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. 2019 Jan 3;234(8):13680–13692. doi: 10.1002/jcp.28047

The long noncoding RNA XIST regulates cardiac hypertrophy by targeting miR‐101

Lili Xiao 1,†, Yulei Gu 1,†, Yunlong Sun 1, Jun Chen 1, Xiaofang Wang 1, Yanzhou Zhang 1, Lu Gao 1,✉, Ling Li 1,✉
PMCID: PMC13483095  PMID: 30605239

Abstract

Cardiac hypertrophy and its resultant heart failure are among the most common causes of mortality, worldwide. Long noncoding RNAs (lncRNAs) are involved in diverse biological processes, and their vital role in the regulation of cardiac hypertrophy is increasingly being discovered. Nevertheless, the biological roles of lncRNA X‐inactive specific transcript (XIST) in cardiac hypertrophy are scarcely reported, and the current study was designed to determine whether cardiac hypertrophy can be regulated by XIST and to elucidate the related mechanism. The animals were randomized to receive either an adeno‐associated virus expressing XIST or control plasmid via a single bolus‐tail vein injection. Two weeks later, hypertrophy was established by transverse aortic constriction (TAC) surgery. In vitro, H9c2 cells were used to explore the potential molecular mechanism of XIST in the regulation of phenylephrine (PE)‐induced cardiomyocyte hypertrophy. A luciferase reporter assay and RNA immunoprecipitation were performed to explore the relationships among XIST, microRNA (miR)‐101, and toll‐like receptor 2 (TLR2). In this study, we demonstrated that the expression of XIST was significantly upregulated in hypertrophic mouse hearts and PE‐treated cardiomyocytes. Then, we observed that knockdown of XIST attenuated PE‐induced cardiomyocyte hypertrophy. Conversely, overexpression of XIST aggravated TAC‐induced cardiac hypertrophy. Finally, we demonstrated that miR‐101 was a direct target of XIST, whereas TLR2 was a target of miR‐101. Rescue assays further confirmed that XIST promoted the progression of cardiac hypertrophy through competitively binding with miR‐101 to enhance the expression of TLR2. Collectively, these in vivo and in vitro findings identify XIST as a necessary regulator of cardiac hypertrophy due to its regulation of the miR‐101/TLR2 axis, suggesting that XIST might act as a therapeutic target for the treatment of cardiac hypertrophy and heart failure.

Keywords: cardiac hypertrophy, lncRNA XIST, miR‐101, TLR2

Our studies identify X‐inactive specific transcript (XIST) as a necessary regulator of cardiac hypertrophy in vivo and in vitro due to its regulation of the miR‐101/TLR2 axis, suggesting that XIST might act as a therapeutic target for the treatment of cardiac hypertrophy and heart failure

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1. INTRODUCTION

Pathological cardiac hypertrophy, induced by prolonged hypertrophic stresses, such as hypertension, ischemia, myocarditis, and valvular heart disease, is the leading risk factor for the development of heart failure (Maron & Maron, 2013; Shimizu & Minamino, 2016; Veselka, Anavekar, & Charron, 2017). Initially, cardiac hypertrophy is beneficial as it maintains cardiac function (Marian, & Braunwald, 2017). However, sustained hypertrophy leads to such effects as deposition of extracellular collagen, loss of adrenergic responsivity, and changes in metabolism (Bernardo, Weeks, Pretorius, & McMullen, 2010; Frey & Olson, 2003). Together, these changes lead to cell death and irreversible structural cardiac remodeling, ultimately resulting in heart failure, and sudden death. Although a variety of specific peptide hormones, growth factors, and microRNAs (miRNAs) have been identified as regulators of cardiac hypertrophy, the underlying molecular mechanisms of cardiac hypertrophy are still not fully understood (Ho, 2010; Rohini, Agrawal, Koyani, & Singh, 2010).

Long noncoding RNAs (lncRNAs) are transcribed RNA molecules greater than 200 nucleotides in length but have no potential for protein coding (Fatica, & Bozzoni, 2014; Jandura, & Krause, 2017; Rinn & Chang, 2012). lncRNAs have been shown to play important roles in various physiological processes, such as RNA processing, modulation of apoptosis and invasion, and chromatin modification and as a competing endogenous RNA (ceRNA; Fatica, & Bozzoni, 2014; Jandura, & Krause, 2017; Mercer, Dinger, & Mattick, 2009). However, to date, most studies on lncRNAs have focused on cancer biology. Recent studies have indicated that lncRNAs are essential for heart development and diseases, whereas only a limited number of lncRNAs have been identified as regulators of cardiac hypertrophy (Lai et al., 2017; Luo, Xu, Liang, Xing, & Zhang, 2018; Sallam, Sandhu, & Tontonoz, 2018; Wo, Guo, Li, Yang, & Wo, 2018). In a recent article, lncRNA CHRF was shown to regulate cardiac hypertrophy by targeting miR‐93 (Wo et al., 2018). Another report has established that lncRNA HOTAIR functioned as a negative regulator of cardiac hypertrophy (Lai et al., 2017). Moreover, MHRT, a cardiac‐specific lncRNA, has been demonstrated to protect the heart from pathological cardiac hypertrophy (Luo et al., 2018). The above studies succeeded in demonstrating the significant potential of newly identified lncRNAs in cardiac hypertrophy.

The lncRNA X‐inactive specific transcript (XIST), derived from the XIST gene, is the master regulator of transcriptional silencing of the X chromosome in mammals (Brockdorff et al., 1991). As one of the first‐identified lncRNAs, XIST has been verified as a tumor regulator in the progression of different kinds of cancers (Yao et al., 2015; Yildirim et al., 2013; Zhu, Kong, Xing, Jin, & Li, 2018). However, studies about the role of XIST in cardiovascular diseases are quite a few, and only a recent study has discovered that XIST is highly expressed in postmyocardial infarction cells; overexpressed XIST could inhibit cell proliferation and promote cell apoptosis in myocardial infarction‐induced mouse hearts (T. Zhou, Qin, Yang, Xiang, & Li, 2017). Another study showed that knockdown of XIST could alleviate oxidative low‐density lipoprotein‐mediated endothelial cell injury (Xu, Ma, Liu, Duan, & Zhang, 2018). However, no further information is available concerning the role of XIST in cardiac hypertrophy with regard to other molecular mechanisms. In this study, we aimed to identify whether XIST is able to modulate the progression of cardiac hypertrophy. Our research provided substantial evidence to demonstrate that increased expression of XIST was a characteristic molecular variation in hypertrophic hearts and cardiomyocytes. Afterward, it was found that overexpression of XIST aggravated cardiac hypertrophy induced by pressure overload. Furthermore, from mechanistic research, it appears that XIST may function as a ceRNA to modulate toll‐like receptor 2 (TLR2) expression levels via competition for miR‐101. A novel hypertrophic modulation model composed of XIST, miR‐101, and TLR2 was provided to study cardiac hypertrophy, shedding new light on the understanding and development of new drugs for heart failure.

2. METHODS

2.1. Animals and treatments

Procedures related to animals conformed with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and received the approval of the Animal Care and Use Committee of the First Affiliated Hospital of Zhengzhou University. Male C57B/L6J mice were procured from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (Beijing, China). A retro‐orbital venous plexus injection of either adeno‐associated virus (AAV9)‐XIST or AAV9‐NC (as a negative control) was administered, and mice were given an intramyocardial injection of 3 × 1011 viral genome particles of AAV9 in 30 μl phosphate‐buffered saline (PBS) in five separate locations in the left ventricle free wall of the heart. Two weeks after injection, a pressure overload mouse model was built by transverse aorta constriction (TAC) as described previously (Li et al., 2018). A Doppler assay was utilized to evaluate whether appropriate aortic constriction had been achieved. Mice that received sham operations were treated similarly but did not undergo aortic constriction. Echocardiography was applied to evaluate the wall thickness and the internal diameter of the left ventricle (LV) at particular time points subsequent to the operation. Four weeks later, the mice were killed, and the hearts, tibiae, and lungs were obtained. Tissues were weighed and evaluated to determine the lung weight/body weight (LW/BW, mg/g), heart weight/tibia length (HW/TL, mg/mm), and HW/BW (mg/g) ratios of the groups.

2.2. Echocardiography and hemodynamic assessment

Echocardiography was conducted on mice anesthetized with 1.5% isoflurane using a MyLab 30CV ultrasound machine (Esaote SpA, Irvine, CA) with a 10‐MHz linear‐array ultrasound transducer as described previously. The LV dimensions were evaluated in the parasternal short‐axis and long‐axis views with a frame rate of 120 hertz. The LV end‐diastolic diameter (LVEDd), LV end‐systolic diameter (LVESd), and fractional shortening (FS) were determined through M‐mode tracing with a sweeping velocity of 50 mm/s at the midpapillary muscle level.

For hemodynamic evaluation, a microtip catheter transducer (SPR‐839; Millar Instruments, Houston, TX) was inserted into the right carotid artery and guided into the murine LV. Subsequent to a 15‐minute stabilization period, heart rate (HR) and pressure and volume signals were measured with a Millar Pressure‐Volume System (MPVS‐400; Millar Instruments). Chart 5.0 Software (Powerlab, AD Instruments, Shanghai, China) was utilized to analyze the results.

2.3. Histological examination

Hearts were resected and immediately placed in a 10% potassium chloride solution to ensure that they were halted in diastole. A saline solution was used to wash the hearts, which were subsequently put into 10% formalin. The hearts were transected near the apex to reveal the LVs and the right ventricles. Some slices with a thickness of four to five µm underwent hematoxylin‐eosin (HE) staining in preparation for histopathology and Picrosirius red staining to enable evaluation of collagen deposition under a microscope. HE staining was used to examine the cross‐sectional area of cardiac muscle cells. A quantitative digital image analysis system (Image‐Pro Plus 6.0, Rockville, MD) was used to examine the cardiomyocytes.

2.4. Plasmid and miRNA transfection

The lncRNA XIST overexpression plasmid and the control vector were synthesized by GeneChem (Shanghai, China). The miR‐101 mimic and the negative control miRNA (miR‐NC), miR‐101 inhibitor (anti‐miR‐101), and miRNA inhibitor negative control (anti‐miR‐NC) were synthesized by RiboBio (Guangzhou, China). The small interfering RNA (siRNA) against XIST (si‐XIST), the siRNA against TLR2 (si‐TLR2), and the siRNA negative control (si‐NC) were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The cells were transfected with the plasmid and miRNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the protocol from the manufacturer.

2.5. Cardiomyocyte culture and treatment

H9c2 cells (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were grown in high‐glucose Dulbecco's modified Eagle's medium (DMEM; C11995; Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (10099; Gibco), penicillin (100 U/ml) and streptomycin (100 mg/ml; 15140; Gibco) in a humidified CO2 incubator (Sanyo 18M; Sanyo, Osaka, Japan) with 5% CO2 at 37°C. Only cells below 15 passages were used. Cells undergoing exponential growth were dissociated with 0.25% trypsin (25200; Gibco) and seeded in six‐well culture plates at 1 × 106 cells/well and then incubated for 24 hr. To induce hypertrophy, phenylephrine (PE) was added to the cells at a concentration of 100 μmol/L for 48 hr. All the in vitro experiments were repeated three times, independently.

2.6. Immunofluorescent staining

Immunofluorescent staining was performed in tissue slices or H9c2 cells with an α‐actinin antibody to evaluate the cell surface area. Briefly, H9c2 cells underwent 24 hr of transduction with various adeno‐associated viruses and were subsequently stimulated for 48 hr with PE. Cells were fixed in 3.7% formaldehyde in PBS before permeabilization with 0.1% Triton X‐100 in PBS. Staining was performed with a 1:100 dilution of α‐actinin. The surface areas were measured using Image‐Pro Plus 6.0 software.

2.7. Western blot analysis

Heart tissues and cultivated cardiac myocytes were lysed in radioimmunoprecipitation assay RIPA buffer. A bicinchoninic acid protein assay kit was used for protein quantification. Protein extracts (50 µg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Invitrogen). The proteins were then transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA) and incubated overnight with multiple primary antibodies at 4°C. The primary antibodies used were TGFβ1, phosphorylated (P‐) and total Smad2 and 3 and were purchased from Cell Signaling Technology (Danvers, MA). TLR2 and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology (Dallas, TX). After a 1‐hr incubation with secondary antibodies at room temperature, the membranes were supplemented with ECL reagents (170–5061; Bio‐Rad, Hercules, CA) and observed with a FluorChem E Imaging System (Cell Biosciences, Lake Franklin, NJ). The expression of the proteins was normalized to that of GAPDH on an identical nitrocellulose membrane.

2.8. Reverse‐transcription polymerase chain reaction (RT‐PCR)

Total mRNA was isolated from cultivated cells or LVs with TRIzol reagent (Invitrogen) and reverse transcribed into complementary DNA with a Transcriptor First Strand cDNA Synthesis Kit (Roche). The relative expression of specific genes was evaluated using quantitative real‐time PCR with SYBR Green (Roche, Diagnostics, Mannheim, Germany). The expression levels were normalized to GAPDH expression.

2.9. Luciferase reporter assay

We used miRanda software (http://www.microrna.org/microrna/getGeneForm.do) and predicted the potential binding sites of miR‐101 on XIST and TLR2. The 3′‐untranslated region (3′‐UTR) of the human XIST and TLR2 genes containing the potential miR‐101 binding sites was cloned into the pRL‐CMV luciferase reporter plasmid, and the mutated sequence was used as the mutated control. For the luciferase reporter gene assay, 293T cells were cultured in 24‐well culture plates and transfected with varying constructs as described in the corresponding figure legends. The firefly and Renilla luciferase activities were measured using the Dual‐Luciferase Reporter Assay System (Promega, Madison, WI) according to the instructions from the manufacturer.

2.10. RNA‐binding protein immunoprecipitation (RIP) assay

The RIP assay was performed using a Magna RIP Kit (EMD Millipore) according to the instructions from the manufacturer. Briefly, 293T cells treated as indicated were lysed in RIP lysis buffer, and the cell lysate was incubated with magnetic beads conjugated to human anti‐Ago2 antibody (Millipore) or isotype‐matched control antibody (normal mouse immunoglobulin G [IgG]; Millipore). Next, the samples were incubated with proteinase K to digest the protein and the immunoprecipitated RNA was isolated. Purified RNA was subjected to quantitative RT‐PCR analysis to detect XIST and miR‐101 levels.

2.11. Statistical analysis

Data are presented as the means ± SEM. Unpaired Student's t tests and two‐way analysis of variance with Bonferroni posttest or Tukey's posttest were used to test for significant differences. p < 0.05 was recognized as significant. SPSS software (version 19.0, SPSS Inc., Chicago, IL) was used to perform the statistical analyses.

3. RESULTS

3.1. Expression of XIST and miR‐101 in pathological cardiac hypertrophy

To identify whether XIST was altered in cardiac diseases, we first detected XIST RNA levels in a mouse model of cardiac hypertrophy induced by TAC. We found that the XIST RNA level was significantly upregulated in hypertrophic hearts (Figure 1a,b). PE has been well documented to induce cardiac hypertrophy. The XIST expression level was also found to be upregulated in PE‐treated cardiomyocytes (Figure 1c,d). As XIST regulates miR‐101, we detected the expression of miR‐101 and found that it was downregulated under these pathological conditions. All these data suggested that XIST and its encoded miR‐101 may participate in the development of pathological cardiac hypertrophy.

Figure 1.

Figure 1

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XIST was upregulated, and miR‐101 was downregulated in pathological cardiac hypertrophy. (a) Detection of XIST and miR‐101 levels in heart samples from sham‐ and TAC‐treated mice by real‐time PCR (n = 6, *p < 0.05 vs. sham group). (b) Histological analyses of hematoxylin and eosin (HE) staining (n = 6) and quantitative analysis of the cell surface area (n = 50 cells) of mouse hearts after TAC surgery (*p < 0.05 vs. sham group). (c) Detection of XIST and miR‐101 levels in PE‐treated cardiomyocytes by real‐time PCR (n = 6). (d) Representative images (n = 6) and quantitative analysis of the cell surface area (n = 100 cells) of cardiomyocytes in response to PE (*p < 0.05 vs. control group). miR: microRNA; PCR: polymerase chain reaction; PE: phenylephrine; TAC: transverse aortic constriction; XIST: X‐inactive specific transcript [Color figure can be viewed at wileyonlinelibrary.com]

3.2. Knockdown of XIST suppresses the hypertrophic response in cultured cardiomyocytes treated with PE

To determine the role of XIST in cardiac hypertrophy, we knocked down XIST in cardiomyocytes with si‐XIST. Infection with si‐XIST in cardiomyocytes successfully decreased the expression of XIST (Figure 2a). Immunostaining revealed that the cell surface area was prominently increased by PE treatment; moreover, cotreatment with PE and si‐XIST notably suppressed PE‐induced cell surface area increases (Figure 2b,c). Accordingly, the PE‐induced expression of hypertrophic hallmarks, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and β‐myosin heavy chain (β‐MHC) was dramatically downregulated in the si‐XIST group compared with the si‐NC group (Figure 2D). These results demonstrated that knockdown of XIST inhibited PE‐induced cardiac hypertrophy.

Figure 2.

Figure 2

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Knockdown of XIST attenuates the hypertrophic response in PE‐treated cardiomyocytes. (a) The expression level of XIST after infection with sh‐XIST or the control (shRNA; n = 6, *p < 0.05). (b) Quantitative analysis of the cell surface area (n = 50 cells, *p < 0.05 vs. control group, # p < 0.05 vs. PE group). (c) Representative images of cardiomyocytes infected with sh‐XIST or shRNA in response to PE (100 μmol/L). (d) The relative mRNA levels of the hypertrophic markers ANP and BNP in cardiomyocytes after treatment with PBS and PE for 48 hr (n = 6, *p < 0.05 vs. control group, # p < 0.05 vs. PE group). ANP: atrial natriuretic peptide; BNP: brain natriuretic peptide; mRNA: messenger RNA; PE: phenylephrine; PBS: phosphate‐buffered saline; shRNA: short hairpin RNA; XIST: X‐inactive specific transcript [Color figure can be viewed at wileyonlinelibrary.com]

3.3. Forced expression of XIST aggravates cardiac hypertrophy in vitro

We next addressed whether elevated levels of XIST in the mice had any effect on pressure overload‐induced cardiac hypertrophy. Mice were subjected to AAV9‐XIST injection to overexpress XIST ( Figure 3a). Four weeks after TAC surgery, the myocardial hypertrophic response was significantly aggravated in XIST‐overexpressing mice, as shown by direct examination of the gross heart, the HE staining, and the cardiomyocyte cross‐sectional area compared with the NC mice (Figure 3b). Moreover, the ratios of HW/BW, HW/TL, and LW/BW were significantly increased in the XIST‐overexpressing mice compared with the NC mice four weeks after TAC (Figure 3c). The cardiac structure and function, as assessed by LVEDd, LVESd, and FS, were significantly impaired in the XIST‐overexpressing mice when compared with the NC group (Figure 3d). Additionally, markers of cardiac hypertrophy (ANP, BNP, and β‐MHC) were dramatically upregulated in the XIST‐overexpressing mice compared with the NC mice (Figure 3e). These gain‐of‐function data indicated that overexpression of XIST exaggerated cardiac hypertrophy in response to chronic pressure overload.

Figure 3.

Figure 3

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Overexpression of XIST mitigates pressure overload‐induced cardiac hypertrophy. (a) Expression level of XIST after 6 weeks of AAV9‐XIST injection (n = 6, *p < 0.05 vs. NC group). (b) Representative histological results and statistical results for the cell cross‐sectional area of the HE staining of the mouse heart tissues (n = 6, n > 100 cells). (c) Statistical results for the ratios of heart weight (HW)/body weight (BW), lung weight (LW)/BW, and HW/tibia length (TL) in the indicated groups (n = 10–12). (d) Echocardiographic measurements of left ventricular end‐diastolic dimension (LVEDd), LV end‐systolic dimension (LVESd), and LV fractional shortening (FS) in different groups (n = 8). (e) Real‐time quantitative PCR was performed to determine the hypertrophic markers of ANP, BNP and β‐MHC in the indicated groups (n = 6). *p < 0.05 vs. sham group, # p < 0.05 vs. TAC group. ANP: atrial natriuretic peptide; BNP: brain natriuretic peptide; NC: negative control; PCR: polymerase chain reaction; TAC: transverse aortic constriction; XIST: X‐inactive specific transcript; β‐MHC: β‐myosin heavy chain [Color figure can be viewed at wileyonlinelibrary.com]

3.4. XIST aggravates cardiac fibrosis in vitro

To investigate the mechanism by which XIST regulates cardiac hypertrophy further, we examined the ability of XIST to modulate fibrosis. Our results showed that interstitial and perivascular fibrosis dramatically increased in the NC mice hearts subjected to TAC and was markedly aggravated in the XIST‐overexpressing mice (Figure 4a,b). Moreover, the mRNA expression levels of fibrotic markers (e.g., collagens I and III, and connective tissue growth factor [CTGF]), which also demonstrated an increased fibrotic response in the XIST‐overexpressing mice compared with the NC group (Figure 4C). To examine the molecular mechanisms of XIST in collagen synthesis, we assessed the effect of XIST on Smad cascade activation. The increased levels of TGF‐β1, Smad2, and Smad3 phosphorylation were markedly increased in XIST‐overexpressing mice after TAC surgery (Figure 4d,e). These results indicated that XIST overexpression aggravated the fibrotic response induced by the chronic pressure overload.

Figure 4.

Figure 4

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Overexpression of XIST attenuates cardiac fibrosis induced by pressure overload in mice. (a) PSR staining of histological sections of the LV in the indicated groups 4 weeks post‐TAC surgery (n = 6). (b) Fibrotic areas were quantified using an image‐analyzing system (n = 33–36 fields). (c) Real‐time PCR analyses of the fibrotic markers (collagens I and III, and CTGF) in the indicated mice (n = 6). (d) Western blots of TGF‐β1, Smad2 phosphorylation, Smad3 phosphorylation, Smad2 and Smad3 from the indicated groups (n = 6). (e) Quantitative western blot analysis. *p < 0.05 vs. vehicle‐sham; # p < 0.05 vs. vehicle‐AB. *p < 0.05 vs. sham group, # p < 0.05 vs. TAC group. CTGF: connective tissue growth factor; LV: left ventricle; PCR: polymerase chain reaction; PSR: Picrosirius red; TAC: transverse aortic constriction; TGF: tumor growth factor; XIST: X‐inactive specific transcript [Color figure can be viewed at wileyonlinelibrary.com]

3.5. XIST directly binds to and controls the expression of miR‐101

Bioinformatics prediction using miRcode indicated that the XIST sequence contained the putative binding site of miR‐101 ( Figure 5a). To further verify this prediction, the wild‐type or mutated miR‐101 binding sites in XIST were cloned into a luciferase reporter plasmid, which were transfected into 293 T cells. A luciferase reporter assay revealed that miR‐101 overexpression was able to inhibit the luciferase activity of XIST‐WT but had no noticeable inhibitory effect on the luciferase activity of XIST‐MUT (Figure 5b), indicating the capability of XIST to directly interact with miR‐101. Furthermore, we performed an RIP assay to confirm the presence of the binding sites between XIST and miR‐101, and we found that XIST and miR‐101 were strikingly more abundant in the Ago2 pellet relative to the anti‐IgG control (Figure 5c). To further investigate the regulatory effect of XIST on miR‐101 expression, we transfected XIST, si‐XIST, or matched controls into cardiomyocytes. RT‐PCR analysis demonstrated that XIST transfection led to a significant increase in XIST expression, whereas si‐XIST introduction resulted in a marked decrease in XIST expression in cardiomyocytes (Figure 5d). However, the enforced expression of XIST dramatically blocked the miR‐101 expression, and XIST knockdown markedly promoted miR‐101 expression in cardiomyocytes (Figure 5e). These data indicated that XIST functioned as a sponge of miR‐101 in cardiomyocytes.

Figure 5.

Figure 5

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XIST directly binds to and controls the expression of miR‐101. (a) The XIST transcript contains the putative miRNA recognition sites complementary to miR‐101. (b) HEK293 cells were transfected with miR‐101 and luciferase constructs of XIST‐UTR (Luc‐XIST‐WT) or mutant (Luc‐XIST‐MUT) (n = 3). (c) The association between XIST and miR‐101 was determined by RIP assay (n = 3). (d) The mRNA level of XIST after transfection with si‐XIST or XIST (n = 3). (e) The relative levels of miR‐101 in the indicated groups (n = 6); *p < 0.05. miRNA: microRNA; mRNA: messenger RNA; MUT: mutant; RIP: RNA‐binding protein immunoprecipitation; si: small interfering; UTR: untranslated region; WT: wild‐type; XIST: X‐inactive specific transcript

3.6. miR‐101 attenuated the prohypertrophic effect of XIST on cardiomyocytes

To further confirm whether the effects of XIST on PE‐induced cardiac hypertrophy were mediated by miR‐101, we transfected anti‐miR‐101, miR‐101, or matched controls into cardiomyocytes. RT‐PCR analysis demonstrated that anti‐miR‐101 transfection led to a marked decrease in miR‐101 expression, whereas miR‐101 transfection resulted in a significant increase in miR‐101 expression in cardiomyocytes (Figure 6a). We then transfected cardiomyocytes with XIST, XIST + miR‐NC, XIST + miR‐101, or miR‐101 subjected to PE treatment. As shown in Figure 6b,c the cell surface area was substantially increased following overexpression of XIST compared with the control group in PE‐treated cardiomyocytes, which was partially decreased by overexpression of miR‐101. Consistently, overexpression of XIST led to a significant increase in the mRNA levels of hypertrophic markers ANP and BNP in PE‐induced cardiomyocytes compared with the control group, which was conspicuously reversed after overexpression of miR‐101 (Figure 6d). Taken together, these results revealed that overexpression of XIST aggravated PE‐induced cardiac hypertrophy by targeting miR‐101.

Figure 6.

Figure 6

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miR‐101 attenuated the prohypertrophic effect of XIST on cardiomyocytes. (a) The mRNA level of miR‐101 after infection with miR‐101 or anti‐miR‐101 (n = 6). (b) Quantitative analysis of the cell surface area in the indicated groups (n = 50 cells). (c) Representative images of cardiomyocytes infected with Ad‐XIST or control adenovirus and treated with or without miR‐101. (d) The relative mRNA levels of the hypertrophic markers ANP and BNP in the indicated groups (n = 6); *p < 0.05. ANP: atrial natriuretic peptide; BNP: brain natriuretic peptide; miR: microRNA; mRNA: messenger RNA; XIST: X‐inactive specific transcript [Color figure can be viewed at wileyonlinelibrary.com]

3.7. TLR2 is a direct target of miR‐101

Among the putative targets of miR‐101, we focused on TLR2, which is involved in the regulation of cardiac hypertrophy. In the present study, compared with the PBS‐treated groups, the protein level of TLR2 increased in PE‐treated cardiomyocytes and partially decreased following knockdown of XIST (Figure 7a). Consistently, mice subjected to TAC displayed increased protein levels of TLR2 in the hearts, and overexpression of XIST significantly promoted the protein levels of TLR2 (Figure 7b). To confirm whether TLR2 is directly modulated by miR‐101 through binding to its 3′‐UTR, we analyzed the 3′‐UTR region of TLR2 with miRcode (http://www.mircode.org) and noticed that miR‐101 has a complementary sequence with the TLR2 3′‐UTR (Figure 7c). Next, we constructed the 3′‐UTR fragment of TLR2, including the miR‐101 targeting site with the corresponding mutant counterpart inserted downstream of the firefly luciferase reporter gene. The luciferase activity assay showed that miR‐101 was able to suppress the luciferase activity of TLR2‐WT‐luc. However, the TLR2‐MUT‐luc showed no response to miR‐101, suggesting that miR‐101 targets TLR2 (Figure 7d). In addition, the TLR2‐WT‐luc construct was then transfected along with miR‐101 and/or XIST. The results from the luciferase assay revealed that the luciferase activity was decreased upon miR‐101 transfection and rescued by XIST, whereas the luciferase activity of the control groups was unchanged (Figure 7e). We then transfected cardiomyocytes with anti‐miR‐101, anti‐miR‐NC, si‐TLR2, anti‐miR‐101 + si‐TLR2, or anti‐miR‐101 + si‐NC. As presented in Figure 7f, transfection of anti‐miR‐101 increased the protein level of TLR2, whereas this effect was weakened following transfection with the si‐TLR2 plasmid.

Figure 7.

Figure 7

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TLR2 is a direct target of miR‐101. (a) Western blots of TLR2 in the indicated groups (n = 6). (b) Quantitative western blot analysis. (c) Sequence alignment showing the binding site between miR‐101 and TLR2. (d) HEK293 cells were transfected with miR‐101 and luciferase constructs of TLR2‐UTR (Luc‐TLR2‐WT) or mutant (Luc‐TLR2‐MUT). (e) The protein level of SIRT1 after transfected with anti‐miR‐101 and/or si‐TLR2. (f) HEK293 cells were transfected with luciferase constructs of TLR2‐Luc, miR‐101, and/or si‐TLR2. *p < 0.05. miR: microRNA; MUT: mutant; si: small interfering; TLR: toll‐like receptor; UTR: untranslated region; WT: wild‐type; XIST: X‐inactive specific transcript

3.8. TLR2 deficiency abolished the prohypertrophic effects of XIST

As miR‐101 mainly mediated the effect of XIST on cardiomyocyte hypertrophy, we then determined whether TLR2 deficiency (Figure 8a) could reverse the prohypertrophic effect of XIST on cardiomyocyte hypertrophy. Cardiomyocytes were transfected with XIST, XIST + si‐NC, XIST + si‐TLR2, or si‐TLR2 with PE treatment. The results showed that overexpressing XIST increased TLR2 expression in PE‐treated cardiomyocytes, whereas this effect was weakened following introduction with the si‐TLR2 plasmid (Figure 8b,c). Moreover, the cell surface area (Figure 8d,e), as well as the hypertrophy markers of ANP and BNP (Figure 8f), were significantly increased by upregulating XIST in PE‐induced cardiomyocytes, whereas knockdown of TLR2 greatly abrogated XIST‐triggered prohypertrophic responses. These data indicated that XIST aggravated PE‐induced cardiac hypertrophy by regulating TLR2.

Figure 8.

Figure 8

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TLR2 deficiency abolished the antihypertrophic effects of XIST. (a) Western blot analysis of TLR2 protein expression after cells were treated with si‐TLR2 (n = 6, *p < 0.05 vs. si‐NC). (b) Western blot analysis of TLR2 protein expression in the indicated groups (n = 6). (c) The relative mRNA level of TLR2 in the indicated groups (n = 6). (d) Representative images of cardiomyocytes in the indicated groups. (e) Quantitative analysis of the cell surface area (n = 50 cells). (f) The relative mRNA levels of the hypertrophic markers ANP and BNP in the indicated groups (n = 6). *p < 0.05. ANP: atrial natriuretic peptide; BNP: brain natriuretic peptide; miR: microRNA; mRNA: messenger RNA; si: small interfering; TLR: toll‐like receptor; XIST: X‐inactive specific transcript [Color figure can be viewed at wileyonlinelibrary.com]

4. DISCUSSION

Noncoding RNAs, including lncRNAs and miRNAs, have been demonstrated to be critical mediators in the initiation and progression of cardiovascular diseases (E. S. et al., 2018). Moreover, increasing evidence suggests that lncRNAs are regulators of cardiac hypertrophy (Lai et al., 2017; Luo et al., 2018; Wo et al., 2018). For instance, lncRNAs Plscr4 and H19 were validated as negative regulators of cardiac hypertrophy (Liu et al., 2016; Lv et al., 2018). The heart‐specific lncRNA Chaer was reported to interact with PRC2, contributing to the activation of pathological cardiac hypertrophy genes (Wang et al., 2016). lncRNA UCA1 was confirmed to be an activator in cardiomyocyte hypertrophy responses via modulating miR‐184 (G. Zhou, Li, Feng, Zhang, & Fang, 2018). lncRNA XIST is located on the X chromosome in the X‐inactivation center (Brockdorff et al., 1991). Many studies have reported that abnormal XIST expression can lead to human cancers, including gastric cancer, lung cancer, pancreatic cancer, hepatocellular carcinoma, and osteosarcoma (Yang, Jiang, Jiang, & Zhao, 2018; Zhu et al., 2018). Although most of the studies on XIST are focused on tumors, the role of XIST in other diseases is gradually being studied. Recently, T. Zhou et al., (2017) reported that XIST could regulate myocardial infarction by targeting miR‐130a‐3p. In addition, Xu et al. (2018) revealed that knockdown of XIST could alleviate oxidative low‐density lipoprotein‐mediated endothelial cell injury through modulation of the miR‐320/NOD2 axis. However, the role of XIST in cardiac hypertrophy has not been studied. In this study, we demonstrated for the first time that XIST is a necessary modulator in the development of pathological cardiac hypertrophy. The XIST‐overexpressing mice exhibited exaggerated hypertrophic growth in response to pressure overload, which was accompanied by aggravated cardiac function and increased cardiac fibrosis.

Recently, ceRNA has been proposed as a novel regulatory mechanism between noncoding RNA and coding RNA. lncRNAs may function as ceRNAs to sponge miRNAs and competitively interact with the miRNA, leading to decreased mRNA degradation (Lai et al., 2017; Wo et al., 2018). In gastric cancer, XIST was found to act as a tumor suppressor by sponging miR‐101 to modulate EZH2 expression (D. L. Chen et al., 2016). Recently, it was reported that XIST could promote malignancies of esophageal squamous cell carcinoma via the regulation of miR‐101/EZH2 (Wu et al., 2017). In accordance with these studies, our study confirmed that XIST functioned as an endogenous sponge of miR‐101 and functioned as a ceRNA to regulate the expression of TLR2 in cardiomyocytes. First, it was verified that miR‐101 was downregulated in PE‐induced hypertrophic cardiomyocytes, and the overexpression of miR‐101 decreased the enlarged cell surface area and the enhanced mRNA and protein levels of the hypertrophic genes in cardiomyocytes induced by PE. Similarly, it was documented that upregulation of miR‐101 inhibited PE‐induced cardiomyocyte hypertrophy. In addition, it was discovered that XIST was a direct target of miR‐101 by using a luciferase reporter assay and RIP assay. Moreover, upregulation of miR‐101 partially abolished the prohypertrophic effect of XIST overexpression on PE‐induced cardiomyocytes. Together, XIST may be involved in regulating cardiac hypertrophy by targeting miR‐101. Studies have reported that XIST protects cardiomyocyte hypertrophy by targeting miR‐330‐3p (Yao et al., 2015). Additionally, lncRNA XIST regulates myocardial infarction by targeting miR‐330a‐3p (Zhou et al., 2017). However, in our study, XIST exerted deteriorating effects in pressure overload‐induced cardiac hypertrophy and PE‐induced cardiomyocyte hypertrophy by targeting miR‐101. We also detected the level of miR‐330‐3p but did not observe any changes (data not given). These inconsistent results may be due to the different disease models and different cell types, which caused variation in the functional role of XIST.

TLR2 is a member of the TLR family and is expressed not only in immune cells but also in nonimmune cells, including cardiomyocytes, fibroblasts, and vascular endothelial cells (Bagchi et al., 2017; Spurthi et al., 2018). TLR2 plays a central role in the pathogenesis of diverse heart disorders and there are various reports indicating that TLR2 is involved in stress responses under pathological conditions (Bagchi et al., 2017; Trentin‐Sonoda et al., 2015; Wang et al., 2017). In a mouse model, Wang et al. (2017) demonstrated that TLR2 deficiency preserved cardiac function and reduced myocardial fibrosis in sustained pressure overload. Another study by Trentin‐Sonoda et al., (2015) reported that knockout of TLR2 prevents renal ischemia‐reperfusion‐induced cardiac hypertrophy in mice. In the current experiment, we demonstrated that the increased protein level of TLR2 was markedly increased by the overexpression of XIST in hypertrophic mouse hearts. Moreover, the results of the luciferase reporter assay suggested that TLR2 was a direct target of miR‐101. Furthermore, we found that miR‐101 overexpression abolished the effects of high TLR2 expression induced by XIST overexpression. All these data indicated that the effect of XIST on cardiac hypertrophy required the activity of miR‐101.

In summary, the current study reveals the crucial regulation of XIST in cardiac hypertrophy by acting as a ceRNA to increase TLR2 expression by sponging miR‐101, suggesting that XIST could be a promising therapeutic approach for the treatment of cardiac hypertrophy.

CONFLICTS OF INTEREST

The authors declare that there are no conflicts of interest.

ACKNOWLEDGMENTS

This study was supported by the Medical Science and Technology Research Project of Henan Province (Grant No. 201702063) and the National Natural Science Foundation of China (Grant No. 81600191).

Contributor Information

Lu Gao, Email: fccgaol5@zzu.edu.cn.

Ling Li, Email: llliling6@126.com.

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