MiR-19a-3p mitigates hypoxia/reoxygenation-induced apoptosis in H9C2 cardiomyocytes by targeting SOCS3

doi:10.21203/rs.3.rs-1658394/v1

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MiR-19a-3p mitigates hypoxia/reoxygenation-induced apoptosis in H9C2 cardiomyocytes by targeting SOCS3

https://doi.org/10.21203/rs.3.rs-1658394/v1

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The available treatment strategies for myocardial ischemia-reperfusion (I/R) injuries have not been so much effective. MicroRNAs (miRNAs) from the miR-19 family, which includes miR-19a and miR-19b, modulate both proliferation and apoptosis in the myocardium. The correlation between I/R injury and miR-19a-3p is unknown. Here, the role of miR-19a-3p in injuries induced by I/R was investigated in H9C2 cardiomyocytes. The MiR-19a-3p Levels were determined to be reduced after hypoxia/reoxygenation (H/R) and miR-19a-3p overexpression reduced apoptosis resulting from H/R, improving the activity of the cells. The opposite effect was observed when miR-19a-3p was inhibited. Potential miR-19a-3p targets were investigated using bioinformatics, identifying Protein Suppressor of cytokine signaling-3 (SOCS3), which was verified by luciferase reporter assays. SOCS3 levels were lower by overexpression of miR-19a-3p. SOCS3 silencing prevented apoptosis induced by miR-19a-3p inhibition, whereas overexpression of SOCS3 blocked a miR-19a-3p mimic's effects on apoptosis. According to these findings, miR-19a-3p reduces apoptosis and injury induced by H/R in cardiomyocytes through targeting SOCS3, and that targeting miR-19a-3p/SOCS3 signalling may present a new strategy in the therapy of myocardial I/R injury.

miR-19a-3p

ischemia-reperfusion(I/R) injury

Suppressor of cytokine signaling-3 (SOCS3)

cardiomyocytes

apoptosis

Myocardial infarction (AMI) is a major cause of death throughout the world. Despite the use of reperfusion therapy, which permits the quick restoration of the myocardium's blood supply, it has been found that reperfusion frequently aggravates the injury, leading to ischemia-reperfusion (I/R) injuries including malignant or even refractory arrhythmias and deteriorating cardiac function. I/R injury thus remains a serious issue in the management of AMI patients [1, 2] and there are still no effective interventions. Thus, the investigation of new treatment directions is needed.

MicroRNAs (miRNAs) are small endogenous nucleic acids with lengths between 21 and 23 nucleotides. They prevent the expression of target genes by degrading or preventing the translation of mRNA[3]. There is evidence that miRNAs are highly expressed in the cardiovascular system and have been associated with various pathological processes, including aberrant development, arrhythmias, and apoptosis[4]. MiR-17-92 is documented to regulate the proliferation of cardiomyocytes, as well as playing a role in the regeneration and repair of the myocardium[5]. miR-19b miRNAs have been found to modulate cardiomyocyte apoptosis[6]. A recent report has shown that miR-19a/19b protects and repairs the myocardium after AMI in mouse models, suggesting that it may be useful as a target for treating heart failure after AMI[7]. In a previous study, we observed significant downregulation of miR-19a-3p in H9C2 cardiomyocytes after hypoxia/reoxygenation (H/R) treatment to mimic myocardial I/R in vitro while miR-19a-3p overexpression led to decreased apoptosis in the cells. In addition, bioinformatic analysis identified potential interaction sites for miR-19a-3p with SOCS3 mRNA. SOCS3 (suppressor of cytokine signaling-3) specifically regulates JAK-STAT3 signaling[8, 9]. SOCS3 expression is normally minimal in cells and may not even be expressed at all; however, expression is markedly increased by the action of a number of cytokines. SOCS3 levels in the plasma of AMI patients were shown to be significantly higher in a previous investigation[10]. SOCS3 levels were also significantly raised in H9C2 cardiomyocytes after H/R-induced injury. A recent study has shown that SOCS3 aggravates apoptosis and adversely affects myocardial I/R[11]. However, the underlying mechanism is not known.

Here, we followed on from previous studies suggesting an association between miR-19a-3p/SOCS3 and myocardial protection after H/R and investigated miR-19a-3p/SOCS3 in myocardial ischemia using H/R to mimic I/R. Ultimately, this could lead to new directions in the treatment of I/R injuries.

Cells and transfection

The Chinese Academy of Sciences Cell Bank (Shanghai, China) provided the rat H9C2 cardiomyocyte cell line. The cells were cultured at 37°C in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA) with fetal bovine serum (FBS, Gibco) at a concentration of 10% and streptomycin/penicillin at a concentration of 1% in an atmosphere of O2 (95%) and CO2 (5%). GenePharma (Shanghai, China) provided the miR-19a-3p mimic, inhibitor, and miR-negative control (miR-NC). Small interfering RNA (SOCS3 siRNA), the negative control (NC siRNA), pc-DNA3.1 vector (NC), and pc-DNA3.1-SOCS3 were all acquired from RiboBio (Guangzhou, China). For overexpression or inhibition of miR-19a-3p expression, cells were transfected for 48 h with the miR-19a-3p inhibitor (100 nM), mimic (50 nM), or respective negative controls using Lipofectamine 3000 (Invitrogen, USA). For silencing or overexpressing SOCS3, small interfering RNA (SOCS3 siRNA) in a concentration of 50 nM, the negative control (NC siRNA) in a concentration of 50 nM, the pc-DNA3.1 vector (NC) in a concentration of 50 nM, and the pc-DNA3.1-SOCS3 plasmid in a concentration of 50 nM were individually mixed with diluted Lipofectamine 3000 before transfection into H9C2 cells. The mixture was incubated in DMEM without glucose or serum for a period of 20 min at room temperature before being added to the cells and then allowed to incubate for 6 h (37℃, 5% CO2). The cells were then grown after the transfection medium was withdrawn for 48 h in normal culture medium before being used for experiments.

Hypoxia/reoxygenation (H/R)

Cells were seeded in Petri dishes and when 70–80% confluent, were split into two groups: a control and a H/R. The normal culture medium was substituted with a serum-free medium for the H/R model, and the cells were grown at 37°C for 3 h in a hypoxic incubator with 95% N2 and 5% CO2. In an oxygenated incubator, the cells were grown in a normal medium for another 3 h after being removed from the serum-free medium.

Bioinformatics predictions and dual-luciferase reporter assay

MiR-19a-3p targets were predicted utilizing TargetScan 4.2 (www.targetscan.org). The dual-luciferase assay was conducted with a kit (Promega, USA) using 293T cells (Chinese Academy of Sciences Cell Bank) in 6-well plates (approximately 70–80% confluent). The cells were transfected with miR-19a-3p or miRNA-NC using the Lipofectamine® RNAiMAX Reagent, and the psiCHECK2 SOCS3-WT (wild-type) or psiCHECK2 SOCS3-MUT (mutant) SOCS3 3'UTR plasmids were transfected into corresponding wells by I Trans® Transfection. Following 36 h, the dual-luciferase assay was performed.

Cell viability measurements

6 replicates per group were used to seed H9C2 cells in the logarithmic growth phase into 96-well plates. CCK-8 (10 µl) reagent was applied to each well and permitted to incubate for 1 to 4 h, after which the absorbance at a wavelength of 450 nm was read and the viability of the cells was determined using the provided formula.

Analysis of quantitative real-time polymerase chain reaction (qRT-PCR)

qRT-PCR was conducted using the provided protocol of the Bulge LoopTM miRNA QRT-PCR Primer (RiboBio, Guangzhou, China) utilizing the specific stem-loop reverse transcription primer set provided by RiboBio. As an internal reference, U6 was employed. RiboBio has patented the primer sequences.

Analysis of apoptosis using flow cytometry

Cells transfected with SOCS3 siRNA, the miR-19b mimic, inhibitor, or the respective controls were analyzed using propidium iodide (PI) and Annexin V-FITC kits (Becton Dickinson, USA), and assessment on a flow cytometer (Beckman Coulter, USA).

Western blotting

A BCA kit(Beyotime Biotechnology,China) was used to determine concentrations of protein in cell lysates. SDS-PAGE was used to run equal amounts of protein, which was then transferred to membranes made of PVDF. Following the blockage of the membranes, they were allowed to incubate at 4°C with primary antibodies overnight. The antibodies used were against α-Tubulin in 1:2000 dilution (Proteintech, USA), SOCS3 (Cell Signaling Technology, USA, 1:1000), cleaved caspase-3 (1:1000, Cell Signaling), p-STAT3 (1:1000, Cell Signaling) STAT3 (1:1000, Cell Signaling), Bax (1:1000, Cell Signaling), and Bcl-2 (Cell Signaling, 1:1000). After that the membranes were permitted to incubate at room temperature for 2 h with the secondary antibody at a dilution of 1:5000, and the bands were observed using a chemiluminescence system (Bio-Rad, USA). The gray scales of the images were evaluated using the Image Lab software provided with the developing system, and the ratios of the band to the internal control were calculated. All experiments were conducted three times.

Statistical analysis

All parameters measured or transformed into ratios were represented as Mean ± SD(), of three different experiments. GraphPad Prism 5 and SPSS 22.0 were employed for statistical analysis. If the variance of measurement data was uniform, one-way ANOVA was used; in the case of non-uniform variance, rank-sum analysis was used. Pyewise comparison of multiple text means was tested by the LSD method. P values less than 0.05 were considered statistically significant.

miR-19a-3p and SOCS3 levels in H/R-treated cardiomyocytes

The optimal conditions for H/R to mimic in vivo I/R injuries were first determined. miR-19a-3p levels were evaluated using RT-qPCR, showing that expression was downregulated after incubation of cells in hypoxic conditions for 3 h in comparison with control cells. The miR-19a-3p expression did not differ between 4-h reoxygenation/3-h hypoxia and 3-h reoxygenation/3-h hypoxia (Fig. 1A). Therefore, the 3-h reoxygenation and 3-h hypoxia conditions were selected for subsequent experiments.

To verify SOCS3 expression during H/R, SOCS3 mRNA levels were determined by RT-qPCR. This showed that SOCS3 expression after H/R treatment was elevated in comparison with untreated cells (Fig. 1C). Furthermore, SOCS3 protein levels were also significantly upregulated in the H/R group (Fig. 1D, E).

These findings implicate raised SOCS3 expression and reduced miR-19a-3p expression in injuries induced by H/R.

miR-19a-3p inhibits apoptosis and viability after H/R treatment

Transfection of the miR-19a-3p mimic dramatically increased miR-19a-3p levels (Fig. 1B). The viability of the H/R-treated cells was also increased (Fig. 2A). Therefore, we postulated that miR-19a-3p overexpression ameliorates apoptosis resulting from H/R. To investigate this, we determined the levels of apoptosis-associated proteins such as Bax, total caspase 3, Bcl-2, and cleaved caspase 3. It was found that the ratios of Bax/Bcl2 and cleaved/total caspase 3 were elevated following H/R therapy in comparison with the controls. In H/R-treated cells (Fig. 3A, B), miR-19a-3p overexpression reduced the cleaved /total caspase 3 (Fig. 3B) and Bax/Bcl2 (Fig. 3E) ratios. In addition, flow cytometry showed that the miR-19a-3p mimic reduced apoptosis (Fig. 2B, C).

For further verification of the part played by miR-19a-3p in apoptosis and injury induced by H/R, we used a miR-19a-3p inhibitor to transfect cells. This aggravated the H/R-induced injury (Fig. 2B, C). In contrast to miR-19a-3p-overexpressing cells, the inhibitor significantly raised the ratios of total cleaved/total caspase 3 (Fig. 3D) and Bax/Bcl-2 (Fig. 3F). In addition, flow cytometry confirmed that the inhibitor aggravated apoptosis in cells after H/R treatment (Fig. 2B, C).

This shows that miR-19a-3p enhances the activity of H9C2 cells after H/R injury while reducing their apoptosis.

Targeting of SOCS3 by miR-19a-3p

Prediction of potential miR-19a-3p targets was carried out with miRanda, TargetScan, RNAhybrid, and Target Gene Prediction at EMBL. SOCS3 expression is anticipated to be regulated by miR-19a-3p. Figure 4A shows the predicted binding sites and the interaction was verified by dual-luciferase reporter assays. psiCHECK2-SOCS3-WT with anticipated mir-19a-3p interaction sites or psiCHECK2-SOCS3-MUT with no expected sites were cloned into luciferase reporter gene vectors. It was found that the psiCHECK2-SOCS3-WT luciferase activity in 293T cells co-transfected with the miR-19a-3p mimic was dramatically lower as compared to cells containing miR-19a-3p NC, while no obvious difference was seen with psiCHECK2-SOCS3-MUT (Fig. 4D). This indicates that miR-19a-3p targets SOCS3.

In addition, qRT-PCR and western blotting revealed that protein (Figs. 4B, C) and SOCS3 mRNA (Fig. 4E) were considerably increased following H/R therapy. SOCS3 expression was considerably lowered by the miR-19a-3p mimic but increased by the inhibitor. Thus, SOCS3 expression is negatively associated with miR-19a-3p after H/R treatment.

The influence of the miR-19a-3p/SOCS3 axis on apoptosis-related protein expression

To examine the effect of the miR-19a-3p/SOCS3 axis on apoptosis-related proteins, we investigated whether SOCS3 silencing counteracted H/R-induced apoptosis in H9C2 cardiomyocytes, examining the levels of the apoptosis-associated proteins Bax, total caspase 3, Bcl-2, and cleaved caspase 3. Bax and Cleaved caspase-3 were found to be significantly upregulated, thus elevating the cleaved/total caspase 3 (Fig. 3A, B) and Bax/Bcl2 (Fig. 3A, C, E) ratios after H/R treatment. Overexpression of miR-191-3p, however, reduced both these ratios (Fig. 5A, B, C). It was also found that SOCS3 overexpression mitigated these effects, reducing the protein ratios (Fig. 5B, C). In contrast, the levels of Bax and cleaved caspase-3 were dramatically increased whereas the cleaved/total caspase 3 (Fig. 5D, E) and Bax /Bcl2 (Fig. 5D, F) ratios were elevated in the H/R + miR-19a-3p inhibitor cells compared with the H/R-only cells. However, SOCS3 silencing reduced the influence of the miR-19a-3p inhibitor on these ratios (Fig. 5E, F). According to these findings, miR-19a-3p reduces apoptosis by regulating SOCS3.

While I/R is effective in restoring blood supply to ischemic tissue, it may also lead to additional damage, including the apoptosis and death of myocardial cells, resulting in cell loss and cardiac dysfunction [12]. Thus, it is extremely important to devise methods of preventing and treating I/R injuries. It has been demonstrated that miRNAs are implicated in I/R damage[3, 13, 14].

Our findings are in agreement with those of several earlier cell and animal model studies that showed the protective roles of miRNA-19a and miRNA19b against the effects of hypoxia, ischemia, and heart failure resulting from endoplasmic stress; in all cases, the miRNAs reduced apoptosis[15]. Here, in H9C2 cells, we found that H/R enhanced apoptosis and lower viability. Notably, H/R treatment reduced the miR-19a-3p levels. However, the effects of H/R on apoptosis were counteracted by elevated miRNA-19a-3p levels.

The miR-19a-3p overexpression was linked to a reduction in cell damage and apoptosis after H/R, suggesting the involvement of miR-19a-3p in the amelioration of myocardial I/R injuries. Overexpression of the miRNA also lowered the levels of proteins related to apoptosis. In contrast, increased damage, measured by reduced cell viability, together with increased apoptosis, measured by raised cleaved caspase 3 levels and elevated ratio of Bax/Bcl-2, was seen after inhibition of miR-19a-3p. This suggests that miR-19a-3p functions as an anti-apoptotic agent. Subsequent investigations indicated that miR-19a-3p targeted and decreased SOCS3 expression in H9C2 cells. Knockdown of SOCS3 also reduced H/R-induced apoptosis, while raised SOCS3 levels counteracted the mitigating action of miR-19a-3p on apoptosis. It thus appears that miR-19a-3p protects H/R-treated cells against apoptosis by modulation of SOCS3 signaling.

SOCS3 is the SOCS family member most closely associated with cardiovascular disease. It also modulates JAK-STAT signaling. Earlier studies have shown that myocardial STAT3 reduces apoptosis after I/R injury[16–18]. It is possible that SOCS3 knockout may activate STAT3 in the JAK-STAT3 pathway. STAT3 has been found to have a protective function in myocardial cells after AMI[19].

Hussain et al. reported that ghrelin has a cardioprotective effect in left ventricular injury induced by myocardial infarction, activating JAK2/STAT3 signaling through the inhibition of SOCS3[17]. Our data suggest that H/R induces H9C2 cardiomyocyte injury by activating SOCS3/STAT3-mediated apoptosis. The miR-19a-3p downregulation seen after H/R also suggests the involvement of miR-19a-3p in H/R-induced cardiomyocyte injury.

To investigate this hypothesis, levels of miR-19a-3p were raised by transfection of a miR-19a-3p mimic. This significantly reduced apoptosis, associated with reduced levels of both SOCS3 and cleaved caspase 3. The abnormal expression of numerous miRNAs in myocardial I/R-injury models has been reported, indicating that miRNAs play significant roles in ischemic injury [20]. miR-494 was shown to modulate PI3K/AKT/mTOR signaling by targeting SIRT1 and to protect cardiomyocytes from I/R injury by decreasing both apoptosis and autophagy[21]. miRNA-15b downregulated both MAPK3 and Bcl-2 expression and aggravated cardiomyocyte apoptosis [22] while miR-181c-5p worsened H/R-induced cellular damage and apoptosis by regulating PTPN4, suggesting that targeting miR-181C-5p/PTPN4 signaling may reduce myocardial I/R injuries [23]. In addition, miR-17-92 was found to mitigate kidney damage caused by H/R through several pathways [24]. Our results suggest that miR-19a-3p protects against cardiac damage through the downregulation of SOCS3 and subsequent reduction in apoptosis. We, therefore, used various bioinformatics software packages, including miRanda, TargetScan, RNAhybrid, and target gene prediction at EMBL, for target prediction. Screening of the miR-19a-3p sequence indicated that it complemented the 3’ UTR of SOCS3. Luciferase assays in H9C2 cells confirmed that miR-19a-3p may interact with the 3'UTR of the human SOCS3 gene and thus inhibit its transcription. SOCS3 has been documented to regulate apoptosis in myocardial I/R injuries, and it was found that knockout of SOCS3 in cardiac tissue resulted in continuous activation of protective signaling pathways, leading to reductions in cell damage and apoptosis and suggesting the importance of SOCS3 in the aggravation of H/R-induced damage[25]. In addition, reduced SOCS3 levels ameliorated the effects of ischemic preconditioning and mitigated myocardial H/R-induced damage [26]. In summary, we observed that overexpression of SOCS3 reduced the protective miR-19a-3p effects on H/R-induced damage and apoptosis, while SOCS3 silencing enhanced cardiomyocyte protection and reduced apoptosis. As a result, our findings revealed that miR-19a-3p protects against damage induced by hypoxia by regulating SOCS3 to reduce apoptosis. Target prediction by bioinformatics software did not predict interactions between miR-19a-3p and cleaved caspase3, Bax, and Bcl-2.

In summary, this is the first demonstration that miR-19a-3p protects against hypoxia-induced cardiomyocyte injury through modulating the function of SOCS3. However, only the function of miR-19-3p was examined on apoptosis through SOCS3 and did not investigate the role of SOCS3-associated signaling in cardiomyocyte apoptosis. The study also only investigated cells in which H/R treatment was used to mimic I/R injuries. Further work is required to verify these results in animal models.

Myocardial infarction

AMI

ischemia-reperfusion

I/R

Suppressor of cytokine signaling-3

SOCS3

hypoxia/reoxygenation

H/R.

Ethical approval and consent to participate

Not applicable

Consent to publish

Availability of data and materials

Data are ethically restricted and cannot be shared publicly. Data are available from the corresponding author by request, and subject to ethical considerations.

Competing-Interest

Authors have no financial ties to the organization that sponsored the study, this work was supported by Science and Technology Plan of Health Commission of Jiangxi Province (NO. 2019216).

Funding

This work was supported by Science and Technology Plan of Health Commission of Jiangxi Province (NO. 2019216).

Author contribution

Xiaoshu Cheng conceived the study and Juxiang Li performed statistical analysis of the data. Zirong Xia wrote the main manuscript and was involved in the whole procedure. All authors critically revised and approved the final version of the manuscript. All authors read and approved the final manuscript.

Acknowledgments

We are grateful to Mingxuan Xu and Zhijian Gong for the partial data collection.

Author details

¹Department of Cardiovascular Medicine, The Second Affiliated Hospital of Nanchang University.

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No competing interests reported.

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MiR-19a-3p mitigates hypoxia/reoxygenation-induced apoptosis in H9C2 cardiomyocytes by targeting SOCS3

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Version 1

Abstract

Figures

Introduction

Materials And Methods

Cells and transfection

Hypoxia/reoxygenation (H/R)

Bioinformatics predictions and dual-luciferase reporter assay

Cell viability measurements

Analysis of quantitative real-time polymerase chain reaction (qRT-PCR)

Analysis of apoptosis using flow cytometry

Western blotting

Statistical analysis

Results

miR-19a-3p and SOCS3 levels in H/R-treated cardiomyocytes

miR-19a-3p inhibits apoptosis and viability after H/R treatment

Targeting of SOCS3 by miR-19a-3p

The influence of the miR-19a-3p/SOCS3 axis on apoptosis-related protein expression

Discussion

Abbreviations

Declarations

References

Additional Declarations

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