Introduction

Acute myocardial infarction (AMI) is a major cause of death in patients with cardiovascular disease [1]. Myocardium subjected to hypoxia and ischemia experiences a series of physical and chemical changes as well as inflammation, apoptosis, necrosis, scarring, and myocardial remodeling [2]. Indeed, accumulating evidence has highlighted the importance of inflammatory response and cardiomyocyte apoptosis in AMI pathogenesis, as they are involved in mediating impaired myocardial function and heart failure [2, 3]. Pro-inflammatory mediators are upregulated in cardiac dysfunction, and, in particular, elevated TNF-α levels in the local infarct myocardium contribute to acute myocardial dysfunction and cause myocardial cell apoptosis [4]. Thus, therapeutic approaches that target components of the inflammatory response and cardiomyocyte apoptosis have been investigated as potential and useful treatments for ischemic myocardial injury.

CD40 is a transmembrane type I receptor that belongs to the tumor necrosis factor receptor (TNFR) family, and is expressed on the surface of dendritic cells and macrophages/monocytes [5, 6]. Apart from immune cells, CD40 has also been identified on endothelial cells [7], hepatocytes [8], epithelial cells, and myocytes [9]. The widespread expression of CD40 accounts for the key role of this co-stimulatory molecule in the regulation of immune response and host defense. Activation of CD40 through ligation to CD40 ligand (CD40L, CD154) induces multiple effects, including the secretion of interleukins, chemokines, and adhesion molecules, which results in the recruitment and activation of immune cells [9]. The CD40/CD40L interaction was found to be crucial in several inflammatory and autoimmune diseases. Aberrant CD40/CD40L signaling in the vasculature is associated with atherosclerosis [10, 11] and myocardial infarction. Recently, it has been shown that CD40L-CD40 signaling modulates inflammatory responses triggered by hepatic I/R injury [12].

MicroRNAs (miRNAs) are an abundant class of highly conserved small (approximately 22 nucleotides) noncoding RNAs which play critical roles in regulating gene expression by binding directly to the 3′-UTR of their target gene mRNA, leading to translational repression or degradation [13]. Additionally, miRNAs are crucial regulators involved in modulating a variety of biological pathways involved in development, growth, homeostasis, immune regulation, and disease progression. Compelling evidence indicates that miRNAs have been involved in the pathogenesis of a number of cardiovascular diseases including AMI [14–16]. Accumulating studies have demonstrated that miR-145 plays a critical role in various cancer cells, including breast cancer [17], esophageal squamous cell carcinoma [18], and bladder cancer [19]. A previous study has indicated that miR-145 is down-regulated in dedifferentiated VSMCs, and is also a critical modulator of VSMC phenotype and proliferation [20, 21]. Additionally, overexpression of miR-143/145 promotes cells differentiation and inhibits the proliferation of VSMCs [22]. A recent report also demonstrated that miR-145 in the heart is rapidly decreased after AMI [23]. MiR-145 was significantly down-regulated in both ischemic heart and cardiac fibroblasts in response to hypoxia [24]. However, the molecular mechanisms involved in hypoxia-induced inflammatory response, and the apoptosis of cardiomyocytes are poorly understood.

In the present study, we took advantage of an in vitro model of cardiomyocyte culture, and determined the biological effects of miR-145-5p in hypoxia-induced inflammation and cardiomyocyte apoptosis to investigate the underlying mechanisms, providing the theoretical support to clarify the role of miR-145-5p in hypoxia of myocardial ischemia.

Materials and methods

Cell culture and hypoxia treatment

H9c2 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA,USA) and were maintained in growth medium composed of Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Rockville, MD, USA) supplemented with fetal bovine serum (FBS, Invitrogen, USA), 100 U/mL of penicillin and 100 mg/mL streptomycin (Sigma, St. Louis, MO, USA), which was cultured in a 5% CO2 humidified atmosphere at 37 °C. The medium was changed every 2 d. After the cells reached 80% confluence, cells were placed in a hypoxia chamber (Thermo, Dreieich, Germany) containing a gas mixture of 94% N2, 5% CO2, and 1% O2 in a humidified incubator (Thermo Fisher Scientific, Waltham, MA) for 48 h.

Cell transfection

H9c2 cells were transfected with miR-145-5p mimic or negative control (NC) (SunBio Medical Biotechnology, China) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. After incubation for 48 h, the transduction efficiency was measured by qRT-PCR.

Quantitative RT-PCR

Total RNA was extracted from cardiomyocytes using TRIzol reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. The concentration of total RNA was determined using an Ultraviolet Spectrophotometer (Eppendorf, Hamburg, German). cDNA was synthesized using the MMLV Reverse Transcriptase kit (Takara Biotechnology, Dalian, China) following the manufacturer’s protocol. Real-time PCR was performed using SYBR Green I (Applied Biosystems, USA), and carried out on an ABI Prism 7700 analyzer (Applied Biosystems, Warrington, UK). U6 and GAPDH were used to normalize the relative abundance of miRNA and mRNA, respectively. The relative expression levels were calculated via the \({{2}^{-\Delta \Delta {{\text{C}}_{t}}}}\) method, and all experiments were analyzed in triplicate.

Western blot analysis

Total proteins were extracted from cultured H9c2 cells using RIPA (Beyotime, Nantong, China), and the concentration was quantified using the BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Equal amounts of protein were separated on 10% SDS-PAGE, and then transferred to a PVDF membrane (Millipore, Billerica, MA). After blocking with 5% nonfat milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) at room temperature for 1 h, the membrane was subsequently incubated with appropriate primary antibodies at 4 °C overnight. The membrane was washed in TBST three times and then incubated with secondary horseradish peroxidase (HRP)-conjugated antibodies (Santa Cruz Biotechnology) at room temperature for 1 h. The signals were developed with enhanced chemiluminescence (ECL; Advansta, Manlo Park, Calif., USA) and visualized on X-ray films. The images were analyzed using Image J software (National Institutes of Health (NIH), Bethesda, MD, USA). The relative protein expression levels were normalized to GAPDH.

MTT assay

Cell survival was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Beyotime, Shanghai, China). Briefly, H9c2 cells transfected with miR-145-5p mimic or negative control (NC) were cultured in a 96-well plates. After incubation under hypoxic conditions for indicated times, MTT (5 mg/mL, Sigma) were added into each well. The plates were cultured for an additional 4 h before 100 μL dimethyl sulfoxide/well (DMSO, Sigma) was added. Absorbance was measured at 490 nm with an automatic microplate reader (Bio-Tek, Winooski, VA, USA). The experiment was repeated three times.

ELISA analysis

The supernatants were collected following the establishment of the hypoxia model, and centrifuged at 3,000 rpm for 15 min before being analyzed using corresponding ELISA kits (R&D Systems, Minneapolis, Minn., USA) according to the manufacturer’s instructions. The experiments were performed three times.

Cell death assessment by DNA fragmentation assay

Apoptotic activity was assessed by detecting fragmented DNA using the Cell Death Detection ELISAPLUS kit (Boehringer Mannheim, Indianapolis, IN, USA) according to the manufacturer’s instructions. Briefly, each culture plate was centrifuged for 10 min at 200×g, the supernatant was removed, and the pellet was lysed for 30 min. After centrifuging the plate again at 200×g for 10 min, and the supernatant that contained the cytoplasmic histone-associated DNA fragments was collected and incubated with an immobilized anti-histone antibody. The reaction products were incubated with a peroxidase substrate for 5 min and measured by spectrophotometry at 405 and 490 nm with a microplate reader (BMG Labtech, Ortenberg, Germany). The signals in the wells containing the substrate alone were subtracted as the background.

Luciferase reporter assay

Luciferase activity assays were performed using the Dual-Luciferase Reporter Assay System (Promega Madison, AL, USA). The cells were co-transfected the pGL3 luciferase reporter vector (Promega, Madison, WI, USA) containing the 3′-UTR of CD40 mRNA or mutated forms (3′-UTR-Mut) with miR-145-5p mimic or negative control (NC) using Lipofectamine 2000 (Invitrogen). After 48 h, cells were harvested and lysed, then the luciferase activity was measured using the Dual-Luciferase reporter assay kit (Promega) according to the manufacturer’s instruction. All experiments were performed at least three times. The relative luciferase activities were normalized to the Renilla control luciferase activity of the controls.

Statistical analysis

All data were presented as the mean ± SD of three independent experiments. Results were analyzed using the SPSS 17.0 software (SPSS, Chicago, IL, USA) and GraphPad Prism 5 Software (San Diego, California, USA). Student’s t tests were used for the comparison of differences between two independent groups. Differences among multiple groups were analyzed using one-way ANOVA. P < 0.05 was considered to indicate statistical significance.

Results

Aberrant expression of miR-145-5p and ALDH2 in cardiomyocytes under hypoxia condition

In an effort to identify the role of miR-145-5p in AMI injury, we utilized H9c2 cell lines treated with hypoxia to simulate ischemia symptoms in vitro, and then evaluated the expression level of miR-145-5p in hypoxic injured cardiomyocytes. The results as shown in Fig. 1a, miR-145-5p was transiently declined in a time-dependent manner by hypoxic stimulation. These results were similar to previously published data [23, 24]. We further determined the expression pattern of CD40 in our hypoxia model. The results of qRT-PCR showed that the mRNA level of CD40 was markedly upregulated in H9c2 cells under hypoxic conditions (Fig. 1b). Western blot evaluations indicated that the protein expression of CD40 was also dramatically enhanced (Fig. 1c). These findings together demonstrated that hypoxia induced a gradual, time-dependent downregulation of miR-145-5p level, along with enhanced CD40 expression in cardiomyocytes, suggesting the potential involvement of miR-145-5p in the development of AMI injury. To examine this notion, H9c2 cardiomyocytes were transfected with miR-145-5p mimic or negative control. Transfection efficiency was confirmed by qRT-PCR assay, and the results showed that the expression level of miR-145-5p was dramatically increased after miR-145-5p mimic transfection (Fig. 1d).

Fig. 1
figure 1

MiR-145-5p and CD40 expression in hypoxia-stimulated cardiomyocytes. a Relative expression level of miR-145-5p was determined by RT-PCR analysis. b The mRNA expression of CD40 was detected using RT-PCR. c The protein level of CD40 was assessed by western blot. d qRT-PCR was carried out to evaluate the transfection efficiency of miR-145-5p in H9c2 cells. MiR-145-5p inhibited hypoxia-induced inflammatory factor production in H9c2 cells. e The relative mRNA levels of IL-1β, TNF-α, and IL-6 were detected using RT-PCR analysis. f The concentrations of IL-1β, TNF-α, and IL-6 measured by ELISA assay. The data were presented as mean ± SD from three separate experiments. *P < 0.05 vs. Untreated, # P < 0.05 vs. Negative control (NC)

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Overexpression of miR-145-5p suppresses hypoxia-induced inflammatory response in H9c2 cells

We next sought to assess the effects of miR-145-5p on cytokine secretion known to play roles in hypoxia-induced inflammation, such as IL-1β, TNF-α, and IL-6. RT-PCR results showed that levels of these inflammatory cytokines were dramatically enhanced following hypoxia stimulation. However, this kind of situation was totally abolished by the addition of miR-145-5p (Fig. 1e). Additionally, the concentrations of these inflammatory cytokines were further measured to confirm the above result using an ELISA assay; the protein expression of cytokines displayed a similar variation tendency of mRNA expression in the hypoxia-injured H9c2 cells (Fig. 1f). These findings indicated that overexpression of miR-145-5p suppressed hypoxia-induced inflammatory response in cardiomyocytes.

Introduction of miR-145-5p protects hypoxia-induced apoptosis in H9c2 cells

To determine the influence of miR-145-5p on cell viability in the setting of acute hypoxia, the MTT assay was subsequently used to analyze the living cells. The data in Fig. 2a implied that cell viability was decreased in H9c2 cells subjected to hypoxia, while it was significantly reversed by the overexpression of miR-145-5p. We further investigated the effect of miR-145-5p on cell apoptosis using the Cell Death Detection ELISAPLUS kit, which measures cytoplasm-associated DNA fragments. The results as shown in Fig. 2b, hypoxia induced cytoplasmic histone-associated DNA fragments, whereas the introduction of miR-145-5p markedly attenuated hypoxia-induced DNA fragments (Fig. 2b). To further examine the anti-apoptotic effect of miR-145-5p, we measured the expression levels of apoptosis downstream targets, including the anti-apoptotic protein Bcl-2 as well as the pro-apoptotic protein Bax. As predicted, western blot assay revealed that miR-145-5p was able to significantly increase the expression of Bcl-2 and decrease the expression of Bax (Fig. 2c, d). In addition, we used western blot for active Caspase-3 and Caspase-9 in H9c2 cells as an indicator of the degree of apoptosis. As shown in Fig. 2e, f, the protein expression levels of Caspase-3 and Caspase-9 were increased in hypoxia-induced cells, which were down-regulated after miR-145-5p mimic transfection. These results collectively suggest that miR-145-5p plays an anti-apoptotic role in cardiomyocytes with hypoxia-induced damage.

Fig. 2
figure 2

MiR-145-5p rescued cardiomyocyte apoptosis under hypoxic conditions, a MTT assay was used to detected cell viability. b The cell apoptosis was measured using the Cell Death Detection ELISAPLUS kit. Representative western blotting and quantitative analysis showing the protein levels of Bax and Bcl-2 (c, d), Caspase-3 and Caspase-9 (e, f). GAPDH was used as the loading control. Data were shown as mean ± SD. from three independently experiments. *P < 0.05 vs. Untreated, # P < 0.05 vs. NC

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MiR-145-5p directly targets CD40 and decreases its expression in hypoxia-induced H9c2 cells

MiRNAs achieve their biological functions via multiple target genes. In the next step of our work, we tried to explore the potential target gene of miR-145-5p in cardiomyocytes by hypoxia treatment. We identified the potential target of miR-145-5p using bioinformatics tools in several databases (TargetScan, PicTar, and miRanda). Bioinformatics analysis verified that CD40, which is known to serve a critical role in inflammation and apoptosis, was predicted to be one of the target genes (Fig. 3a). To experimentally validate whether CD40 is indeed directly regulated by miR-145-5p, we carried out a dual-luciferase reporter assay. As illustrated in Fig. 3b, miR-145-5p strongly suppressed the luciferase activity of the wild-type (WT) CD40-3′-UTR reporter gene, whereas no effect was observed with the corresponding mutant (Mut) reporter. We further investigated the regulation of miR-145-5p on the expression of CD40 in cardiomyocytes exposure to hypoxia. RT-PCR and western blot analysis were performed to assess the expression level of CD40, and the results showed that both mRNA (Fig. 3c) and protein (Fig. 3d, e) levels of CD40 were remarkably upregulated in cardiomyocytes under hypoxic conditions, which was apparently abrogated by the overexpression of miR-145-5p. Our data, therefore, suggest that miR-145-5p binds directly to the 3′-UTR of CD40 to repress its expression in hypoxia-stimulated cardiomyocytes.

Fig. 3
figure 3

CD40 is a direct target gene of miR-145-5p in H9c2 cells subjected to hypoxia. a 3′-UTR of the CD40 gene is predicted to be bound by miR-145-5p. b The interaction between the miR-145-5p and 3′-UTR of CD40 mRNA detected by Dual-Luciferase Reporter Assay. c qPCR assay was performed to evaluate the mRNA expression of CD40. d The level of CD40 protein was determined using western blot. e Statistical analyses of CD40 protein. The data were expressed as means ± SD from three independent experiments. *P < 0.05 vs. Untreated, # P < 0.05 vs. NC

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CD40 contributes to the effect of miR-145-5p on hypoxia-induced inflammatory response and cell apoptosis

The above results indicated that miR-145-5p has anti-apoptotic and anti-inflammatory roles in hypoxia-induced cardiomyocytes; therefore, we questioned whether CD40 has a functional role. To investigate the biological importance of CD40 as a target of miR-145-5p, we depleted the CD40 protein by siRNA and assayed the effect of miR-145-5p inhibition. The elimination of CD40 expression by siRNA treatment efficiently repressed the CD40 expression at both the mRNA (Fig. 4a) and protein level (Fig. 4b). Moreover, depletion of CD40 notably decreased the secretion of inflammatory cytokines IL-1β, TNF-α, and IL-6 (Fig. 4c), indicating that CD40 is involved in the process of inflammatory response under hypoxic conditions. Meanwhile, silencing of CD40 remarkably suppressed cell apoptosis induced by hypoxia treatment (Fig. 4d), which also effectively down-regulated Bax expression while enhancing Bcl-2 expression (Fig. 4e, f), thus suggesting a protective effect of CD40 in hypoxia-stimulated cardiomyocytes. These effects are similar to those of miR-145-5p on hypoxia-induced inflammatory response and cell apoptosis. Taken together, our data demonstrate that miR-145-5p contributes to hypoxia-induced inflammatory response and apoptosis by blocking the expression of CD40.

Fig. 4
figure 4

CD40 contributes to the effect of miR-145-5p on inflammatory response and cell apoptosis in hypoxia-injured H9c2 cells. The mRNA and protein expression level were detected using RT-PCR, a and western blotting (b, c), respectively. d The cell apoptosis were measured using Cell Death Detection ELISAPLUS kit. e Representative western blotting showing the protein levels of Bax and Bcl-2. f Quantitative results. Values are presented as mean ± SD. At least three independent experiments were performed. *P < 0.05 vs. Untreated, # P < 0.05 vs. si-NC.

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Discussion

The continuous inflammatory response and necrosis of ischemic tissue are two of the most marked characteristics that could mutually enhance during the process of MI-induced heart damage, eventually leading to heart failure. Therefore, elucidating the molecular mechanisms in cardiomyocytes is of great importance for the establishment of therapeutic strategies against ischemic heart disease.

Accumulating evidence pinpoints a critical role of miRNAs in ischemic heart disease. Important work to date has focused on the role of miR-145-5p in various cancers. However, it is not clear whether these processes contribute meaningfully to repair of the human heart, so the physiologic significance of altered miR-145-5p levels in conditions such as ischemic damage remains to be elucidated. To further evaluate the effect and underlying mechanism of miR-145-5p in AMI, we used hypoxia-induced H9c2 cells as a cell model of ischemic injury to investigate the specific roles of miR-145-5p in inflammatory responses and cardiomyocyte apoptosis in response to hypoxia. In this study, we demonstrated that a hypoxic environment directly induces an inflammatory response and myocardial cell apoptosis. Recently, it has been reported that hypoxia stimulation led to the downregulation of miR-145-5p in cardiac fibroblasts, but has no effect on aortic smooth muscle cells [24]. In this regard, we first determined the expression levels of miR-145-5p and CD40 in hypoxia-stimulated cardiomyocytes, and observed a decreased level of miR-145-5p accompanied by the up-regulation of CD40 in the setting of hypoxia. These results are in agreement with previous studies showing that miR-145 in the heart is rapidly decreased after AMI [23]. Above all, these findings indicate that miR-145-5p may have the potential to become a biomarker for AMI.

Increasing evidence indicates that AMI is accompanied by inflammatory responses. The inflammatory response immediately triggered by AMI is a key determinant for restoring homeostasis in an ischemic myocardial injury [3]. The increased expression of a variety of endogenous inflammatory cytokines can lead to myocardial dysfunction. Our results are identical to those of a previous study, which demonstrated that hypoxia aggravated inflammatory response as reflected by an increase in the secretion of cytokines IL-1β, TNF-α, and IL-6. It is noteworthy that the introduction of miR-145-5p efficiently alleviated hypoxia-induced inflammatory factor production. These findings suggest that miR-145-5p may play an anti-inflammatory role to protect H9c2 cells from ischemic injury.

Cardiomyocyte apoptosis is a key cellular event of ischemic/hypoxic cardiomyopathy [25]. It has been demonstrated in the literature that AMI is defined as myocardial cell death following severe ischemia. Inflammation is an integral part of the response to any kind of cell death. Cardiomyocyte apoptosis induced by hypoxia is an important pathological phenomenon in the heart [26]. In the current study, we observed that hypoxia-induced cardiomyocyte apoptosis accompanied by a decrease in the expression of Bcl-2 and an increase in the expression of Bax, Caspase-3, and Caspase-9. However, overexpression of miR-145-5p led to a striking decrease in cardiomyocyte apoptosis following exposure to acute hypoxia. Cardiomyocyte apoptosis significantly influenced the prognosis of ischemic heart disease and heart failure [27]; hence, the present findings implicate that miR-145-5p may play an important role in the progression of ischemic/hypoxic cardiomyopathy and may represent a potential therapeutic target. These observations demonstrate that miR-145-5p inhibits apoptosis, which is also consistent with the reduction of inflammation. Together, all these findings demonstrate that the augmentation of miR-145-5p attenuated an inflammatory response, indicating a decreased accumulation of inflammatory cytokines, thereby resulting in a reduction in the deleterious effects of inflammation and apoptosis in hypoxia-injured cardiomyocytes.

Numerous studies have indicated the vital role of the CD40/CD40L system in inflammation-associated disease. Activation of CD40 through ligation to CD40L induces multiple effects, including the secretion of pro-inflammatory molecules. CD40 serves as a link between inflammation, atherosclerosis, and thrombosis. Subsequent research studies have demonstrated that CD40 was enhanced in adult HL-1 cardiomyocytes after IFN-γ-stimulation [9]. It also has been demonstrated that CD40 activation induces apoptosis [8, 28]. It might also be possible that miR-145-5p works in the finer regulation of hypoxia-induced inflammation and apoptosis. Future studies may be directed to exploit these possibilities. Based on a computational analysis, we predicted that CD40 was a direct target of miR-145-5p. Then, the dual-luciferase reporter system was constructed to confirm that the introduction of miR-145-5p remarkably repressed the luciferase activity of CD40-3′-UTR reporter vector. Moreover, the overexpression of miR-145-5p notably down-regulated CD40 at both mRNA and protein expression levels under hypoxic conditions. These observations are consistent with a recent study showing that miR-145-5p is directly targets CD40 [29–31]. Another important finding in the present study is that the downregulation of CD40 may confer the anti-inflammatory and anti-apoptotic properties of miR-145-5p. It is interesting to note that the inhibition of CD40 by CD40-siRNA alleviated cardiac dysfunction by suppressing inflammatory responses and apoptosis; these effects are similar to those of miR-145-5p on hypoxia-induced inflammation and cell apoptosis. Thus, it is reasonable to get the conclusion that miR-145-5p mediates the protective role in apoptosis and inflammation response following hypoxic treatment by targeting CD40.

In conclusion, our data highlight the protective role of miR-145-5p inflammatory response and apoptosis in the setting of acute hypoxia treatment, possibly by targeting CD40. Based on our results, miR-145-5p may serve as a potential diagnostic marker and therapeutic target for ischemic heart disease, although further study of the in vivo effect is needed.