MiR-21 attenuates FAS-mediated cardiomyocyte apoptosis by regulating HIPK3 expression

Abstract MicroRNA-21 (miR‐21) plays an anti-apoptotic role following ischemia–reperfusion (I/R) injury (IRI) in vivo; however, its underlying mechanism remains unclear. The present study explored the effects of miR-21 and homeodomain interacting protein kinase 3 (HIPK3) on cardiomyocyte apoptosis induced by hypoxia/reoxygenation (H/R) in vitro. To this end, the rat cardiomyocyte H9C2 cell line was exposed to H/R and the roles of miR-21 and HIPK3 in regulating cell viability and apoptosis were evaluated by cell counting kit-8 assay, terminal-deoxynucleotidyl-transferase-mediated dUTP nick end labeling, and flow cytometry. Immunofluorescence and Western blotting were performed to detect the expression/phosphorylation of apoptosis-related proteins. miR‐21 expression was measured with quantitative real‐time polymerase chain reaction. The putative interaction between miR-21 and HIPK3 was evaluated using the luciferase reporter assay. Our results showed that (i) miR-21 overexpression or HIPK3 down-regulation significantly attenuated H9C2 cells apoptosis after H/R, (ii) suppression of miR-21 expression promoted apoptosis, (iii) miR-21 overexpression inhibited HIPK3 expression, (iv) HIPK3 was the direct and main target of miR-21, (v) miR-21/HIPK3 formed part of a reciprocal, negative feedback loop, and (vi) HIPK3 down-regulation decreased FAS-mediated apoptosis by inhibiting the phosphorylation of FADD, which subsequently inhibited the expression of BAX and cleaved caspase-3 and increased the expression of BCL2. Our study indicates that miR-21 attenuates FAS-mediated cardiomyocyte apoptosis by regulating HIPK3 expression, which could eventually have important clinical implications for patients with acute myocardial infarction.


Introduction
Cardiovascular disease is the leading cause of death worldwide.It is usually attributed to the negative effects of acute myocardial ischemia-reperfusion (I/R) injury (IRI), for which there is still no effective prevention method [1].Certain micro (mi)RNAs are implicated in IRI and their expression in cardiomyocytes changes markedly after this type of injury [2][3][4].The role of miRNA-21 (miR-21) in cardiovascular disease has gained special attention [5,6].Indeed, one study [7] identified miR-21 as one of the most important miRNAs implicated in numerous IRI-associated cardiovascular disease states.The authors of the present study used the online database miRDB to identify miR-21 target genes following IRI and found that it regulated several apoptotic genes.Of the 469 genes identified, the top target gene encoded FAS ligand (FASL) [7].This finding suggests that miR-21 is associated with IRI-induced, FAS-mediated apoptosis.
Although both miR-21 and HIPK3 are associated with cardiomyocyte apoptosis, there is little information on how miR-21 and HIPK3 interact during myocardial IRI.Myocardial IRI can be simulated in vitro using myocardial hypoxia/reoxygenation (H/R).Thus, in the present study, we exposed H9C2 cells to H/R to determine the how miR-21 and HIPK3 interacted to regulate apoptosis in the context of IRI.

Cell counting kit (CCK)-8 assay
Cells were seeded in 96-well culture plates at 6×10 4 cells/ml.After undergoing transfection, the cells were exposed to H/R.About 10 μl CCK-8 reagent (Dojindo Molecular Technologies, Japan) was then added to each well.After 2 h, the optical density was determined spectrophotometrically at a wavelength of 450 nm.

Lactate dehydrogenase (LDH) assay
Culture supernatant was collected.The LDH activity was measured with a LDH activity assay kit (Jiancheng Biotechnology, China), according to the manufacturer's instructions.

Flow cytometry
Cells were harvested with trypsin (Gibco, U.S.A.), washed with phosphate-buffered saline, and suspended in 1× binding buffer.Next, 5 μl AnnexinV-PE (BD Biosciences, U.S.A.) and 5 μl 7-amino-actinomycin D (7AAD) (BD Biosciences, U.S.A.) were added to the cell suspension, followed by a 15-20 min incubation at room temperature in the dark.About 400 μl of 1× binding buffer was then added to each tube.Fluorescence was detected by a flow cytometer (BD Biosciences, U.S.A.) and apoptosis was quantified using Flowjo software.The total apoptosis rate was determined by AnnexinV-PE and 7AAD assay Q2 (early apoptosis) + Q3 (late apoptosis).

Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted using the Trizol total RNA reagent (TIANGEN, China) and reverse-transcribed into cDNA using the FastKing RT Kit (TIANGEN, China).qRT-PCR was performed using the SuperReal PreMx Plus reagent (TIANGEN, China), with the following primers (GENERAL BIOL, China): HIPK3 (NM 031144.3;137 bp), forward: 5 -TCACAGAGGCTTGGAGACTG-3 and reverse: 5 -ACAACATGTGCGATGCCTAC-3 ; beta-actin (NM 031787.2;173 bp), forward: 5 -CACCATGTACCCAGGCATTG-3 and reverse: 5 -CCTGCTTGCTGATCCACATC-3 .Reaction conditions were as follows: pre-denaturation at 95 • C for 15 min, followed by 40 cycles of denaturation at 95 • C for 10 s, and annealing and extension at 60 • C for 32 s.Beta-actin served as an internal control.The miRcute miRNA Isolation Kit was used to extract miRNA and cDNA was generated with miRcute Plus miRNA First-Strand cDNA Kit (TIANGEN, China).qRT-PCR was carried out using the miRcute Plus miRNA qPCR Kit (TIANGEN, China) using the following reaction conditions: pre-denaturation at 95 • C for 15 min, followed by 45 cycles of denaturation at 94 • C for 20 s, and annealing and extension at 60 • C for 34 s.Data were normalized to U6 spliceosomal RNA.The upstream and downstream miR-21 and U6 primers were designed by RiboBio Corporation (China).

Statistical analysis
Data were presented as means+ − standard deviation (SD) and analyzed using SPSS 25.0 software.One-way ANOVA and Welch variance analysis were used for comparisons.P-values<0.05 were considered as a measure of statistical significance.

Effect of miR-21 levels on H9C2 cell viability and LDH activity after H/R
We found that miR-21 greatly affected H9C2 cell viability and LDH activity after H/R (Figure 1A,B).miR-21 overexpression increased cell viability and decreased the release of LDH, while suppression of miR-21 expression had the opposite effect.These results indicate miR-21 greatly increases cell viability and decreases cell injury after H/R.

Down-regulation of HIPK3 increases H9C2 cell viability and decreased LDH activity after H/R
We confirmed that the expression of HIPK3 mRNA was effectively down-regulated following the transfection of H9C2 cells with si-HIPK3 (Figure 1C).Cells transfected with si-HIPK3 exhibited increased cell viability (Figure 1A) and decreased LDH activity than H/R+si-NC group (Figure 1B).These findings indicate that HIPK3 down-regulation greatly increases cell viability and decreases cell injury after H/R.

Effect of miR-21 levels on H9C2 cell apoptosis after H/R
We found that apoptosis and BAX expression increased and BCL2 expression decreased after H/R.However, miR-21 overexpression decreased apoptosis and BAX expression and increased BCL2 expression, while the suppression of miR-21 expression had the opposite effect (Figures 2A-D and 3A-C).These results indicate that miR-21 has a protective effect by limiting apoptosis after H/R.

HIPK3 down-regulation suppresses H9C2 cell apoptosis after H/R
Transfection of H9C2 cells with si-HIPK3 significantly decreased their rate of apoptosis (Figure 2A-D).This result indicates that HIPK3 down-regulation inhibits apoptosis after H/R.

miR-21/HIPK3 form part of a reciprocal, negative feedback loop
We found that miR-21 overexpression inhibited HIPK3 expression, while suppression of miR-21 expression had the opposite effect (Figure 4A-C).Meanwhile, HIPK3 down-regulation increased miR-21 expression (Figure 4D).These results indicate that miR-21 interacts with HIPK3, forming part of a reciprocal negative feedback loop.

HIPK3 is a direct and main target of miR-21
TargetScan 7.2 predicted that HIPK3 was a direct target of miR-21 (Figure 5A).We found that the relative luciferase activity of H9C2 cells co-transfected with miR-21 mimic and psiCHECK™-2 vector containing HIPK3-WT-3 -UTR  was significantly lower than that of the HIPK3-WT+NC group; however, this effect was not observed in cells co-transfected with miR-21 mimic and psiCHECK™-2 vector containing HIPK3-MUT-3 -UTR (Figure 5B).The results prove that miR-21 directly targets HIPK3.After co-transfecting H9C2 cells with si-HIPK3 and miR-21 inhibitor, the effect of the miR-21 inhibitor on apoptosis and cell viability was significantly diminished (Supplementary Figure S1A-C).This finding confirms that HIPK3 is the main target of miR-21.

Effect of HIPK3 on FAS-mediated apoptosis
Down-regulation of HIPK3 decreased FAS-mediated apoptosis by inhibiting the phosphorylation of FADD.The reduced phosphorylation of FADD inhibited the expression of BAX and cleaved caspase-3 (C-caspase-3), while promoting the expression of BCL2 (Figure 6A-F).

Discussion
miR-21 is an abundant miRNA in cardiomyocytes and is tightly associated with myocardial IRI [5,6].The up-regulation of miR-21 has been confirmed in various pathological conditions [15][16][17][18][19]; however, several studies have reported the down-regulation of miR-21 in myocardial IRI [20,21].Consistent with the results of these myocardial IRI studies, we also showed that miR-21 was down-regulated after H/R in H9C2 cells.Since apoptosis is a common pathological phenomenon in the I/R-induced death of cardiomyocytes, it is often used to assess the extent of IRI [22].Sayed et al. [21] found that the overexpression of miR-21 in mouse hearts with IRI pathology resulted in a smaller infarct size and a lower cardiomyocyte apoptosis rate.Moreover, Cheng et al. [23] found that ischemic preconditioning (IP)-induced cardiac protection against I/R was eliminated, while cardiomyocyte apoptosis rate was increased, in miR-21-deficient rats.Our study showed that miR-21 overexpression attenuated cardiomyocyte apoptosis after H/R, while the inhibition of miR-21 expression had the opposite effect, which suggests that miR-21 plays an anti-apoptotic role in cardiomyocytes after H/R.
HIPK3 is aberrantly expressed in malignant tumors and is associated with apoptosis [10][11][12][13].Researchers confirmed that FADD is a substrate of HIPK3, and that HIPK3 affects the phosphorylation of FADD at serine 194 [8,10]; this modification is the key to FAS-mediated apoptosis and the subsequent nuclear translocation of FADD [24].In addition, FAS is required for the stabilization of the HIPK3-FADD complex [8].Moreover, the phosphorylated FADD enhances the activation of MEK kinase 1 (MEKK1) and c-jun NH 2 -terminal kinase 1 (JNK1), which further promotes apoptosis [25].In our study, HIPK3 down-regulation decreased FADD phosphorylation and cardiomyocyte apoptosis after H/R; however, whether the phosphorylation of FADD drives cardiomyocyte apoptosis by enhancing MEKK1 and JNK1 activation remains unclear.
Both miR-21 and HIPK3 play a role in cardiomyocyte apoptosis; however, it was unclear whether they interacted with each other.Our study showed that miR-21 interacted with HIPK3 to protect cardiomyocytes against H/R-induced apoptosis, likely via the miR-21-mediated inhibition of HIPK3 expression.Specifically, we showed that  We found that miR-21 and HIPK3 functioned as part of a reciprocal, negative feedback loop, whereby, miR-21 overexpression inhibited HIPK3 expression, while HIPK3 downregulation increased miR-21 expression.We speculate that this feedback loop helps maintain high miR-21 levels in H9C2 cells during H/R and enables it to exert its anti-apoptotic effect; this phenomenon has also been found in other studies of miR-21.For instance, Liang et al. [26] reported a reciprocal miR-21/transforming growth factor β-receptor III (TGFβRIII) loop in cardiac fibroblasts, whereby miR-21 up-regulation resulted in the down-regulation of TGFβRIII, which increased TGF-β1 expression; conversely, the increase in TGF-β1 expression up-regulated the expression of miR-21.Sun et al. [27] also reported a reciprocal, negative feedback loop involving miR-21/programmed cell death 4 (PDCD4)/activation protein-1 (AP-1) in renal fibrogenesis, whereby miR-21-mediated PDCD4 inhibition enhanced AP-1 activity, and AP-1 in turn promoted miR-21 transcription.Because reciprocal, negative feedback loops involving miR-21 have been identified in a variety of diseases, we believe that such a mechanism is crucial for the biological function of miR-21.
miR-21 regulates multiple mRNAs by altering their transcriptional stability or impacting on their translation [28].For instance, the overexpression of miR-21 inhibited the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in a mouse model of IRI [29] and PDCD4 expression in an IRI rat model and H/R-exposed H9C2 cells, which subsequently inhibited apoptosis [30].However, by co-transfecting H9C2 cells with si-HIPK3 and miR-21 inhibitor, we observed that HIPK3 down-regulation effectively reversed the pro-apoptotic action of the miR-21 inhibitor after H/R.Therefore, we believe that miR-21 mainly targets HIPK3 in H/R H9C2 cells but cannot exclude the possibility that other miR-21 targets may also play a role.
In conclusion, we confirmed that miR-21 attenuated cardiomyocyte apoptosis after H/R by inhibiting HIPK3 expression.Reduced HIPK3 levels in turn inhibited FAS-mediated apoptosis by preventing FADD phosphorylation.We also confirmed that miR-21 directly and predominantly targets HIPK3 and forms part of a reciprocal, negative miR-21/HIPK3 feedback loop.
The data from the present study provide a potential therapeutic approach for preventing IRI-associated apoptosis, which could be applied in a clinical setting to improve the prognosis of patients with acute myocardial infarction.However, because all of our data were generated using H9C2 cells in vitro, they will need to be validated in animal IRI models and clinical trials.

Figure 6 .
Figure 6.HIPK3 down-regulation affects the expression of proteins relative to Fas-mediated apoptosis (A) Representative images of Western blot analysis.(B-F) Statistical analysis of protein levels: HIPK3 down-regulation decreased the phosphorylation of FADD and the expression of BAX and C-Caspase-3, and increased the expression of BCL2 (n=3).In panels B,D,E, the gray values of bands were normalized to that of beta Actin.C-Caspase-3, cleaved caspase-3; p-FADD, phosphorylated FADD.Data are presented as mean + − SD; # P<0.05 vs. CTL; ## P<0.01 vs. CTL; P<0.05 vs. H/R+ si-NC; P<0.01 vs. H/R+ si-NC.