miR‐31‐5p suppresses myocardial hypertrophy by targeting Nfatc2ip

Abstract Cardiac hypertrophy, worldwide known as an adaptive functional compensatory state of myocardial stress, is mainly believed to proceed to severe heart diseases, even to sudden death. Emerging studies have explored the microRNA alteration during hypertrophy. However, the mechanisms of microRNAs involved in cardiac hypertrophy are still uncertain. We studied young rats to establish abdominal aorta coarctation (AAC) for 4 weeks. With the significant downregulated cardiac function and upregulated hypertrophic biomarkers, AAC‐induced rats showed enlarged myocardiocytes and alterations in microRNAs, especially downregulated miR‐31‐5p. miR‐31‐5p targets the 3′UTR of Nfatc2ip and inhibits myocardial hypertrophy in vitro and in vivo. Furthermore, we verified that Nfatc2ip is necessary and sufficient for cardiac hypertrophy in neonatal rat cardiomyocytes. Moreover, we found miR‐31‐5p inhibited the colocalization of Nfatc2ip and hypertrophic gene β‐Mhc. Luciferase assay and ChiP‐qPCR test demonstrated that Nfatc2ip binded to the core‐promoter of β‐Mhc and enhanced its transcriptional activity. Above all, our study found a new pathway, mir‐31‐5p/Nfatc2ip/β‐Mhc, which is involved in cardiac hypertrophy, suggesting a potential target for intervention of cardiac hypertrophy.

5][6][7][8] Nfatc2ip is one of the NFAT families, which has been thought to promote cardiac hypertrophy and pathological remodelling, 9 and disrupt NFAT signalling to attenuate cardiac hypertrophy. 10Interestingly, one published study has observed that Nfatc2ip significantly upregulated in the third month after ST-elevation myocardial infarction, 11 which is close to LV hypertrophy. 12creasing evidence reported that microRNAs (miRNAs), mainly involved in post-transcriptional regulation of gene expression, play crucial roles in the regulatory network in cardiovascular disease, including cardiac hypertrophy.Two categories defined as prohypertrophic and anti-hypertrophic microRNAs are involved in cardiac hypertrophy.MicroRNAs, including miR-155, miR-22, miR-217 etc., are reported to promote cardiac hypertrophic processes, 7,[13][14][15][16][17] whereas miR-1, miR-133a, miR-142-3p, etc., are found to attenuate cardiac hypertrophy. 18,19Though the mechanism of microRNAs involved in cardiomyocyte remodelling has been researched, the mechanism of microRNA involved in overload-induced cardiac hypertrophy in the early stage remains uncertain.
In this study, microarrays and qPCR revealed that miR-31-5p was dominantly increased in 4-week abdominal aorta coarctation (AAC)inducted rats.Then, we verified that miR-31-5p suppresses cardiac hypertrophy in vivo and in vitro.Nfatc2ip was the target of miR-31-5p, confirmed by dual-luciferase assay.Moreover, overexpressed Nfatc2ip induced myocardial hypertrophy in the primary cell, which is rescued by si-Nfatc2ip RNA.Further, we found that Nfatc2ip activates β-Mhc transcription by targeting the core-promoter of β-Mhc, and miR-31-5p inhibits Nfatc2ipβ-Mhc signalling to prevent cardiac hypertrophy.

| Construction of a rat model of pressure-overload myocardial hypertrophy
Male Sprague-Dawley (SD) rats (80-100 g) were provided by Shanghai SLAC Laboratory Animal Co., Ltd.Rats were housed with free access to food and water at a constant temperature of 24 ± 1°C, a humidity of 55 ± 5% and a 12 h light/dark cycle, with adequate food and water.
Rats' cardiac hypertrophy model (n = 8) was induced by AAC for 4 weeks.The abdominal aorta was occluded at the suprarenal level using a 2-0 silk suture over a blunt 24-G needle (the external diameter was 0.55 mm). 20Rats were anaesthetised using 2% isoflurane (R510-22-16, RWD Life Science) and oxygen with a flow rate of 0.5 L/min and maintained with 1.5% isoflurane with an airflow of 0.3 L/min in an induction chamber connected with an animal anaesthesia machine.The sham groups (n = 8) underwent similar surgery without placement of the aortic band.
To evaluate the miR-31-5p effect on cardiac hypertrophy, the rats' AAC model was performed on Day 7, and the injection of miR-31-5p agomir, antagomir and the scramble was performed on Days 10 and 21 respectively.The sham group was processed in the same way with the same value of saline sodium injected.
The rats were anaesthetized with pentobarbital sodium (30 mg/ kg, intraperitoneal injection), killed, blood taken from the abdominal aorta and tissues harvested.

| Agomir and antagomir were administered by tail vein injection
The miR-31-5p Agomir (5 mg/kg, RiboBio Co., Ltd.), antagomir (5 mg/ kg), and Scramble (5 mg/kg) were injected via tail vein separately on Day 10 (the third day after AAC modelling) and administered twice using the exact dosage at Day 21.The sham group and AAC group rats were injected with the same volume of normal saline.

| MicroRNA microarray assay
The heart tissues were taken for the miRNA microarray assay, which was performed by Guangzhou RiboBio Co., Ltd.The experiment included pre-hybridization, hybridization, washing and imaging.
GustomArray™ microarray was assembled with a hybridization cap and clips.The microarray was rinsed to decrease the unspecific hybridization background, was covered with the imaging solution and was loaded into the Genepix 400B microarray scanner to scan.
Based on the manufacturer's protocol, bands were visualized using a chemiluminescence kit (WBKLS0500, Millipore).

| Immunohistochemical staining
Immunofluorescence was conducted following a two-step protocol in which the sections or wells were first incubated with Nfatc2ip (1:500, sc-377461, Santa Cruz).The secondary antibodies used were Alexfluor488 (1:1000, ab150113, Abcam); subsequently, the sections were incubated with DAPI.As Nfatc2ip and β-Mhc are from the same species (mouse), which may affect their co-staining, we used the CF-647 dye to conjugate to β-Mhc antibody.After staining with Nfatc2ip and Alexfluor 488, and washing three times, β-Mhclabelled CF647 (1:100) were incubated for 1 h at room temperature followed by incubation with DAPI.The images were captured using a confocal microscope (LSM 880, Zeiss), and the area of the cells was measured by ImageJ.

| Real-time quantity PCR analysis
The total RNA lysates were prepared using Trizol Reagent (139505, Life) for 30 min on ice and then isolated using Direct-zolTM RNA MiniPrep Plus (ZRC200606, Zymo Research).The quantity and the quality of RNAs were analysed on a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific).cDNAs were prepared using Transcriptor First Strand cDNA Synthesis Kit (TAKARA), with a mix of dT primers for mRNA and stem-loop primers (Sangong, Shanghai) for miRNA, at 50°C for 1 h.The qPCR was performed using SYBR Green master mix (TAKARA) with appropriate primers.Relative quantification of gene expression was conducted with the Bio-Rad CFX96 (Bio-Rad) and Real-Time PCR System.Relative quantification was carried out with the 2 −∆∆Ct method.The GAPDH mRNA was used to control mRNA expression and U6 for miRNA.The primers are shown as follows:

| Transfection of miR-31-5p mimics and inhibitors to NRCMs and H9C2 cells
The NRCMs were transfected with miR-31-5p mimics and inhibitors with Scramble as normal control using Lipofectamine RNAiMAX (13778075, Invitrogen) transfection reagent after plating for 18 h.
After 24 h, NRCMs were treated with ANGII (500 nM) containing medium for another 24 h.NRCMs were harvested by fixing with 4% PFA or stored at −80°C.
H9C2 cells were plated into 6-well cell culture clusters and transfected with 100 nM miR-31-5p mimics or inhibitors for 6 h according to the protocol of Lipofectamine 2000.The cells were collected after the terminal transfection for 24 h with 0.025% trypsin digestion and lysis with RIPA buffer for western blot analysis.

| Nfatc2ip-overexpressed H9C2 cell line
H9C2 cells were infected with lentivirus generated with the plasmid containing EF1a-Nfatc2ip-eGFP-CMV-puro and packaging plasmids (OBiO Technology Corp., Ltd., Shanghai).After 8h infection, 1 μg/mL puromycin (P8230, Solarbio) was added to the culturing medium to screen the positive cells for later use in the experiments.

| Co-transfection of plasmid into H9C2 cells
H9C2 cells at a density of 2 × 10 4 cells per well were transfected using Lipofectamine 3000 (Invitrogen) with pisCHECK-Nfatc2ip construct plasmid (200 ng) and miR-31 mimic or inhibitor (100 nM) for 6-8 h in a 24-well cell.They were then altered with fresh medium for another 24-h culture.Thereafter, cells were collected and assayed with the Dual-Luciferase Reporter Assay System to quantify the luminescent signal.In the cloning vector of pisCHECK-Nfatc2ip, microRNA target sites were inserted after the Rellina luciferase (hRluc) region and Firefly luciferase (hluc) was taken as an internal reference.
We set up five groups as follows: Nfatc2ip WT +S, Nfatc2ip WT +M,

| Chromatin immunoprecipitation
The nucleoprotein extracts were acquired using a kit (78833, Thermo).The chromatin was sheared with sonication equipment in an ice bath, with 70% adequate power, 5 s on and 10 s off, total of 50 cycles.Thereafter, the sheared DNA fragment was loaded onto 1% agarose gel with a running condition of 140 V, 40 min.

| Statistical analysis
All data in our study were expressed at mean ± standard deviation (SD).
Analysis of variance was carried out with GraphPad prism9 (Version

| miR-31-5p is downregulated in AAC-induced myocardial hypertrophy and dysfunction
Rats underwent surgery the AAC model on Day 7, followed by killing on Day 35 (Figure 1A).The hearts of AAC-induced rats were larger than those in the sham group; moreover, the cardiomyocytes revealed by WGA staining and the relative area in AAC-induced heart tissue were increased than the sham group rats (Figure 1B,C).
Consequently, the cardiac function was detected by echocardiography, with rats in the AAC group showing decreased EF and FS but increased LV wall thickness (Figure 1D-F).Meanwhile, the protein and mRNA levels of cardiac hypertrophy biomarkers Anp and β-Mhc were significantly upregulated in AAC rats (Figure 1G,H).The above results indicated a successful early cardiac hypertrophy in rats by AAC induction.Then, we explored the alterations in microRNAs in the induced heart tissue using microRNA microarray assay.The top10 increased or decreased microRNAs were as follows: miR-31-5p, mir-20b-3p, let-7a-5p and so on (Figure 1B,C).For this study, we verified six more microRNAs (shown in Figure 1J) through qPCR testing, which showed a consistent decrease, particularly miR-31-5p.
Taken together, miR-31-5p may play a key role in cardiomyocyte remodelling during cardiac hypertrophy processing.

| miR-31-5p suppress cardiac hypertrophy in vitro and in vivo
Because the miR-31-5p expression downregulated in AAC-induced rats significantly, we hypothesized that miR-31-5p might play a suppressive role in cardiac hypertrophy.To verify this notion, we performed gain and loss approaches for miR-31-5p in vitro and in vivo.

| miR-31-5p targets Nfatc2ip by binding to its 3′-UTR
To explore the mechanism of miR-31-5p suppressing cardiac hypertrophy, we analysed its potential targets using bioinformatic

| Nfatc2ip contributes to myocardiocyte hypertrophy
To determine whether Nfatc2ip is a crucial gene for the hypertrophic remodelling of myocardiocytes, we applied the lentivirus containing the Nfatc2ip gene to make up the Nfatc2ip-overexpressed H9C2 cell line.si-RNAs of Nfatc2ip, as a rescue trail, were transfected into cells, showing that the fourth si-RNA of Nfatc2ip (si-4) was the most effective (Figure 4A).Cells infected with lentivirus-containing vector and transfected using scrambles were defined as negative control

| miR-31-5p inhibits myocardial hypertrophy through blocking Nfatc2ip transcriptional function
Nfatc2ip, known as a transcriptional factor, belongs to the NFATC family, and has been demonstrated to be involved in the transcription of the hypertrophic-related genes in the early stage of hypertrophic remodelling of myocardiocytes, which is thought to be an important marker for cardiac hypertrophy. 9,10Overexpression of β-Mhc has been verified to be disadvantageous in cardiac function under severe cardiovascular stress and accelerated LV hypertrophy with isoproterenol stimulation, 21 suggesting that β-Mhc may probably be a potential target downstream of Nfatc2ip.To verify this notion, Nfatc2ip and β-Mhc were co-stained with immunofluorescence in AAC rats, which were treated with miR-31-5p mimics, miR-31-5p inhibitors and scrambles.
The colocalized cells were increased in AAC and AAC+S group rats compared to the sham group.While miR-31-5p agomir significantly decreased the colocalized cells, miR-31-5p antagomir increased that compared to AAC+S, suggesting a positive correlation between Nfatc2ip and β-Mhc (Figure 5A).Always, the 0-2000 bp before the initiation transcriptional site is defined as the gene's promoter.To determine whether Nfatc2ip will transactivate β-Mhc, we further designed the luciferase plasmids derived from the fragmental promoter of β-Mhc (Figure 5B), which were co-transfected with the TK-derived plasmid as inner control.Figure 5C shows that Fragment 2 had the highest relative luciferase activity among the four fragments, suggesting the co-promoter (CP) is in the −1000 to −1500 bp range.By bioinformatic programme, the potential CP was predicted, and the mutant sites were designed as shown in Figure 5D.Consequent relative luciferase activity was found to be increased in cells transfected with WT plasmid and decreased with Mut plasmid, which confirmed the CP promoter of β-Mhc is localized at 1165-1214 bp of β-Mhc.Moreover, we sheared the DNA from the nuclear protein isolated from rat hearts into the 250-bp segment, then incubated with a magnetic bead containing the According to Wenjun Dai's work, 20 we used rats weighing 80-120 g to make up a 4-week AAC-induced cardiac hypertrophic model, which are younger rats rather than adults.Our data also showed that Despite miR-31-5p, Nfatc2ip is predicted to be targeted by various microRNAs, and one piece of evidence has proved that miR-301b-5p targets Nfatc2ip to regulate inflammation in the liver. 27ough studies about Nfatc2ip in cardiac hypertrophy are scarce, Nfatc2ip is significantly upregulated in the third month after STelevation myocardial infarction, 11 some of which have proposed that Nfatc2ip is involved in common variable immunodeficiency 28 or calcium channel TRPV6-mediated breast cancer. 29In this study, we verified that miR-31-5p targets Nfatc2ip by binding to its 3′-UTR.
Moreover, we first found out that Nfatc2ip is upregulated in the early stage of myocardial hypertrophy, and the overexpression of Nfatc2ip-induced myocardial hypertrophy is rescued by si-Nfatc2ip RNA.Our results established a mechanistic link between miR-31-5p and hypertrophy, with Nfatc2ip serving as a mediator of the antihypertrophic signal of miR-31-5p.
Our another significant finding is that Nfatc2ip promotes the transcriptional activity of β-Mhc.Mammalian cardiac muscle expresses two genes encoding myosin heavy chains (Mhc), including α-and β-Mhc.Cardiac hypertrophy is accompanied by the remodelling of myocardiocytes, which decreases α-Mhc and increases β-Mhc. 30β-Mhc is an essential and multifunctional target of cardiac hypertrophy. 21Patients with defective β-Mhc gene mutation were more vulnerable to cardiac hypertrophy 31 ; however, overexpressed β-Mhc augmented the hypertrophic level. 21ile found to promote the length of myocardiocytes, 32 others thought that β-Mhc is predominantly involved in fibrosis. 33Our data first showed elevated β-Mhc corresponding to decreased miR-31-5p and increased Nfatc2ip protein level.The fibrosis in our study has not been evaluated, which may be investigated further.
Moreover, recent studies have focused on functional analyses of the β-Mhc gene promoter, [34][35][36] suggesting the interplay of cis The overview of this study.The pressure overload-induced mechanical stress on the cardiomyocytes results in downregulated miR-31-5p, which inhibits the Nfatc2ip translation through binding to the 3′-UTR of Nfatc2ip mRNA, followed by more core-promoters of β-Mhc leading to upregulated β-Mhc transcription that accelerate cardiac hypertrophy.

First, we verified
the expression efficiency of miR-31-5p in mimicor inhibitor-treated NRCMs (FigureS1A).We then transfected miR-31-5p mimics, miR-31-5p inhibitors or scrambles (as a negative control) in the AngII-induced NRCM model.Phalloidin staining showed that AngII exposure and transfection with scrambles significantly increased the cell size and relative area of NRCMs compared to the control.While miR-31-5p mimics reversed the increase, miR-31-5p inhibitors enhanced the increase (Figure 2A,B).Moreover, the mRNA and protein levels of the hypertrophic biomarkers Anp and β-Mhc were upregulated in AAC rats, which was largely reversed by transfection with miR-31-5p mimics.However, transfection with the miR-31-5p inhibitor made an upregulation (Figure 2C,D).Further, the efficiency of miR-31-5p expression treated with agomir and antagomir was confirmed in rats (FigureS1B).Rats underwent surgery to induce the AAC model at Day 7; then, miR-31-5p Agomir, miR-31-5p Antagomir and scrambles were injected twice on Days 10 and 21, respectively (FigureS1C).Echocardiography showed impaired cardiac function, including decreased EF and FS but increased LV wall thickness of AAC rats compared with control rats.There was no significant difference between the AAC and scrambles groups.Compared to the scrambles, miR-31-5p agomir reversed the impaired cardiac function while miR-31-5p antagomir enhanced that (FigureS1D-F).In AAC rats injected with scrambles, similar to the cells induced with AngII, levels of Anp and β-Mhc were increased along with an increase in cell size (Figure2E,H).These results indicated that miR-31-5p could suppress cardiac hypertrophy.
programmes, including miRbase, microRNA.organd miRcode, predicting that miR-31-5p may effectively target Nfatc2ip through binding its 3′-UTR (Figure3A).Therefore, we detected the mRNA and protein levels in the NCRMs mentioned above.Nfatc2ip expression was increased in AngII-induced cells compared to the control group, whereas it was downregulated by miR-31-5p mimics and upregulated by miR-31-5p inhibitors (Figure3B,C).To experimentally validate Nfatc2ip as a direct target of miR-31-5p, we constructed a luciferase reporter vector containing 3′-UTR of Nfatc2ip (Nfatc2ip WT ) and the mutant 3′-UTR of Nfatc2ip (Nfatc2ip MT ) (Figure3A,D).The Nfatc2ip WT and Nfatc2ip MT plasmids were co-transfected with miR-31-5p mimics or inhibitors, respectively, into H9C2 cells.As shown in Figure3E, miR-31-5p strongly inhibited the luciferase activity of Nfatc2ip WT , and its inhibitors increased the luciferase activity.Unsurprisingly, no effect was observed with the corresponding mutant construct.The above results demonstrated that Nfatc2ip is the target of miR-31-5p.

F I G U R E 4
Nfatc2ip antibody, followed by qPCR with primers designed for the CP of β-Mhc.The results revealed a significant increase in the enrichment of β-Mhc promoter in AAC, AAC+S and AAC+Anta groups; however, AAC+A remarkably decreased that.These results demonstrated that Nfatc2ip activated transcription of β-Mhc through binding to the CP of β-Mhc, and miR-31-5p inhibited the β-Mhc expression by targeting Nfatc2ip which blocked the transcription of β-Mhc.Nfatc2ip is necessary and sufficient for cardiac hypertrophy.(A) The Nfatc2ip inhibition by siRNA fragments s (si1-si4) transferred into H9C2 cells is revealed by WB. (B) The protein level of Anp, β-Mhc and Nfatc2ip was detected by WB (n = 3).NC presents cells infected with vector lentivirus and transfected with scramble fragments.Ove-Nfatc2ip means cells infected with lentivirus-based overexpression of Nfatc2ip, and Ove−/ si-Nfatc2ip is defined as infected with Nfatc2ip lentivirus and then transfected with the si-Nfatc2ip fragment.(C, D) The cell surface area was illustrated with Phalloidin (green), and the relative area was calculated (n = 3, 3 fields/ sample).(E) The mRNA level of Anp, β-Mhc and Nfatc2ip was detected by qPCR (n = 3, 3 repeats).One-way ANOVA was used to analyse the statistics, and the significance is expressed as follows: *p < 0.05, ***p < 0.001.This study revealed significantly downregulated miR-31-5p in AACinduced cardiac hypertrophic rats.Mechanistically, miR-31-5p suppresses cardiac hypertrophy in vivo and in vitro by targeting Nfatc2ip.Nfatc2ip overexpression induced myocardial hypertrophy, which is rescued by si-Nfatc2ip RNA.Moreover, β-Mhc, known as a biomarker of cardiac hypertrophy and reported to be involved in remodelling myocardiocytes in the early stage of overload pressure-induced cardiac hypertrophy, was transactivated by Nfatc2ip binding to its core-promoter.Taken together, our findings showed that miR-31-5p was downregulated in AAC-induced rats, leading to the suppression of Nfatc2ip removed, resulting in upregulated b-Mhc, which initiates cardiomyocyte remodelling in the early stage of cardiac hypertrophy.Cardiac hypertrophy is an adaptive response to pressure overload, clinically, which is correlated with an increase in the incidence of cardiovascular disease and is often the initial step in the progression to congestive heart failure.Animal models of cardiac hypertrophy are crucial for researching the pathogenesis and developing therapeutic strategies for preventing cardiac hypertrophy.Many researches have revealed the various mechanisms of hypertrophic remodelling of myocardiocytes.Most of them used an induction duration of more than 6 weeks, while a few focused on the early stage of cardiac hypertrophy.
4-week induction by AAC showed significantly impaired cardiac function, increased heart size and cardiomyocyte size, and increased cardiac F I G U R E 5 miR-31-5p blocks Nfatc2ip transcriptional function ameliorating myocardial hypertrophy.(A) Representative pictures of co-staining of Nfatc2ip (green) and β-Mhc (red) in rat heart tissue.The nucleus is stained with DAPI (blue) (n = 3).(B) The representative fragments of Nfatc2ip promoter from −500 to −2000 bp.The dual-luciferase reporter reveals the most responsible fragment of the promoter.(C) The fragments mentioned were inserted into the pGL3-basic plasmid promoting Luc gene, and a control plasmid expressed Rellina luciferase (hRluc) initiated with TK promoter.They were co-transfected into H9C2 cells (n = 6, 3 replicates).(D) Sequences of core-promoter (WT) and the related mutant (MT).(E) The transactivational function of WT or MT of core-promoter revealed by the dualluciferase re-porter (n = 6, 3 replicates).(F) Representative picture of the DNA shearing fragments (around 250 bp) with sonication followed by performed chromatin.(G) Chip-qPCR detection revealed the enrichment of core-promoter of β-Mhc infected with Nfatc2ip antibody (n = 3).One-way ANOVA was used for statistical analysis, and the significance is expressed as follows: *p < 0.05, **p < 0.01, ***p < 0.001.hypertrophic biomarkers Anp and b-Mhc.These results demonstrate that 4-week AAC-induced young rats are an appropriate animal model for studying cardiac hypertrophy in the early stage.Accumulative miRNAs have been characterized mainly by prohypertrophic and anti-hypertrophic miRNAs.Our microarray assay has shown that miR-22, miR-195 and miR-377 were upregulated, and let-7 microRNA families, miR-133 and miR-26 were downregulated in the cardiac hypertrophy, as observed previously.7,[22][23][24][25][26]Interestingly, miR-31-5p was first found to be significantly downregulated in 4week AAC-induced young rats in our study.miR-31-5p has been reported to be involved in cancer and T cells.Moreover, downregulated miR-31-5p was reported to promote aortic aneurysm/dissection, which implies miR-31-5p plays a role in cardiac-associated diseases.Although the miR-31-5p inhibitor reversed the increased cardiomyocyte (H9C2 cell line) size induced by sh-LncRNA Tincr, this induction may be caused by many complex factors, which cannot be equated with the induction of overload pressure.Due to the diversity of miRNA targets, the impact on a disease is determined by the combined effects of multiple targets.Our data have demonstrated that miR-31-5p mimic or agomir can suppress the hypertrophic cell in vivo and in vitro along with the downregulated protein and mRNA levels of cardiac hypertrophic biomarkers.Together with all these findings, our study first found that miR-31-5p is an antihypertrophic miRNA that is downregulated in the early stage of cardiac hypertrophy.