Abstract
Heart failure (HF) induced by ischemia myocardial infarction (MI) is one of the major causes of morbidity and mortality all around the world. Atorvastatin, a hydroxymethylglutaryl coenzyme A reductase inhibitor, has been demonstrated to benefit patients with ischemic or non-ischemic-induced HF, but the mechanism is still poorly understood. Increasing evidence indicates that lncRNAs play important role in variety of human disease. However, the role and underlying molecular mechanisms remain largely unclear. In our work, we applied 0.5% O2 to generate a hypoxia cardiac progenitor cell (CPC) model. Then, CCK8 and EdU assays were employed to investigate the role of atorvastatin in hypoxia CPC cell model. We found that hypoxia inhibits CPC viability and proliferation through modulating MEG3 expression, while atorvastatin application can protect CPCs from hypoxia-induced injury through inhibiting MEG3 expression. Then, we demonstrated that repression of MEG3 inhibited the hypoxia-induced injury of CPCs and overexpression of MEG3 inhibited the protective effect of atorvastatin in the hypoxia-induced injury of CPCs. Furthermore, our study illustrated that atorvastatin played its role in CPC viability and proliferation by modulating the expression of HMGB1 through the MEG3/miR-22 pathway. Our study, for the first time, uncovered the molecular mechanism of atorvastatin’s protective role in cardiomyocytes under hypoxia condition, which may provide an exploitable target in developing effective therapy drugs for MI patients.
Introduction
Heart failure (HF), which is a leading cause of death worldwide, is mainly caused by loss of contracting cardiac muscle cells known as cardiomyocytes [1]. Hypoxia-induced cardiomyocyte death is the chief reason of cell death in heart ischemia injury [2]. Atorvastatin, a hydroxymethylglutaryl coenzyme A reductase inhibitor, has been demonstrated to contribute to anti-apoptotic and anti-arrhythmic effects in the heart [3]. Atorvastatin has the ability to improve left ventricular ejection fraction and attenuate adverse left ventricular remodeling in HF patients induced by ischemia [4]. A previous study indicated that atorvastatin can inhibit the apoptosis of cardiomyocytes after heart ischemia injury by down-regulating ER stress in rats [5]. However, the molecular mechanism of atorvastatin in myocardial protection after ischemia is still poorly known.
Recently, increasing evidence indicates that noncoding transcripts are functionally active regulation molecules in many physiological and pathological processes. These noncoding RNAs include microRNAs (miRNAs, about 20–22 bp) and long noncoding RNAs (lncRNAs, >200 bp) [6]. It has been shown that the lncRNAs can act as molecular sponges for miRNAs and thereby regulating miRNA activity [7]. It has also been demonstrated that the miRNAs and lncRNAs play an important role in cardiovascular diseases. LncRNA-MALAT1 can abrogate the cardioprotective effects of fentanyl through negatively regulating miR-145/Bnip3 pathway in heart ischemia-reperfusion injury [8]. H19 can mediate CTGF expression by sponging miR-455 in cardiac fibrosis process [9]. LncRNA maternally expressed gene 3 (MEG3) is a tumor suppressor and regulator of tumor suppressors, such as p53 [10]. In cardiovascular diseases, MEG3 was reported to promote hypoxia-induced human pulmonary artery smooth muscle cell proliferation and migration through reducing PTEN expression via sponging miR-21 [11]. However, whether MEG3 contributes to CPC proliferation and function in the heart repair process after ischemia injury is still unknown. Several reports have documented that miR-22 is involved in the hypoxia [12–14]. Mathia et al. demonstrated that miR-22 is a HIF repressor constitutively expressed in the adult kidney and up-regulated in AKI [12].
High mobility group box 1 (HMGB1) is a non-histone chromosome binding protein [15]. HMGB1 contains two DNA-binding domains (A-box and B-box) and a highly acidic C-terminal tail, which plays an important role in many biological processes, such as gene transcription and DNA damage repair [16–19]. Overexpression of HMGB1 can promote tumor proliferation and therefore induce tumor metastasis effect [20]. When locally administered into the infarcted mouse heart tissue, HMGB1 can promote myocardial regeneration and improve heart function in ischemia mouse heart. Restoration of the injured heart tissue was reportedly due to the proliferation of resident CPCs and their differentiation into myocytes as well as angiogenesis [21]. However, the mechanism through which HMGB1 contributes to CPC proliferation in the infarcted myocardium is still unknown.
In the present study, we demonstrated that atorvastatin can protect CPCs from hypoxia-induced injury through the MEG3/miR-22/HMGB1 pathway, which may provide an opportunity for the development of novel therapeutic target for myocardium repair after MI.
Materials and Methods
CPC isolation and culture
All the isolation and culture process of CPCs were performed under sterile condition. Briefly, the hearts of C57BL/6 mice (Biocytogen, Beijing, China) were chopped into small pieces and then digested with collagenase (Sigma-Aldrich, St Louis, USA). After the digested small pieces of heart tissue were cultured for 2 weeks in 6-well plates coated with Matrigel (Coring Co., Corning, USA), c-Kit (Santa Cruz Biotech, Santa Cruz, USA)-coated beads (Thermo Fisher Scientific, Waltham, USA) were used to collect the ‘small, round and bright’ CPCs which were migrated on the edge of the heart tissue. After purification, the CPCs were cultured in DMEM/F12 medium (Hyclone, Logan City, USA) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin G and 100 μg/ml streptomycin. Next, the cells were maintained at 37°C in the cell culture incubator containing 95% air and 5% CO2.
Plasmid construction
The MEG3 and HMGB1 constructs were generated in pcDNA (Invitrogen, Carlsbad, USA). The full-length human MEG3 and HMGB1 cDNA sequences were amplified by PCR and inserted into the EcoRI and XhoI sites of the pcDNA vector, respectively. The PCR amplification was performed with a TC1000-G gradient magnifier (DLAB Scientific, Los Angeles, USA) and done with the following conditions: 95°C for 10 min; 32 cycles of 95°C for 30 s, 58°C for 30 s and 72°C for 1 min; 72°C for 10 min. The primers used were: MEG3: 5′-CCGGAATTCGGCAGACGGCGGAGAGCAGAGAGG-3′ (forward) and 5′-CGCTCGAGTATTGAGAGCACAGTGGGGTGC-3′ (reverse); HMGB1: 5′-CCGGAATTCATGGGCAAAGGAGATCCTAAG-3′ (forward) and 5′-CGCTCGAGTTATTCATCATCATCATCTTC-3′ (reverse).
RNA interference and cell transfection
MEG3 and HMGB1 specific siRNAs were synthesized by GenePharma (Shanghai, China). The sequences of siRNAs are followings: siMEG3-1: 5′-GCUCAUACUUUGACUCUAUTT-3′; siMEG3-2: 5′-GAUCCCACCAACAUACAAATT-3′; siMEG3-3:5′-CCCUCUUGCUUGUCUUACUTT-3′. siHMGB1-1: 5′-CUGCGAAGCUGAAGGAAAATT-3′; siHMGB1-2: 5′-AUCACAGUGUUGUUAAUGUTT-3′; siHMGB1-3: 5′-CACAAACTGCCATTCAACAGGTATT-3′. miR-22 mimics and inhibitors were purchased from RiboBio (Guangzhou, China): miR-22 mimics: 5′-AAGCUGCCAGUUGAAGAACUGU-3′; miR-22 inhibitors: 5′-ACAGUUCUUAACUGGCAGCUU-3′. Lipofectamine-2000 (Invitrogen) was used to transfect the siRNA into CPCs using the opti-MEM culture medium (Gibco, Waltham, USA). After 48 h of culture, the cell samples were collected and analyzed for the subsequent experiments.
RNA isolation and quantitative RT-PCR
Trizol reagent (Invitrogen) was used to extract total RNA from CPCs with different treatments following the manufacturer’s instructions. A total of 2 μg RNA was used for reverse transcription reactions using the cDNA Synthesis SuperMix (Transgen Biotech, Shanghai, China). cDNA (10 ng) was applied for quantitative real-time RT-PCR amplification using the SYBR Green reagent (Donghuan Biotech, Shanghai, China) and performed on the Prism 7500 SDS system (Applied Biosystems, Waltham, USA). Relative mRNA and microRNA expression levels were normalized to β-Actin or U6 snoRNA. The primers for various genes were as follows: MEG3 (forward: 5′-GGCAGGATCTGGCATAGAGG-3′; reverse: 5′-CGAGTCAGGAAGCAGTGGGTT-3′); miR-22 (forward: 5′-GGCCGTAAGCTGCCAGTTGAAG-3′; reverse transcriptional primers: 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACAGTTC-3′); HMGB1 (forward: 5′-GATGGGCAAAGGAGATCCTA-3′; reverse: 5′-CTTGGTCTCCCCTTTGGGGG-3′); β-actin (forward: 5′-GTCATTCCAAATATGAGATGCGT-3′; reverse: 5′-GCTATCACCTCCCCTGTGTG-3′); U6 (forward: 5′-CTCGCTTCGGCAGCACA-3′; reverse: 5′-AACGCTTCACGAATTTGCGT-3′).
Western blot analysis
Cells with different treatments were harvested, washed with PBS, and lysed in lysis buffer with protease inhibitors cocktail (Sigma, St Louis, USA). A total of 20 μg whole-cell lysates were fractionated by 10% SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membrane, blocked in PBS containing 5% fat-free milk and immunoblotted with various antibodies. The anti-HMGB1 antibody (Ab79823) and anti-β-Actin (Ab6276) were obtained from Abcam (Cambridge, UK). All antibodies were used according to the manufacture’s protocol. β-Actin was used as the loading control. Immunoreactivity was detected using a commercial Enhanced Chemiluminescence detection kit (Donghuan Biotech) and analyzed using the ImageJ software (NIH, Bethesda, USA).
Cell viability assay
Cell Count kit-8 (Transgen Biotech, Shanghai, China) was used to detect the viability of CPCs following the manufacturer’s instructions. Briefly, cells were seeded in 96-well plates. After treatments, CCK-8 solution (10% of the medium, 10 μl) was added into each well and incubated for 4 h prior to analysis. Then the absorbance at 490 nm was measured.
EdU incorporation assay
CPCs with different treatments were seeded in 48-well plates at a density of 1000 cells per well. 5-Ethynyl-2′-deoxyuridine (EdU) (Donghuan Biotech) were applied to detect the cell proliferation. The cells were incubated with EdU for 2 h. Then, cells were fixed with 4% paraformaldehyde for 30 min at room temperature, permeabilized with 0.3% Triton X-100, followed by staining with Apollo Staining reaction liquid (Donghuan Biotech). Hoechst was employed to mark the cells. The results were detected using a fluorescent microscope (CKX53; Olympus, Tokyo, Japan).
Luciferase reporter assay
The wild-type MEG3-3′UTR, mutant MEG3-3′UTR, wild-type HMGB1-3′UTR, and mutant HMGB1-3′UTR containing the putative binding site of miR-22 were cloned into the psiCHECK-2 luciferase reporter plasmid (Promega, Madison City, USA). The plasmids and miR-22 mimics were co-transfected into cells using lipofectamine-2000 (Invitrogen) according to manufacturer’s protocol. After 48 h, the cells were harvested and prepared for further luciferase activity detection (Donghuan Biotech) according to the manufacturer’s instructions.
Statistical analysis
Data are presented as the mean ± standard error of mean (SEM) of three or more biological replicates. Two-tailed Student’s t-test or one-way ANOVA was employed to determine the P-values. Results were considered to be statistically significant at P value <0.05.
Results
Hypoxia inhibits the cell viability and proliferation and atorvastatin restores that in CPCs
It has been reported that ischemia can result in hypoxia [22]. Therefore, to explore the mechanism of CPC proliferation in ischemia heart tissue, we first established a hypoxia CPC model. We cultured CPCs with 0.5% O2 at different time points. As shown in Fig. 1A,B, Hif-1α expression was significantly increased at both mRNA level and protein level after hypoxia treatment. CCK8 analysis and EdU incorporation assay illustrated that hypoxia inhibited cell viability and proliferation of CPCs (Fig. 1C,D). Meanwhile, atorvastatin could block hypoxia-induced inhibition of the cell viability and proliferation of CPCs (Fig. 1C,D). These data confirmed that atorvastatin alleviated the hypoxia-induced inhibition of biological functions of CPCs.
Hypoxia reduces the cell viability and inhibits the cell proliferation in CPCs (A) qPCR analysis of the mRNA level of Hif-1α in CPCs under hypoxia condition. (B) Western blot analysis of the protein level of Hif-1α in CPCs under hypoxia condition. (C) CCK8 analysis of the cell viability of CPCs with or without Ator treatment under hypoxia condition. (D) EdU incorporation assay analysis of the CPC proliferation potential with or without Ator treatment under hypoxia condition. β-Actin was detected as the control. Each experiment was repeated at least three times. **P < 0.01. Ator, Atorvastatin.
Atorvastatin protects CPCs from hypoxia-induced injury through inhibiting MEG3 expression
A previous study demonstrated that lncRNA MEG3 mainly acts as a growth suppressor in tumor cells [23]. We wondered whether MEG3 affects the proliferation of CPCs under hypoxia condition. qRT-PCR analysis demonstrated that MEG3 mRNA level was significantly increased under hypoxia condition (Fig. 2A). Meanwhile, atorvastatin treatment could reverse MEG3 expression after hypoxia stimuli (Fig. 2A), which suggested that hypoxia indeed affected MEG3 expression.
Atorvastatin protects CPCs from hypoxia-induced injury through regulating MEG3 expression (A) qRT-PCR assay analysis of the mRNA level of MEG3 in CPCs with or without Ator treatment under hypoxia condition. (B) qRT-PCR assay analysis identified the effectiveness of siRNA against MEG3 in CPCs. (C) qRT-PCR assay analysis identified the effectiveness of pcDNA3.1-MEG3 in CPCs. (D) CCK8 analysis of the cell viability of CPCs after MEG3 knockdown and overexpression with or without Ator treatment under hypoxia condition. (E) EdU incorporation assay analysis of the cell proliferation ability in CPCs hypoxia cell model after MEG3 knockdown and overexpression with or without Ator treatment under hypoxia condition. β-Actin was detected as the control. Each experiment was repeated at least three times. **P < 0.01. Ator, Atorvastatin.
To further explore whether MEG3 is involved in hypoxia-induced CPC injury, we analyzed the biological function of CPCs after MEG3 suppression and overexpression. siRNAs specific to MEG3 was designed to repress MEG3 expression and pcDNA3.1-MEG3 plasmid was designed to overexpress MEG3 in CPCs, respectively (Fig. 2B,C). siMEG3-3 was selected for further research because it has the best suppression activity. Interestingly, cell viability and proliferation potential of CPCs were elevated after MEG3 suppression (Fig. 2D,E). Meanwhile, MEG3 overexpression blocked the atorvastatin protective effect in CPC biological function under ischemia condition (Fig. 2D,E). These data suggested that MEG3 was involved in the role of atorvastatin in hypoxia-induced inhibition of CPC cell viability and proliferation.
Atorvastatin-regulated MEG3 targets miR-22 to affect the cell viability and proliferation of CPCs under hypoxia condition
As is known, lncRNAs can act as miRNA sponges and sequester miRNAs to regulate biological processes [9]. To further figure out the underlying mechanism of the MEG3 under hypoxia condition, we then performed bioinformatics analysis to screen the miRNA targets of MEG3. qRT-PCR results demonstrated that miR-22 and miR-361 were significantly elevated after MEG3 knockdown (Fig. 3A). As miR-22 was reported to be involved in the pathological process of heart disease [24], we next focused on the biological function of miR-22 in CPCs. As depicted in Fig. 3B, dual-luciferase reporter assay illustrated that MEG3 and miR-22 indeed had intermolecular binding. In addition, miR-22 expression level was increased after MEG3 suppression in CPCs under hypoxia condition and further, the overexpression of MEG3 inhibited the atorvastatin-induced restoration of miR-22 expression in CPCs under hypoxia condition (Fig. 3C). These data confirmed that miR-22 was a target of MEG3 in the role of atorvastatin in CPCs under hypoxia condition.
Atorvastatin regulates the viability and proliferation of CPCs via modulating miR-22 expression through MEG3 in hypoxia (A) qRT-PCR analysis of the expression levels of predicted miRNAs after MEG3 knockdown. U6 was used as the control. (B) Luciferase assay was used to verify the binding between miR-22 and MEG3. (C) qRT-PCR assay analysis of the miR-22 expression level in CPCs transfected with siMEG3 and pcDNA3.1-MEG3 with or without Ator treatment after hypoxia stimuli. (D) qRT-PCR assay analysis of the miR-22 expression level in CPCs in hypoxia with or without Ator treatment. U6 was used as the control. (E) CCK8 assay analysis of the cell viability in CPCs after transfected with miR-22 mimics and inhibitors with or without Ator treatment under hypoxia condition. (F) EdU incorporation assay analysis of the cell proliferation ability after transfected with miR-22 mimics and inhibitors with or without Ator treatment under hypoxia condition. U6 was detected as the control. Each experiment was repeated at least three times. **P < 0.01. Ator, Atorvastatin.
To further identify the role of miR-22 in CPCs after hypoxia stimuli, qRT-PCR was applied to measure the expression level of miR-22 under hypoxia condition. It was found that miR-22 level was significantly decreased after hypoxia treatment (Fig. 3D). Meanwhile, atorvastatin treatment could increase miR-22 level under hypoxia condition (Fig. 3D). In addition, the cell viability and proliferation potential of CPCs under hypoxia condition were effectively increased by miR-22 mimics (Fig. 3E,F). However, atorvastatin treatment can reverse the effects of miR-22 mimics and inhibitors in CPCs after hypoxia stimuli (Fig. 3E,F). These data clearly suggested that atorvastatin regulated the cell viability and proliferation of CPCs under hypoxia condition via modulation of MEG3/miR-22 pathway.
Atorvastatin regulates the viability and proliferation of CPCs via modulating HMGB1 expression through miR-22 under hypoxia condition
Given that miRNAs can play its function by targeting mRNAs, next we applied bioinformatics analysis to predict the potential target mRNAs of miR-22. HMGB1 mRNA was then predicted as a target of miR-22. Both qRT-PCR and western blot analysis results illustrated that HMGB1 expression level was increased in CPCs after transfection with miR-22 inhibitors, but reduced after transfection with miR-22 mimics (Fig. 4A,B). Dual-luciferase reporter assay further verified the molecular binding between HMGB1 and miR-22 (Fig. 4C,D).
miR-22 regulates the expression of HMGB1 in CPCs (A) qRT-PCR assay were used to detect the HMGB1 expression in CPCs transfected with miR-22 mimics and inhibitors. GAPDH was used as the control. (B) Western blot analysis were used to detect the HMGB1 expression in CPCs transfected with miR-22 mimics and inhibitors. GAPDH was used as the control. (C) and (D) Luciferase reporter assay analysis of the binding between miR-22 and HMGB1. Each experiment was repeated at least three times. **P < 0.01. Ator, Atorvastatin.
We then further investigated whether miR-22-targeted HMGB1 contributes to the inhibition of biological function of CPCs in response to hypoxia injury. Indeed, both the mRNA level and protein level of HMGB1 were increased under hypoxia condition, but reversed after atorvastatin treatment (Fig. 5A). Furthermore, HMGB1 expression level under hypoxia condition was significantly decreased by miR-22 mimics (Fig. 5B). In addition, Atorvastatin treatment can significantly increase HMGB1 expression in CPCs after transfection with miR-22 inhibitors under hypoxia condition (Fig. 5C). To further explore whether HMGB1 is involved in hypoxia-induced inhibition of the biological function of CPCs, we analyzed the biological functions of CPCs after HMGB1 suppression and overexpression. HMGB1 siRNAs and pcDNA3.1-HMGB1 overexpression plasmid were designed to repress or overexpress HMGB1 in CPCs, respectively (Fig. 5D). The cell viability and proliferation potential of CPCs were elevated after HMGB1 suppression (Fig. 5E,F). Meanwhile, HMGB1 overexpression blocked the protective effect of atorvastatin in CPC biological function under hypoxia condition (Fig. 5E,F). These data suggested that miR-22 indeed regulates the viability and proliferation of CPCs via modulating HMGB1 expression in the role of atorvastatin under hypoxia condition.
Atorvastatin regulates the viability and proliferation of CPCs via modulating HMGB1 expression via miR-22 in hypoxia (A) qRT-PCR and western blot analysis of the mRNA and protein level of HMGB1 in CPCs after Ator treatment under hypoxia condition. GAPDH was used as the control. (B) qRT-PCR and western blot analysis of the mRNA and protein level of HMGB1 in CPCs after transfection with miR-22 mimics under hypoxia condition. GAPDH was used as the control. (C) qRT-PCR and western blot analysis of the mRNA and protein level of HMGB1 in CPCs after transfection with miR-22 inhibitors and Ator treatment under hypoxia condition. GAPDH was used as the control. (D) qRT-PCR assay analysis identified the effectiveness of siRNA against MEG3 and pcDNA3.1-MEG3 in CPCs. (E) CCK8 assay analysis of the cell viability in CPCs after transfected with siMEG3 and pcDNA3.1-MEG3 with or without Ator treatment under hypoxia condition. (F) EdU incorporation assay analysis of the cell proliferation potential in CPCs hypoxia cell model transfected with siMEG3 and pcDNA3.1-MEG3 with or without Ator treatment under hypoxia condition. All experiments were performed in three biological repeats. **P < 0.01. Ator, Atorvastatin.
Atorvastatin exerts its role via modulating the MEG3/miR-22/HMGB1 axis in CPCs under hypoxia condition
As described above, MEG3 can regulate miR-22 expression and miR-22 can then regulate the HMGB1 expression in CPCs after hypoxia. Based on these results, we thus hypothesized that MEG3 might regulate the proliferation and migration of CPCs through the MEG3/miR-22/HMGB1 axis signaling pathway. Indeed, both the mRNA level and protein level of HMGB1 were significantly decreased after MEG3 suppression, and meanwhile increased after MEG3 overexpression under hypoxia condition (Fig. 6A,B). Furthermore, modulation of the MEG3/miR-22/HMGB1 axis affected the cell proliferation and migration of CPCs under hypoxia condition. CCK8 and EdU analysis demonstrated that overexpression of MEG3 inhibited the cell viability and proliferation of CPCs treated with atorvastatin under hypoxia condition. However, the miR-22 alleviated the overexpressed MEG3-induced inhibition of cell viability and proliferation of CPCs treated with atorvastatin under hypoxia condition. Ultimately, the repression of HMGB1 inhibited the role of MEG3/miR-22 pathway in cell viability and proliferation of CPCs treated with atorvastatin under hypoxia condition (Fig. 6C,D). In summary, these results clearly demonstrated that atorvastatin played its role via modulating the MEG3/miR-22/HMGB1 axis in CPCs under hypoxia condition.
Atorvastatin regulates the proliferation and migration of CPCs in hypoxia through the MEG3/miR-22/HMGB1 axis (A) qRT-PCR and western blot analysis of mRNA and protein level of HMGB1 in CPCs after MEG3 suppression. GAPDH was used as the control. (B) qRT-PCR and western blot analysis of mRNA and protein level of HMGB1 in CPCs after pcDNA3.1 transfection with Ator treatment under hypoxia condition. (C) CCK8 assay analysis of the cell viability in CPCs after different treatments under hypoxia condition. (D) EdU incorporation assay analysis of the cell proliferation potential in CPCs after different treatments under hypoxia condition. All experiments were performed in three biological repeats. **P < 0.01. Ator, Atorvastatin.
Discussion
Ischemia-induced HF is the leading cause of death worldwide. Heart ischemia is usually caused by narrowing or obstruction of the coronary artery, which then leads to insufficient blood flow to provide adequate oxygenation and then results in heart hypoxia [25]. The hypoxia-induced heart damage is caused by the accumulation of metabolic waste products, mitochondrial dysfunction, and damage to cell membranes, which lead to cardiomyocytes death and heart dysfunction [26,27]. The application of atorvastatin, a clinical statin drug, can improve heart function and attenuate left ventricular remodeling [4].
In our research, we applied low O2 concentration (only 0.5% O2) to treat the CPCs to create a hypoxia CPCs model. Elevated Hif-1α expression suggested our hypoxia CPCs model is successfully established. Atorvastatin treatment can block hypoxia-induced Hif-1α elevation. In addition, decreased viability and proliferation of CPCs induced by ischemia injury is consistent with previous report that hypoxia can inhibit cell proliferation and function [28,29].
LncRNA MEG3 is a maternally expressed, imprinting gene [15]. We further found that MEG3 was significantly increased in CPCs after hypoxia injury. Knockdown or overexpression of MEG3 can effectively affect the proliferation and cell viability of CPCs, which is consistent with a previous study showing that lncRNA MEG3 mainly acts as a growth suppressor in tumor cells [23]. However, atorvastatin application can significantly block the inhibitory effect of MEG3 on CPCs under hypoxia conditions, indicating that atorvastatin may play its protective role through inhibiting MEG3.
The role of miR-22 in cell proliferation is still controversial. Some evidence demonstrated that miR-22 promoted cell proliferation, while others supported that miR-22 inhibited cell proliferation [7–10]. In our study, we found that miRNA-22 mimics restored the hypoxia-induced inhibition of cell viability and proliferation and meanwhile miRNA-22 inhibitor repressed the atorvastatin-induced promotion of cell viability and proliferation. Because miRNAs may possess multiple target genes, thus we believe that the role of miRNAs is up to its target gene which exerts function in specific cellular progresses. Furthermore, Zhu et al. [11] revealed that angiotensin II-induced hypertrophic cardiomyocytes and the administration of atorvastatin reversed the angiotensin II-induced up-regulation of miRNA-22. However, we found that atorvastatin inhibited the hypoxia-induced promotion of miR-22 expression in our study. Mathia et al. [12] demonstrated that overexpression of miR-22 inhibits Hif-1α expression, and conversely knockdown of endogenous miR-22 enhances hypoxia-induced Hif-1α expression. Thus, these results suggested that atorvastatin regulates miR-22 via different pathways under different condition. Nevertheless, more researches are needed to further dissect the underlying mechanism.
LncRNAs can act as microRNA sponges and alter microRNA levels by sequestering targeted microRNAs [7]. By using bioinformatics analysis as well as luciferase experiment, we validated that miR-22 is a target of MEG3. The miR-22 expression pattern was negatively correlated with the MEG3 expression level. miR-22 mimics and inhibitors significantly affected the proliferation and cell viability of CPCs, suggesting that miR-22 is indeed a down-stream target of MEG3. Of note, atorvastatin can significantly elevate miR-22 expression level. Meanwhile, atorvastatin effectively reversed the effect of miR-22 inhibitors on CPCs. These data suggested that atorvastatin might regulate the biological function of CPCs through miR-22 via MEG3.
It has been suggested that HMGB1 can protect the ischemia mouse heart by inducing myocardial regeneration and improving heart function through promoting the proliferation and differentiation of resident CPCs [21]. It is quite interesting that HMGB1 is a down-stream target of miR-22. In addition, MEG3 suppression or overexpression can significantly affect the mRNA level as well as the protein level of HMGB1. Meanwhile, the cell viability and proliferation of CPCs are influenced by MEG3 through miR-22/HMGB1. Most importantly, atorvastatin can significantly reverse the negative regulatory effect of MEG3 and positive effect of miR-22 on HMGB1, indicating that atorvastatin may function on CPCs through the MEG3/miR-22/HMGB1 axis.
In conclusion, we revealed that atorvastatin protected CPCs from hypoxia-induced injury through modulating the MEG3/miR-22/HMGB1 axis, thus, for the first time, we uncovered the molecular mechanism through which atorvastatin regulates the proliferation of CPCs under hypoxia condition, which provided an exploitable target for the development of effective therapies for MI patients.
Funding
This work was supported by the grants from the Shanghai Pudong New Area Health Planning Commission Industry Special Project (No. PW2013E-2), the Shanghai Municipal Health Planning Commission Key Specialist Project (No. ZK2015A13), the Shanghai Pudong New Area Science Committee Project (No. PKJ2017-Y42), and the National Natural Science Foundation of China (No. 81560077).
References
Author notes
Jinwen Su and Ming Fang contributed equally to this work.
