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Introduction

Myocardial ischemia is a pathological condition characterized by interruption of blood supply to myocardium. Reperfusion has become a standard therapy but may lead to irreversible damage to heart tissue, known as myocardial ischemia/reperfusion (MI/R) injury (1), which is a leading cause of mortality worldwide (2). MI/R injury contributes to a range of cardiovascular outcomes, including systolic myocardial dysfunction, cardiac electrophysiology disorder and myocardial death (3,4). Therefore, it is key to determine the underlying mechanisms and develop novel agents to protect myocardial cells during MI/R injury.

Previous studies have identified that microRNAs (miRNAs or miRs) play essential roles in MI/R injury (5-7). miRNAs are small non-coding RNAs 19-22 nucleotides in length. They regulate target mRNAs by binding the sequences in the 3'untranslated regions (3'-UTRs) to facilitate degradation or suppress translation (8). MI/R injury is aggravated by dysregulation of miRNAs. Hinkel et al (9) reported that miR-92a is significantly upregulated in pigs subjected to percutaneous I/R. With the utilization of locked nucleic acid-modified antisense miR-92a treatment, infarct size was significantly decreased (9). Similarly, miR-15 family, including miR-15a, miR-15b, miR-16-1, miR-16-2, miR-195 and miR-497, is upregulated in the infarcted region of the heart in response to I/R injury in mice and pigs (10). On the other hand, miRNAs serve as an antagonist in cardiovascular disease. For example, miR-103/107 repairs I/R injury by antagonizing receptor-interacting serine/threonine-protein kinase 1 and 3-dependent necrosis and binding to Fas-associated protein with death domain (11). Overexpression of miR-494 activates the Akt/mitochondrial signaling pathway, resulting in cardioprotective effects against I/R-induced injury in a mouse model (12). Despite many studies (13-15) demonstrating that MI/R injury is associated with microRNAs, the mechanisms are unknown because of the complexity of cellular events. Therefore, it is key to understand the role of miRNAs in MI/R injury.

SRY-related high mobility group-Box gene 9 (SOX9) is a transcription factor of the SRY family, which is involved in cell processes, including oxidation and tumor growth (16-18). SOX9 is expressed in multiple types of tissue, including lung, brain, and cardiomyocytes (19). SOX9 involved in hepatic (20) and cerebral (21) I/R injury. Suppressing SOX9 markedly decreases hepatic I/R and concurrent injury by inhibiting TGF-β1 activation (20). Moreover, SOX9 plays a vital role in myocardial disease. For example, the loss or inactivation of SOX9 may reduce the initiation of cardiac hypertrophy, fibrosis and inflammation. SOX9 may act as a regulator of myocardial infarction and is involved in crosstalk between myocytes and fibroblasts (22,23). For example, in patients with ischemic heart disease, the loss of SOX9 may delay the cardiac fibrotic response (24). SOX9 exacerbates hypoxia-induced cardiomyocyte apoptosis by promoting miR-223-3p expression (19). SOX9 suppression by miR-30e elevation protects cardiac function and decreases ventricular remodeling (25). The aforementioned data indicate that SOX9 may be involved in the onset of MI/R injury. However, the underlying molecular mechanism remains unknown. Therefore, the present study aimed to determine the role of miRNAs in regulating MI/R injury and the underlying mechanisms.

Materials and methods

Animal model

Animal experiments were approved [approval no. KLLY(A)-2019-008] and performed in accordance with animal welfare regulations (26) of Zunyi Medical University Committee (Zunyi, Guizhou, China). All experimental procedures were fully compliant with the guidelines for animal research from the National Institutes of Health (27). A total of 84 adult male Sprague-Dawley rats (age, 3 months; weight, ~200 g) that were purchased from Laboratory Animal Center of Third Military Medical University (Chongqing, China) were used in the present study. Animals were housed at 18-22˚C, relative humidity of 50-70% and a 12/12-h light/dark cycle. Food and water were available ad libitum. At the end of experiments, all animals were euthanized by intraperitoneal injection of pentobarbital (150 mg/kg).

To establish the MI/R model, rats were divided into I/R or Sham group, n=15 in each group, or I/R + control (n=5), I/R + miR-30c mimics (n=5), I/R + miR-30c inhibitor (n=5). Rats were anesthetized with 45 mg/kg sodium pentobarbital. The rats undergone endotracheal intubation and ventilation (tidal volume, 7 ml; ratio of exhalation/inhalation time, 1.25; breathing rate, 80/min). The thorax was opened by left thoracotomy to expose the heart. A 6.0 prolene suture was used to induce myocardial ischemia by ligating the proximal left anterior descending coronary artery (LAD) with a slipknot. The slipknot was removed after 30 min to allow reperfusion for 30 min. The sham group underwent the same procedure but was not subjected to ligation. Heart samples were obtained for biological and molecular analyses.

Hematoxylin and eosin (H&E) staining

Myocardial tissue samples were collected, fixed with 4% polyformaldehyde at room temperature overnight, dehydrated and then embedded in paraffin. Then the tissues were cut into 5-µm sections. The sections were stained using H&E staining kit (C0105S, Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Images of stained sections were obtained by light microscope at 400x (Nikon Corporation 80i).

ELISA

Serum samples of rats after I/R injury or sham surgery, or cell culture supernatant of isolated RPM cells was collected and centrifugated at 200 x g for 5 min at 4˚C. The serum levels of lactate dehydrogenase (LDH; cat. no. A020-2-2), cardiac troponin I (cTnI; cat. no. H149-2) and creatine kinase isoenzymes (CK; cat. no. A032-1-1) were detected using ELISA kits (all Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's instructions. Levels of IL-18 (cat. no. H015) and IL-1β (cat. no. H002-1-1) in cell culture supernatant and serum samples were determined with commercial kits (both Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's instructions.

Cell culture

293T cells have high transfection efficiency and are easy to obtain and culture in vitro (28). 293T cells were obtained from Nanjing Synthgene Medical Technology, Co., Ltd. Cells were cultured using DMEM (HyClone; Cytiva) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 1 mM sodium pyruvate and 1% antibiotic-antimycotic (15240-112, Invitrogen, Gibco) at 37˚C in a humidified incubator (Thermo Fisher Scientific, Inc.) with 95% atmosphere and 5% CO2.

Isolation of rat primary myocardial (RPM) cells and hypoxia/reoxygenation (H/R) treatment

The ventricular tissue of 39 rats heart was excised immediately at the end of reperfusion and cut into small pieces, followed by 5 min digestion with 0.25% pancreatin at 37˚C three times. After centrifugation at room temperature at 1,000 x g for 5 min, RPM cells were collected and cultured according as previously described (29,30). To simulate MI/R injury, FBS/glucose-free DMEM was used and cells were cultured at 37˚C in hypoxic condition (5% CO2, 94% N2 and 1% O2) for 4 h. Then, cells were maintained in normoxic conditions at 37˚C (5% CO2 and 95% air) for 2 h, as previously described (31). Cells under normoxic conditions at 37˚C for 2 h served as the control group.

Western blotting

RPM cells or myocardium tissue were lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology) supplemented with 1% PMSF (cat. no. 329-98-6, Roche Applied Science) and 1% phosphatase inhibitor (Thermo Fisher Scientific, Inc.). The bicinchoninic acid protein assay kit was used to determine the total protein concentration. Equal amounts of protein (20 mg) from each sample were resolved on 10% SDS-PAGE (Bio-Rad Laboratories, Inc.) and transferred onto polyvinylidene fluoride membrane (Bio-Rad Laboratories, Inc.). Following blocking with 5% milk for 2 h at room temperature, the membranes were incubated with primary antibodies against SOX9 (cat. no. ab185966; 1:1,000), NF-κB p65 (cat. no. ab239882; 1:1,000), NLRP3 (cat. no. ab263899; 1:2,000), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC; cat. no. ab180799; 1:5,000), caspase-1 (cat. no. ab179515; 1:1,000) and GAPDH (cat. no. ab181602; 1:10,000) overnight at 4˚C. PVDF membranes were incubated at room temperature with horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit IgG H&L; cat. no. ab6721; 1:10,000) for 1 h, then Amersham ECL Western Blotting Detection Reagent (Cytiva, Amersham). The antibodies were all from Abcam. The blots were scanned with a ChemiDoc MP system (Bio-Rad Laboratories, Inc.). The density of each specific band was measured using ImageJ software V1.52a (National Institutes of Health).

miRNA sequencing

The total RNA was extracted from the heart of MI/R model and sham group rats with TRIzol reagent (Invitrogen, USA). After construction of small RNA libraries using the NEB Next Multiplex Small RNA Library Prep Set for Illumina (catalog # E7330L, New England Biolabs, Inc.), index codes were added to attribute sequences for each sample. Agilent 2100 Bioanalyzer and DNA 1000 chip kit (Agilent, part # 5067-1504) were used to determine the quality of the sequencing library. The length of the library is ~135-155 bp. After PCR products were purified, 140-160 bp DNA fragments were recovered. The concentration of the desired products was determined from the Qubit Fluorometer (Thermo Fisher Scientific, Inc.) and used to prepare an equimolar pool of 7 pM solution for cluster generation on an Illumina flow cell. Finally, single-end reads were sequenced using NovaSeq 6000 SP reagent kit (100 cycles; cat. no. 2002746; Illumina Inc.) with Illumina MiSeq (Illumina, Inc.), as previously described (32). miRDeep2 software (github.com/rajewsky-lab/mirdeep2) was used to predict the novel miRNAs. Then, the trimmed reads were aligned to merged pre-miRNA databases (known pre-miRNA from miRBase v21 plus the newly predicted pre-miRNAs) using Novoalign software (v2.07.11) with at most one mismatch.

Analysis of differentially expressed miRNAs

The expression of miRNAs was determined by the number of reads per million clean tags. Differentially expressed miRNAs were analyzed using DEGseq software v.3.16 (bioconductor.org/packages/release/bioc/html/DESeq.html). For data analysis, differentially expressed miRNA profiles between sham group and I/R group were compared with unpaired t-test. The miRNAs with fold-change >1.5 and P-value <0.05(33) were defined as differentially expressed miRNAs. Then, hierarchical clustering was performed. miRNAs were selected to verify expression by reverse transcription-quantitative (RT-q)PCR.

RT-qPCR

Total RNA was extracted from RPM cells or heart tissue using TRIzol (Invitrogen, USA) according to the manufacturer's instructions for the analysis of miRNA expression. cDNA was synthesized using a TaqMan™ Advanced miRNA cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. RT-qPCR was performed using TaqMan™ Fast Advanced Master Mix (Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: Initial denaturation for 10 min at 95˚C, followed by 40 cycles of 2 sec (95˚C), 20 sec (60˚C) and 10 sec (70˚C), as previously described (34). Quantitative measurements were determined via the 2-ΔΔCq method (31). β-actin and U6 snRNA were used as internal controls for RNA and miRNA, respectively. The primers were designed with the miRNA Design V1.01 (Vazyme Biotech Co., Ltd.) with a universal reverse primer sequence. Primer sequences were as follows: miR-328a-3p: Forward (F), 5'-GCTGGCCCTCTCTGCCC-3' and reverse (R), 5'-AGTGCAGGGTCCGAGGTATT-3'; miR-128-3p: F, 5'-CGCGTCACAGTGAACCGGT-3' and R, 5'-AGTGCAGGGTCCGAGGTATT-3'; miR-148a-3p: F, 5'-GCGCGTCAGTGCACTACAGA-3' and R, 5'-AGTGCAGGGTCCGAGGTATT-3'; miR-676: F, 5'-CGCCGTCCTGAGCTTGTC-3' and R, 5'-AGTGCAGGGTCCGAGGTATT-3'; miR-30d-5p, F: 5'-TGTAAACATCCCCGACTGGA-3' and R, 5'-GAACATGTCTGCGTATCTC-3'; miR-30c-5p: F, 5'-GCGCGTGTAAACATCCTACACT-3' and R, 5'-AGTGCAGGGTCCGAGGTATT-3'; SOX9, F: 5'-GTCGGTGAAGAATGGGCAAG-3' and R, 5'-GACCCTGAGATTGCCCGGA-3'; β-actin: F, 5'-CGCGAGTACAACCTTCTTGC-3' and R, 5'-CGTCATCCATGGCGAACTGG-3' and U6: F, 5'-CTCGCTTCGGCAGCACATATACT-3' and R, 5'-ACGCTTCACGAATTTGCGTGTC-3'.

Luciferase assay

For reporter assay, pMIR-SOX9-3'-UTR-wild-type (WT) and pMIR-SOX9-3'-UTR-mutant (MUT) luciferase reporter plasmids (Nanjing Synthgene Medical Technology, Co., Ltd.) were transfected into 293T cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The transfected cells were treated with miR-30c mimics (3'-UCACAUCCUACAAAUGU-5', 100 pmol) or miR-NC (sequence: 5'-CUAACGCAUGCACAGUCGUACG-3'), both from Nanjing Synthgene Medical Technology, Co., Ltd. Luciferase activity was measured 48 h after miR-30c mimics transfection using a Dual-Luciferase Reporter Assay (Promega Corporation). Data were normalized to Renilla luciferase activity.

Cell transfection

For in vitro transfection, 100 nM miR-30c-5p mimic (sequence: 5'-UGUAAACAUCCUACACUCUCAGC-3'), miR-30c-5p inhibitor (sequence: 5'-GCUGAGAGUGUAGGAUGUUUACU-3'), miR-30c-5p mimics control (sequence: 5'-CUAACGCAUGCACAGUCGUACG-3') (35), miR-30c-5p inhibitor control (sequence: 5'-CAGUACUUUUGUGUAGUACAA-3') and, which were from Nanjing Synthgene Medical Technology Co., Ltd, were transfected into RPM cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 48 h at 37˚C according to the manufacturer's instructions. miR-30c mimic was co-transfected with 5 µg plasmid pcDNA3.1-SOX9-Flag (Nanjing Synthgene Medical Technology, Co., Ltd.). A total of 5 µg plasmid vector was transferred. For in vivo transfection, 5 µg miR-30c mimic, miR-30c inhibitor or controls was transferred into the left ventricle anterior wall of rat myocardium by 8 µl Entranster (Nanjing Synthgene Medical Technology Co., Ltd.). After injection, the chest was closed and the rat was permitted to recover. I/R was performed 48 h later.

Cell viability assay

RPM cells were harvested at 48 h after transfection. Then, 200 µl 0.5 mg/ml MTT solution (Nanjing Synthgene Medical Technology Co., Ltd.) was added to each group and incubated for 4 h at 37˚C. DMSO was used to dissolve purple formazan. Absorbance was detected at a wavelength of 450 nm, as previously described (30).

Statistical analysis

Graphpad Prism 8.0 (GraphPad Software, Inc.) and SPSS 19.0 (IBM Corp.) were used for statistical analysis. Data are presented as the mean ± SD of three independent experiments. Two-tailed unpaired Student's t-test was used to compare two groups. One-way ANOVA followed by Tukey's post hoc test was used to compare >2 groups. P<0.05 was considered to indicate a statistically significant difference.

Results

MI/R injury induces pyroptosis and inflammation in vivo

To investigate the effect of MI/R injury on myocardium pyroptosis and inflammation, a rat model of MI/R injury was established. Infiltrating inflammatory cells, myocardial cell swelling, degeneration, cardiac necrosis and loss of transverse striations were observed in the heart subjected to I/R (Fig. 1A). Compared with the sham group, the concentrations of serum CK, LDH, and cTnI were significantly increased in the I/R group (Fig. 1B-D), which suggested successful establishment of the MI/R injury model. Western blotting showed that the expression of pyroptosis-associated protein markers, including ASC, NLRP3 and caspase-1 were significantly upregulated in the rats subjected to I/R injury compared with the sham group. Moreover, the levels of inflammatory marker NF-κB p65 also increased almost 2-fold in the I/R compared with the sham group (Fig. 1E-F). Consistently, following I/R treatment, the expression levels of IL-18 and IL-1β were significantly increased ~2-fold compared with the sham group (Fig. 1G-H). These results indicated that MI/R injury induced myocardium pyroptosis and inflammation in vivo.

Figure 1 MI/R induces pyroptosis and inflammation in vivo. (A) Hematoxylin and eosin staining of rat tissue in Sham and I/R groups (400x). Serum levels of (B) CK, (C) LDH and (D) cTnI were assessed by ELISA following I/R injury. Data are normalized to the Sham group. (E) Protein expression of NF-κB p65, ASC, NLRP3 and caspase-1 was (F) analyzed using western blotting. Serum levels of (G) IL-8 and (H) IL-1β were assessed by ELISA following I/R injury. Data are expressed as the mean ± SD (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. Sham. MI/R, myocardial ischemia/reperfusion; CK, creatine kinase; LDH, lactate dehydrogenase; cTn, cardiac troponin; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain.

miR-30c is downregulated and SOX9 is upregulated after I/R treatment in vivo

MI/R induced significant changes in expression of candidate miRNAs, including miR-328a-3p, miR-128-3p, miR-148-3p, miR-676, miR-30d-5p and miR-30c-5p (Fig. 2A-B). Expression levels of miR-328a-3p and miR-128-3p were significantly increased, while expression of miR-30c-5p significantly decreased following MI/R injury (Fig. 2B). In the present study, RNA sequencing showed that fold-change of expression of miR-30c-5p was 1.52 (P=0.0176). The reasons for choosing miR-30c-5p are its high expression following I/R (TagCount, 7,078 vs. 10,736). miRNA-30c-5p has two precursors, MI0000866 and MI0000871, which are slightly different judging from the tags detected in miRNA sequencing. Therefore, two expression data are given. Moreover, the expression levels calculated with the two precursors is almost the same, therefore, the two miRNAs are both miR-30c-5p (Table SI). Expression of SOX9 in rats was detected by western blotting and RT-qPCR. There was significant upregulation of SOX9 expression in the MI/R group at the protein level but no significant change at the mRNA level (Fig. 2C and D).

Figure 2 miR-30c expression is downregulated and SOX9 is upregulated in MI/R heart in vivo. (A) Expression profile of miRs in sham and I/R model. (B) Expression of miR-328a-3p, miR-128-3p, miR-148-3p, miR-676, miR-30d-5p and miR-30c-5p was measured by RT-qPCR. (C) mRNA level of SOX9 in heart tissue was measured using RT-qPCR. (D) Protein levels of SOX9 in heart tissue were (E) measured using western blotting. Data are expressed as the mean ± SD (n=3). *P<0.05, ***P<0.001 vs. Sham. miR, microRNA; MI/R, myocardial ischemia/reperfusion; RT-q, reverse transcription-quantitative; SOX9, SRY-related high mobility group-box gene 9.

SOX9 is a target of miR-30c

To confirm the association between miR-30c and SOX9, bioinformatics analysis was performed, which predicted a binding site for miR-30c in the 3'-UTR of WT SOX9 (Fig. 3A). Luciferase activity was significantly reduced by miR-30c transfection in with SOX9 WT, but not MUT UTR (Fig. 3B). Overexpression of miR-30c in RPM cells significantly downregulated the protein expression of SOX9. Conversely, following treatment with miR-30c inhibitor, the protein expression of SOX9 was increased ~2-fold. However, compared with the control, the mRNA expression of SOX9 was not significantly altered following miR-30c mimics or inhibitor transfection (Fig. 3C and D). These data suggested that SOX9 was a direct target of miR-30c.

Figure 3 SOX9 is a target gene of miR-30c. (A) Predicted binding sequence for the SOX9 3'UTR with miR-30c. (B) Following transfection with plasmid containing WT SOX9 3'-UTR (pMIR-SOX9-WT) or pMIR-SOX9-MUT, the luciferase activity was measured using dual-luciferase reporter assay. ***P<0.001 vs. NC. (C) SOX9 protein expression was detected by western blotting following miR-30c mimics, miR-30c inhibitor and control transfection and (D) quantified. (E) mRNA expression of SOX9 was assessed by reverse transcription-quantitative PCR following miR-30c mimics, miR-30c inhibitor and control transfection. Data are expressed as the mean ± SD (n=3). ***P<0.001. miR, microRNA; UTR, untranslated region; WT, wild-type; MUT, mutant; SOX9, SRY-related high mobility group-box gene 9; NC, negative control.

miR-30c inhibits cell pyroptosis in vitro

To investigate the function of miR-30c, H/R exposed cells were transfected with miR-30c mimics or inhibitor. Expression of miR-30c was notably decreased following H/R (Fig. 4A) but increased ~200-fold in RPM cells following transfection with miR-30c mimics. Conversely, expression decreased following transfection with miR-30c inhibitor. After H/R treatment, the protein levels of NF-κB p65, ASC, NLRP3 and caspase-1 in the RPM cells were significantly upregulated. Compared with the H/R group, miR-30c mimics treatment significantly inhibited expression of these proteins. However, inhibition of miR-30c restored the expression of these proteins (Fig. 4B-C). Effects of miR-30c on expression of pro-inflammatory factors IL-18 and IL-1β secreted by RPM cells following H/R exposure was determined using ELISA. Overexpression of miR-30c markedly decreased IL-18 and IL-1β levels in the H/R exposed cells, while these levels were further increased after miR-30c inhibitor transfection (Fig. 4D-E). H/R significantly inhibited primary myocardial cell proliferation. miR-30c mimics treatment reversed the effect of H/R on cell viability (Fig. 4F). Overexpression of miR-30c restored downregulation of SOX9 caused by H/R (Fig. 5A-B). Moreover, overexpression of SOX9 before miR-30c mimics transfection could abolish effects of SOX9. Consistent with these results, the pyroptosis protein markers showed a similar trend (Fig. 5A-D). Expression of IL-1β and IL-18 showed no difference between the H/R + mimics control and H/R + miR-30c mimics + SOX9 overexpression group (Fig. 5B).

Figure 4 miR-30c inhibits cardiomyocyte pyroptosis under H/R in vitro. (A) Expression of miR-30c was assessed by reverse transcription-quantitative PCR following miR-30c mimics, miR-30c inhibitor and control transfection under H/R exposure. (B) Protein expression of (C) NF-κB p65, ASC, NLRP3 and caspase-1 was analyzed by western blotting. Levels of (D) IL-8 and (E) IL-1β were assessed by ELISA after miR-30c mimics, miR-30c inhibitor and control transfection under H/R exposure. (F) Viability of H/R-exposed cells after transfection with miR-30c mimics, miR-30c inhibitor and control. Data are expressed as the mean ± SD (n=3). *P<0.05, **P<0.01, ***P<0.001. NC, negative control; miR, microRNA; H/R, hypoxia/reoxygenation; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain.

Figure 5 miR-30c decreases pyroptosis via SOX9 in vitro. (A) Protein expression levels of NF-κB p65, ASC, NLRP3, caspase-1 and SOX9 were analyzed by western blotting and (B) quantified following miR-30c mimics, miR-30c mimics + SOX9 OE and control transfection. Expression levels of (C) IL-8 and (D) IL-1β were assessed by ELISA following H/R. Data are expressed as the mean ± SD (n=3). *P<0.05 and ***P<0.001. OE, overexpression; miR, microRNA; H/R, hypoxia/reoxygenation; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; SOX9, SRY-related high mobility group-box gene 9.

SOX9/miR-30c axis regulates MI/R injury in vivo

To confirm the effect of the SOX9/miR-30c axis in vivo, MI/R injury rats were treated with miR-30c mimics and miR-30c inhibitor. Compared with the sham group, more infiltrating inflammatory cells, myocardial cell swelling, degeneration, cardiac necrosis and loss of transverse striations were observed in the I/R and I/R + miR-30 inhibitor group. Conversely, it was observed in the present study that the I/R injured heart showed obvious integrity of the myocardial membrane, a normal myofibrillar structure with striations, branched appearance and continuity with adjacent myofibrils after miR-30c mimics treatment (Fig. 6A) (36). According to the results of western blotting, there was notable upregulation in expression of NF-κB p65, ASC, NLRP3, SOX9 and caspase-1 when the rats were subjected to I/R. Elevated expression levels of these proteins were further upregulated by the miR-30c inhibitor transfection. However, the upregulated expression levels of these proteins in the heart of I/R injury rats were restored by miR-30c mimics treatment. (Fig. 6B). Consistently, the levels of cTnI, CK and LDH, key diagnostic markers of MI/R injury (30), were increased in I/R-treated rats compared with the sham group. There was a significant reduction in all these markers following miR-30c mimics transfection. However, following miR-30c inhibitor treatment, the levels of these markers were not significantly decreased (Fig. 6D-F). Similarly, miR-30c mimics decreased serum levels of IL-18 and IL-1β, while miR-30c inhibitor did not significantly affect production of IL-18 and IL-1β (Fig. 6G and H).

Figure 6 SOX9/miR-30c axis regulates cardiac I/R injury in vivo. (A) Hematoxylin and eosin staining of rat tissue (400x magnification). (B) Protein expression of (C) NF-κB p65, ASC, NLRP3, caspase-1 and SOX9 in rat heart tissue. Serum levels of (D) CK, (E) LDH and (F) cTnI were assessed by ELISA. Expression levels of (G) IL-8 and (H) IL-1β were assessed by ELISA. Data are expressed as the mean ± SD (n=3). **P<0.01, ***P<0.001 vs. Sham; #P<0.05, ##P<0.01, ###P<0.001 vs. I/R + control. CK, creatine kinase; LDH, lactate dehydrogenase; cTn, cardiac troponin; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; SOX9, SRY-related high mobility group-box gene 9; I/R, ischemia/reperfusion.

Discussion

The present study aimed to investigate the function and the underlying mechanisms of miRNAs on I/R injury-induced pyroptosis. miR-30c was downregulated, while the expression of SOX9 was upregulated in the MI/R rat model. Overexpression of miR-30c inhibited pyroptosis both in vivo and in vitro. Furthermore, miR-30c negatively regulated SOX9 expression by binding its 3'UTR.

In the present study, NLRP3 was activated following MI/R injury, which initiates adaptor protein (ASC) cleavage of caspase-1 and IL-1β and IL-18 generation (37). There was a high level of pyroptosis in rats subjected to MI/R or cells treated with H/R. I/R causes cell death, including apoptosis, autophagy, necrosis and pyroptosis. A number of studies have demonstrated that pyroptosis is triggered by NLRP3, AIM2-like receptor proteins and tripartite motif-containing protein and other inflammasomes (38,39). Following the stimulation of these inflammasomes, downstream inflammatory caspase-1 or caspase-11 is cleaved, leading to secretion of pro-inflammatory cytokines IL-1β and IL-18(40). This process is involved in various types of heart disease, including MI/R injury, myocardial infarction and heart failure (41). For examples, inflammasomes activated by I/R lead to IL-1β production in the heart (42). The accumulation of inflammasomes ASC, cryopyrin and caspase-1 has been observed in an acute myocardial infarction mouse model: Following cryopyrin inhibitor treatment, the formation of inflammasome and infarct size are limited (43). Sandanger et al (44) found that compared with WT hearts, a marked improvement of cardiac function and decreased hypoxic damage were present in the hearts of NLRP3-deficient mice subjected to I/R. Therefore, targeting components of pyroptosis may decrease cardiac I/R injury.

miRNAs modulate target genes by binding the 3'UTR of specific mRNAs and may serve a role in the regulation of cardiovascular disease (45). In a rat in vivo I/R model, upregulating miR-30c-5p decreases myocardial injury, histopathological changes and apoptosis (46). The reasons for investigating miR-30c-5p are its high expression following I/R and its target gene SOX9 is a transcription factor of the SRY family, which is involved in various cell processes, including oxidation and tumor growth (47). Studies (19,24) indicate that SOX9 may be involved in the onset of MI/R injury, although the underlying molecular mechanism remains unknown.

Consistent with the present results, Sun et al (48) found that in a rat MI/R model with LAD ligation, miR-30c-5p expression is downregulated. In addition, overexpression of miR-30c inhibits apoptosis, oxidative stress and inflammation both in vivo and in vitro (48). In addition, overexpression of miR-30c-5p-induced protective effects are associated with its target gene Bach1 and subsequent activation of Nrf2(48). However, another study found that miR-30c expression is increased in a rat model of MI/R injury and overexpression of miR-30c-5p promotes MI/R injury by activating NF-κB pathway and targeting sirtuin 1(49). In the aforementioned study (49), LAD was ligated for 30 min before reperfusion for 2 h, while in the present study, following 30 min LAD ligation, hearts were reperfused for 30 min before sample collection. Therefore, both the reperfusion and the sample collection timepoint may be key for miRNA expression profile. miR-30c-5p can target different signaling pathways during MI/R injury (48,49). The present study reported the association between miR-30c, MI/R injury and pyroptosis. The present study demonstrated that under MI/R injury, SOX9 expression was significantly increased and miR-30c was notably downregulated in vivo. miR-30c directly targeted SOX9 by binding the 3'UTR of SOX9. There was no significant difference in mRNA expression of SOX9 however, the protein expression of SOX9 was significantly increased by I/R. It was hypothesized that miR-30c-5p in cardiomyocytes bound to SOX9 mRNA and inhibited SOX9 protein expression but did not directly degrade SOX9 mRNA. This translational suppression mechanism has been discussed by Huntzinger and Izaurralde (50). Overexpression of miR-30c in vitro suppressed H/R-induced pyroptosis. Conversely, miR-30c inhibitor treatment promoted the effect of H/R treatment. In addition, overexpression of SOX9 abolished the effect of miR-30c on pyroptosis. Following overexpression of miR-30c in vivo, pyroptosis was suppressed and MI/R injury was alleviated. This suggested that regulation of miR-30c expression may contribute to pyroptosis and may serve as a therapeutic target to decrease MI/R injury.

miR-30c decreases the production of reactive oxygen species (ROS) (51,52), which are key for tissue damage and myocardial protection. Furthermore, it is widely reported that overactivation of ROS pathway signaling increases pyroptosis (53,54). During MI/R injury, excessive production ROS and increased pyroptosis of cardiomyocytes may be induced by decreased expression of miR-30c.

In summary, miR-30c was notably downregulated in rats subjected to I/R and directly targeted SOX9. Moreover, miR-30c may serve as a suppressor of cell pyroptosis to decrease I/R-induced heart disease. In the future, gene therapy targeting miR-30c-5p overexpression and SOX9 knockdown may serve a role in MI/R injury clinically.

Supplementary Material

miRNA expression for I-R_vs_N (based on TagCount_ALL_Isoform data).

Acknowledgements

Not applicable.

Funding

Funding: The present study was supported by the National Natural Science Foundation of China (grant no. 81860062) and Guizhou Science and Technology Planning Project [grant no. QianKeHeJiChu(2018)1195].

Availability of data and materials

The datasets generated and/or analyzed during the current study are available in the Gene Expression Omnibus repository: ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE221780.

Authors' contributions

JN, WZ, SY and SC performed the experiments, wrote the manuscript and constructed figures. HW and TY conceived and designed the experiments and provided reagents. JN and HW confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

Animal experiments were approved [approval no. KLLY(A)-2019-008] by Zunyi Medical University Committee (Guizhou, China).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References