MiR-489 aggravates H2O2-induced apoptosis of cardiomyocytes via inhibiting IGF1

Abstract Myocardial infarction (MI) is a major type of cardiovascular disorder worldwide. In the present study, we established a new microRNA (miRNA)–mRNA cross-talk network by integrating data obtained from The National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO). In addition, functional assays, including Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) analyses, were conducted using the Database for Annotation, Visualization, and Integration Discovery (DAVID). In our study, we generated a new differentially expressed miRNA (DEmiRNA)-differentially expressed gene (DEG) cross-talk network of MI composed of three miRNA (miR-489, miR-375, and miR-142-3p) nodes and 163 mRNA nodes. In vitro experiments demonstrated that miR-489 expression was increased in H2O2-treated H9c2 cardiomyocytes in vitro, mimicking myocardial injury. We observed that down-regulation of miR-489 reduced H2O2-induced apoptosis, while overexpression of miR-489 had the opposite effects, as revealed by flow cytometry and Western blot analyses. Furthermore, we confirmed the relationship between miR-489 and IGF1 through double luciferase reporter gene assays, which partly explains the antiapoptotic mechanism of miR-489. In conclusion, the experimental results of the present study could provide important clues for investigating the mechanism of MI.


1.Introduction
Myocardial infarction (MI) is a common and catastrophic cardiovascular disorder characterized by myocardial necrosis induced by long-term ischemia [1]. Acute MI (AMI) is the primary cause of cardiovascular disorders and is a highly common cause of mortality and disability [2]. Nearly 550,000 first-episode and 200,000 recurrent AMIs occur each year, causing major social and economic burdens on healthcare systems [3]. In the clinic, the diagnosis and treatment of AMI is primarily based on clinical symptoms.
At present, the clinical treatment of acute myocardial infarction primarily involves the recanalization of large blood vessels, which results in myocardial ischemia-reperfusion injury. There has been a great deal of work to elucidate the cause of such injuries, but reperfusion injury is still unavoidable once revascularization is performed [4,5,6].
Currently, although extensive efforts have been made to elucidate the molecular mechanisms that contribute to AMI [7,8,9,10], the etiology and pathogenesis of this condition remains unclear. Therefore, there is an urgent need to identify biomarkers and mechanisms for this condition for the prediction and treatment of MI.
MicroRNAs (miRNAs) are a type of small noncoding RNAs with fewer than 22 nucleotides that negatively regulate transcription by inhibiting protein translation or degradation. Increasing numbers of studies have shown that abnormal miRNA expression can lead to various cardiovascular disorders, including cardiac ischemia and vascular atherosclerosis [11,12]. For example, miR-342-5p in circulating exosomes induced by long-term exercise can have an endogenous cardioprotective role in Downloaded from https://portlandpress.com/bioscirep/article-pdf/doi/10.1042/BSR20193995/892296/bsr-2019-3995.pdf by guest on 09 September 2020 myocardial ischemia/reperfusion injury by inhibiting hypoxia/reoxygenation-induced cardiomyocyte apoptosis by targeting caspase 9 and Jnk2 and activating p-Akt signaling through regulation of Ppm1f [13].
The role of bioarray techniques in basic research has been increasingly recognized by scientists, as it allows for the identification of new genes and noncoding RNAs linked to multiple diseases, providing potential clues and scientific evidence for further studies using bioinformatics tools [14]. In the present study, the gene expression profile of GSE34198 and the miRNA expression profiles of GSE61741 and GSE31568 were obtained from the Gene Expression Omnibus (GEO) [15,16,17]. The limma package and metaMA package in R were used to identify differentially expressed (DE) genes (DEGs) and DE miRNAs (DEmiRNAs) of MI, respectively. Enrichment analyses were conducted to elucidate the functions of the DEGs in MI. We constructed a protein-protein interaction (PPI) network of the DEGs and defined the genes with a high values in the network as hub genes. We predicted the target genes of DEmiRNAs by searching the miRWalk database and establishing a new miRNA-mRNA regulatory network that is involved in the development and occurrence of MI; these findings will provide new insights for the diagnosis and treatment of MI.
We identified several DEmiRNAs associated with MI, including miR-489, miR-1274b, miR-142-3p, and miR-375. The results of a previous study proved that inhibition of both endothelial miR-92a and miR-489 can reduce chronic kidney disease-associated atherosclerosis, repressing the elevated expression of Fam220a and Downloaded from https://portlandpress.com/bioscirep/article-pdf/doi/10.1042/BSR20193995/892296/bsr-2019-3995.pdf by guest on 09 September 2020 Tgfb2, respectively [18]. In a rat model of gentamicin injury, elevation of miR-489 in urine preceded elevation of urinary creatinine and blood urea nitrogen, suggesting that urinary miR-489 could be a new biomarker of kidney injury [19]. Interestingly, intrarenal miR-489 and urinary miR-489 were both elevated in a kidney model of ischemia-reperfusion injury [20]. Previous research has shown that extracellular and cellular levels of miR-489-3p are related to cardiovascular and kidney disorders, but the function, mechanism and therapeutic potential of miR-489-3p in MI have not been reported. Therefore, this study, we generated an in vitro MI model using H9c2 cardiomyocytes to assess MI and verify the effects of miR-489 in myocardial damage.

Gene expression profile data
The gene expression profiles of GSE34198 and the miRNA expression profiles of GSE61741 and GSE31568 were obtained from the GEO database. In these GSEs, 10 samples were randomly selected for differential expression analysis.

Screening for DEGs and DEmiRNAs
The DEGs were identified using an empirical Bayesian method through the R "limma" package. The threshold of DEGs was defined with |logFC| > 1 and P-value < 0.05. In addition, the metaMA package, which can handle missing data and eliminate batch effects, was used to integrate the different platforms [21]. We used the limma and metaMA packages to confirm the DEmiRNAs, and |ES| > 6 and FDR < 0.05 was used to filter the identified DEmiRNAs.

Construction of a PPI network
To uncover the core regulatory genes, we used the STRING database [22] to construct a PPI network. These interaction networks were visualized using Cytoscape [23].

Prediction of target genes of DEmiRNAs
To predict targets of DEmiRNAs, we used the online database miRWalk. In addition, to screen for predicted target genes, we constructed a regulatory network between DEmiRNAs and DEGs.

H9c2 cell culture and treatment
Mouse H9c2 cardiomyocytes (Chinese Academy of Sciences, Shanghai, China) were cultured at 37°C in a humidified incubator under an atmosphere with 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum

Cell Counting Kit-8 (CCK8) assays
Cell viability was examined by CCK8 (Dojindo, Japan) assays. We seeded 100 μL of cell suspension per well into 96-well plates at a density of 5000 cells/well. At specific time points, we added 10 μL of CCK-8 solution to the cells and incubated the samples for 2 h at 37°C. Then, the reaction product was quantified following the manufacturer's instructions.

Western blot analysis
H9c2 cell extracts were prepared in RIPA lysis buffer (Suo Lai Bao, Beijing, China) containing 1% phenylmethanesulfonyl fluoride (PMSF). Total proteins were quantified with a BCA protein assay kit (Vazyme, China). The proteins were electrophoresed in sodium dodecyl sulfate (SDS)-polyacrylamide gels and then transferred to PVDF membranes (Millipore, MA, USA). The membranes were incubated for 2 h with 5% nonfat milk in Tris-buffered saline for blocking. Subsequently, the membranes were and anti-β-actin (1:5000, Sigma, A2228). Then, the membranes were incubated with the appropriate secondary antibodies for 2 h on a shaker at room temperature. The protein levels were quantified by relative densitometry and normalized to that of βactin as an internal control.

RNA extraction and RT-qPCR
The miR-489 levels in H9c2 cells were measured using RT-qPCR. After H2O2 treatment, the total RNA of the samples was extracted using TRIzol reagent (Tiangen, China), after which 1 μg of RNA was used to synthesize cDNA using a RevertAid First

TUNEL assay
The apoptosis of H9c2 cells was assessed using a one-step TUNEL assay kit (KeyGen, Nanjing, China). After being fixed with 2% formaldehyde and permeabilized on ice with 0.1% Triton X-100, samples were then incubated at 37°C with TUNEL reaction buffer according to the manufacturer's protocol. Subsequently, fluorescence microscopy was used to observe the apoptotic cells.

Cell apoptosis analysis
The apoptosis rate of cells was evaluated using an Apoptosis Detection Kit (Vazyme Biotech) following the manufacturer's instructions. The apoptotic rate was obtained by calculating the sum of the ratio of the right upper quadrant and the right lower quadrant.

Double luciferase reporter gene assay
The binding domain of miR-489 and the 3' untranslated region (UTR) of IGF1 was obtained from the online database miRWalk. The 3'-UTRs of IGF1 with wild-type and mutant binding sites for miR-489 were generated by RiboBio (Guangzhou, China) and cloned into pmiR-RB-REPORT (RiboBio). The vectors were cotransfected with miR-489 mimic and control into 293T cells, after which the cells were incubated for 48 h and then analyzed for luciferase activity. Next, the Dual-Glo® Luciferase Assay System (Promega, Madison, USA) was used to calculate the relative luciferase activity following the manufacturer's protocol

Statistical analysis
The results are presented as the means ± SEM. Statistical comparisons among different groups were conducted by one-way ANOVA, while differences between two groups were assessed by Student's t-test. P < 0.05 was defined as significant.

Screening for DEGs
We identified 688 DEGs (including 500 upregulated genes and 188 downregulated genes) from the gene expression datasets (criteria of P < 0.05 and |logFC| ≥ 1). In addition, we obtained 4 DEmiRNAs based on |combined ES| > 6 and FDR < 0.05 using the metaMA package and the miRNA expression dataset. The top 10 DEGs and DEmiRNAs are shown in Tables S1 and S2, respectively.

Functional analysis results
DAVID was used for GO functional analysis and KEGG pathway enrichment analysis. The GO analysis results indicated that the DEGs were primarily enriched in "histone acetyltransferase activity", "positive regulation of transcription from RNA polymerase II promoter", and "signal transduction involved in regulation of gene expression" ( Table S3). The primary KEGG pathways were "Cytokine-cytokine receptor interaction", "Glycosphingolipid biosynthesis -lacto and neolacto series", and "Intestinal immune network for IgA production" (Table S3).

Construction of a DEmiRNA-DEG crosstalk network
First, the targets of three DEmiRNAs were predicted from the miRWalk database,

MiR-489 is upregulated in H2O2-induced H9c2 cells
The viability of H2O2-treated H9c2 cells was assessed using a CCK8 kit. Cell viability decreased in dose-dependent manner and reached approximately 50% at 450 μm H2O2 (Fig. 2A). Therefore, we cultured H9c2 cells in 450 μm H2O2 for 1 h to simulate hypoxia induced by MI in vitro. As shown in Fig. 2B, the levels of proapoptotic proteins (cleaved caspase3 and Bax) were increased, whereas that of the anti-apoptotic protein Bcl-2 was decreased, demonstrating that H2O2 triggered cell injury. The RT-qPCR results effectively showed miR-489 expression in cardiomyocytes under normal or H2O2 conditions. As shown in Fig. 2C, miR-489 expression was increased in H9c2 cardiomyocytes treated with H2O2 compared to those cultured under normal conditions (P < 0.05), suggesting that miR-489 may be involved in mediating H2O2-induced H9c2 cell damage.

MiR-489 regulates H2O2-induced H9c2 cell apoptosis in vitro
To further elucidate the effect of miR-489 on H2O2-induced apoptosis, we silenced miR-489 with miRNA inhibitors. Fig. 3A shows that miR-489 expression in the miR-489 inhibitor group was lower than that observed in the NC group (P < 0.05). The results of Tunel assays (Fig. 3B), flow cytometry (Fig. 3C) and Western blot analyses to detect the expression of apoptosis-related proteins (Fig. 3D), including Bax, Bcl-2 and cleaved caspase 3, revealed that miR-489 downregulation decreased H2O2-induced

MiR-489 inhibits the expression IGF1 in H2O2-treated H9c2 cells
To investigate the effect of miR-489 on H2O2-induced apoptosis, we used miRWalk to predict candidate targets of miR-489 in H9c2 cells. The predicted binding site of the IGF1 3'-UTR and the complementary sequence of miR-489 are shown in Fig. 4A. Furthermore, our hypothesis was confirmed by dual luciferase reporter assays. As shown in Fig. 4B, the luciferase activity notably decreased after cotransfection with the miR-489 mimic and pGL3-IGF1-wt vector. In contrast, this result was not observed in the group transfected with pGL3-IGF1-mut. Our experimental data showed that miR-489 may participate in H2O2-induced cardiomyocyte apoptosis by regulating IGF1. In addition, as shown in Fig. 4C, the IGF1 protein level decreased after H9c2 cells were treated with H2O2. In H9c2 cells treated with H2O2 and the miR-489 inhibitor, the IGF1 level increased compared to that observed in the H2O2 group. These findings indicate that IGF1 may be a target of miR-489. In the current study, many DE RNAs had a high values in the PPI network, suggesting that they may have important roles in the pathogenesis of MI. Various previous studies have proven that the abnormal expression of ESR1, which had the highest value in our PPI network, was closely associated with cardiovascular diseases.

Discussion
Dysfunction of ESR1 may be one of the causes of acute coronary events [24]. In a previous study, female mice overexpressing ERα underwent LAD coronary ligation followed by reperfusion, and the myocardial fibrosis of the mouse heart muscle was relieved, indicating that ERα could protect against ischemic injury in the hearts of female rodents [25]. A study by Zhai et al. showed that ERα could protect male mice from ischemia/reperfusion (I/R) injury and that ERα knockout may aggravate I/R damage by impeding the calcium influx and disrupting the mitochondrial function [26].
In an I/R injury model of adult female rabbits, acute pretreatment with estrogen or an Erα activator substantially reduced the area of infarction, indicating that estrogen may exert a protective role during I/R through the ER [27]. Furthermore, ERα was shown to play protective roles in the female heart by regulating the activation of p38 MAPK, proapoptotic signaling and the expression of proinflammatory cytokines [28]. The results of these studies suggested that regulation of ESR1 could be a possible strategy for the treatment of MI.
In this study, a functional analysis of the 688 identified DEGs showed that they were enriched in various cellular functions, especially histone acetyltransferase activity, the Wnt signaling pathway and other functions.
This finding suggests that the alterations of cardiomyocyte metabolism could have important roles in MI. The reinitiation of blood flow after myocardial ischemia could lead to additional injury to cardiomyocytes, potentially causing MI and heart failure by inducing oxidative stress [29]. The oxidative stress response of cardiomyocytes may be regulated by lysine acetylation. In a rat myocardial I/R model, HDAC6 could deacetylate peroxiredoxin 1 and reduce its activity, increasing ROS production and exacerbating oxidative damage in cardiomyocytes [30].
Additionally, the Wnt pathway inhibitor DKK-1 was shown to relieve atherosclerosis by affecting the proliferation of vascular smooth muscle cells (VSMCs) cultured in hyperlipidemic serum [38]. When N-cadherin was overexpressed, inhibition of classical Wnt signaling could reduce VSMC proliferation by 50% [39]. Kaga et al. observed that intracellular β-catenin was aggregated after the addition of the GSK-3β inhibitor lithium, resulting in decreased cardiomyocyte and vascular endothelial cell apoptosis [40]. In another study, Wnt11 expression was shown to improve the survival of MI and cardiac function by suppressing inflammatory cytokine expression through regulation of NF-κB [41]. Overall, these studies demonstrated that histone Our experiments also showed that the expression of miR-489 was increased in cardiomyocytes treated with H2O2, suggesting that miR-489 may be involved in H9c2 cell apoptosis induced by H2O2. Consequently, inhibition of miR-489 expression may be a possible therapeutic approach to prevent MI. To further explore the functional and molecular mechanisms of miR-489, we used bioinformatics analysis to predict the potential targets of miR-489 and performed luciferase reporter assays to verify the relationship between these molecules.
In our study, IGF1 expression was reduced in H2O2-induced H9c2 cells, which contrasted with that observed for miR-489. We proved that miR-489 directly binds to the 3'-UTR of IGF1 to inhibit the expression of IGF1. Furthermore, according to previous studies, IGF1 promotes heart survival, which may accelerate protein metabolism, promote cardiomyocyte growth and regulate myocardial contraction, while inhibiting apoptosis during cardiac ischemia [42]. Clinical studies have shown that low IGF1 levels may be positively associated with all-cause mortality and recurrence of MI [43]. In addition, IGF1 may have a protective effect on the heart in animal models [44]. Thus, our experimental data are consistent with those of previous studies showing that the reduction of IGF1 may be associated with myocardial injury.
In summary, our data showed that miR-489 may be a cardiomyocyte injury factor in MI. First, our results demonstrated that miR-489 expression is increased in H2O2treated H9c2 cells. Moreover, miR-489 could regulate apoptosis by targeting IGF1 in myocardial cells. Taken together, these results indicate that miR-489 is a potential therapeutic target for myocardial injury and that IGF1 may be a potential novel myocardial protective factor. Xiong, Xinquan Yang and Yongqing Mao contributed to the conception and design of experiments and performed data analysis.

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