.

Funding None. Conflicts of interest The authors declare that they have no conflict of interest. Acknowledgement None. References Al-Shudiefat, A.A., Sharma, A.K., Bagchi, A.K., Dhingra, S., Singal, P.K., 2013. Oleic acid mitigates TNF-alpha-induced oxidative stress in rat cardiomyocytes. Mol. Cell Biochem. 372, 75–82. Bajaj, G., Sharma, R.K., 2006. TNF-alpha-mediated cardiomyocyte apoptosis involves caspase-12 and calpain. Biochem. Biophys. Res. Commun. 345, 1558–1564. Bartel, D.P., 2004. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297. Braun, J.S., Novak, R., Murray, P.J., Eischen, C.M., Susin, S.A., Kroemer, G., Halle, A., Weber, J.R., Tuomanen, E.I., Cleveland, J.L., 2001. Apoptosis-inducing factor mediates microglial and neuronal apoptosis caused by pneumococcus. J. Infect. Dis. 184, 1300–1309. Cande, C., Cecconi, F., Dessen, P., Kroemer, G., 2002. Apoptosis-inducing factor (AIF): key to the conserved caspase-independent pathways of cell death? J. Cell Sci. 115, 4727–4734. Chen, G., Goeddel, D.V., 2002. TNF-R1 signaling: a beautiful pathway. Science 296, 1634–1635. Dai, R., Ahmed, S.A., 2011. MicroRNA, a new paradigm for understanding immunoregulation, inflammation, and autoimmune diseases. Transl. Res. 157, 163–179. Daugas, E., Susin, S.A., Zamzami, N., Ferri, K.F., Irinopoulou, T., Larochette, N., Prevost, M.C., Leber, B., Andrews, D., Penninger, J., Kroemer, G., 2000. Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis. FASEB J. 14, 729–739. Engel, D., Peshock, R., Armstong, R.C., Sivasubramanian, N., Mann, D.L., 2004. Cardiac myocyte apoptosis provokes adverse cardiac remodeling in transgenic mice with targeted TNF overexpression. Am. J. Physiol. Heart Circ. Physiol. 287, H1303–H1311. Farazi, T.A., Hoell, J.I., Morozov, P., Tuschl, T., 2013. MicroRNAs in human cancer. Adv. Exp. Med. Biol. 774, 1–20. Finkel, T., 2003. Oxidant signals and oxidative stress. Curr. Opin. Cell Biol. 15, 247–254. Franke, W.W., Schumacher, H., Borrmann, C.M., Grund, C., Winter-Simanowski, S., Schlechter, T., Pieperhoff, S., Hofmann, I., 2007. The area composita of adhering junctions connecting heart muscle cells of vertebrates—III: assembly and disintegration of intercalated disks in rat cardiomyocytes growing in culture. Eur. J. Cell Biol. 86, 127–142. Haunstetter, A., Izumo, S., 1998. Apoptosis: basic mechanisms and implications for cardiovascular disease. Circ. Res. 82, 1111–1129. Huang, T., She, K., Peng, G., Wang, W., Huang, J., Li, J., Wang, Z., He, J., 2016. MicroRNA-186 suppresses cell proliferation and metastasis through targeting MAP3K2 in non-small cell lung cancer. Int. J. Oncol. 49, 1437–1444. Kang, P.M., Izumo, S., 2000. Apoptosis and heart failure: a critical review of the literature. Circ. Res. 86, 1107–1113. Kleinbongard, P., Schulz, R., Heusch, G., 2011. TNFalpha in myocardial ischemia/reperfusion, remodeling and heart failure. Heart Fail. Rev. 16, 49–69. Kocher, A.A., Schuster, M.D., Szabolcs, M.J., Takuma, S., Burkhoff, D., Wang, J., Homma, S., Edwards, N.M., Itescu, S., 2001. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat. Med. 7, 430–436. Lee, R.C., Feinbaum, R.L., Ambros, V., 1993. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75, 843–854. Lin, L., Wu, X.D., Davey, A.K., Wang, J., 2010. The anti-inflammatory effect of baicalin on hypoxia/reoxygenation and TNF-alpha induced injury in cultural rat cardiomyocytes. Phytother. Res. 24, 429–437. Moe, G.W., Marin-Garcia, J., Konig, A., Goldenthal, M., Lu, X., Feng, Q., 2004. In vivo TNF-alpha inhibition ameliorates cardiac mitochondrial dysfunction, oxidative stress, and apoptosis in experimental heart failure. Am. J. Physiol. Heart Circ. Physiol. 287, H1813–H1820. Narula, J., Haider, N., Virmani, R., DiSalvo, T.G., Kolodgie, F.D., Hajjar, R.J., Schmidt, U., Semigran, M.J., Dec, G.W., Khaw, B.A., 1996. Apoptosis in myocytes in end-stage heart failure. N. Engl. J. Med. 335, 1182–1189. Nian, M., Lee, P., Khaper, N., Liu, P., 2004. Inflammatory cytokines and postmyocardial infarction remodeling. Circ. Res. 94, 1543–1553. Pillai, R.S., 2005. MicroRNA function: multiple mechanisms for a tiny RNA? RNA 11, 1753–1761.

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Ries, J., Vairaktaris, E., Agaimy, A., Kintopp, R., Baran, C., Neukam, F.W., Nkenke, E., 2014. miR-186, miR-3651 and miR-494: potential biomarkers for oral squamous cell carcinoma extracted from whole blood. Oncol. Rep. 31, 1429–1436. Shanmugam, G., Narasimhan, M., Sakthivel, R., Kumar, R.R., Davidson, C., Palaniappan, S., Claycomb, W.W., Hoidal, J.R., Darley-Usmar, V.M., Rajasekaran, N.S., 2016. A biphasic effect of TNF-alpha in regulation of the Keap1/Nrf2 pathway in cardiomyocytes. Redox Biol. 9, 77–89. Sugano, M., Koyanagi, M., Tsuchida, K., Hata, T., Makino, N., 2002. In vivo gene transfer of soluble TNF-alpha receptor 1 alleviates myocardial infarction. FASEB J. 16, 1421–1422. Susin, S.A., Lorenzo, H.K., Zamzami, N., Marzo, I., Snow, B.E., Brothers, G.M., Mangion, J., Jacotot, E., Costantini, P., Loeffler, M., Larochette, N., Goodlett, D.R., Aebersold, R., Siderovski, D.P., Penninger, J.M., Kroemer, G., 1999. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397, 441–446. Xiao, T., Zhang, Y., Wang, Y., Xu, Y., Yu, Z., Shen, X., 2013. Activation of an apoptotic signal transduction pathway involved in the upregulation of calpain and apoptosis-inducing factor in aldosterone-induced primary cultured cardiomyocytes. Food Chem. Toxicol. 53, 364–370.

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Yao, K., He, L., Gan, Y., Zeng, Q., Dai, Y., Tan, J., 2015. MiR-186 suppresses the growth and metastasis of bladder cancer by targeting NSBP1. Diagn. Pathol. 10, 146. Yu, S.W., Wang, H., Poitras, M.F., Coombs, C., Bowers, W.J., Federoff, H.J., Poirier, G.G., Dawson, T.M., Dawson, V.L., 2002. Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor. Science 297, 259–263. Yu, L., Zhao, Y., Xu, S., Jin, C., Wang, M., Fu, G., 2014. Leptin confers protection against TNF-alpha-induced apoptosis in rat cardiomyocytes. Biochem. Biophys. Res. Commun. 455, 126–132. Zhang, X., Chen, J., Graham, S.H., Du, L., Kochanek, P.M., Draviam, R., Guo, F., Nathaniel, P.D., Szabo, C., Watkins, S.C., Clark, R.S., 2002. Intranuclear localization of apoptosis-inducing factor (AIF) and large scale DNA fragmentation after traumatic brain injury in rats and in neuronal cultures exposed to peroxynitrite. J. Neurochem. 82, 181–191. Zhu, X., Shen, H., Yin, X., Long, L., Xie, C., Liu, Y., Hui, L., Lin, X., Fang, Y., Cao, Y., Xu, Y., Li, M., Xu, W., Li, Y., 2016. miR-186 regulation of Twist1 and ovarian cancer sensitivity to cisplatin. Oncogene 35, 323–332.

Please cite this article in press as: Xu, H., et al., TNF␣-induced downregulation of microRNA-186 contributes to apoptosis in rat primary cardiomyocytes. Immunobiology (2017), http://dx.doi.org/10.1016/j.imbio.2017.02.005