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. 2019 Jan 16;234(8):14319–14329. doi: 10.1002/jcp.28131

SP1‐SYNE1‐AS1‐miR‐525‐5p feedback loop regulates Ang‐II‐induced cardiac hypertrophy

Ye Wang 1,†,✉, Rongyi Cao 2,†, Wenwen Yang 3, Bangruo Qi 4,✉
PMCID: PMC13482839  PMID: 30652310

Abstract

Cardiac hypertrophy (CH) has become a huge threat to human health. Recent years, long noncoding RNAs (lncRNAs) have been studied in human diseases, including CH. According to bioinformatics analysis, 10 lncRNAs possibly involved in the progression of CH were screened out. Among which, lncRNA SYNE1 antisense RNA 1 (SYNE1‐AS1) could be upregulated by Angiotensin II (Ang‐II) in cardiomyocytes. Thus, we chose SYNE1‐AS1 to do further study. To identify the biological function of SYNE1‐AS1 in CH, SYNE1‐AS1 was silenced in Ang‐II‐induced cardiomyocytes. Results of immunofluorescence staining demonstrated that increased cell surface area in Ang‐II‐induced cardiomyocytes was reduced by SYNE1‐AS1 knockdown. Moreover, the hypertrophic responses were attenuated by SYNE1‐AS1 knockdown. Mechanically, SYNE1‐AS1 positively regulated Sp1 transcription factor (SP1) by sponging microRNA‐525‐5p (miR‐525‐5p). On the basis of previous reports, SP1 can transcriptionally activate lncRNAs. Therefore, we investigated the interaction between SP1 and SYNE1‐AS1 promoter. Intriguingly, SYNE1‐AS1 was activated by SP1. At last, rescue assays demonstrated the function of SP1‐SYNE1‐AS1 axis in CH. In conclusion, SP1‐induced upregulation of lncRNA SYNE1‐AS1 promoted CH via miR‐525‐5p/SP1 axis.

Keywords: Ang‐II, cardiac hypertrophy, miR‐525‐5p, SP1, SYNE1‐AS1

Long noncoding RNA SYNE1 antisense RNA 1 (SYNE1‐AS1) was upregulated in cardiac hypertrophy in vivo and in cardiomyocytes treated with Angiotensin II knockdown of SYNE1‐AS1 attenuated cardiac hypertrophy SYNE1‐AS1 acted as a molecular sponge of microRNA‐525‐5p (miR‐525‐5p). MiR‐525‐5p attenuated the hypertrophic responses.

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1. INTRODUCTION

Cardiac hypertrophy (CH) is an organic response of the heart to multiple stresses to protect and sustain normal cardiac function at the early stage (Liu et al., 2016). Nevertheless, continuous CH with undesirable cardiac reprogramming often results in increased risk for heart failure and cardiac death (Frey & Olson, 2003; Harvey & Leinwand, 2011). Despite that various specialized peptide hormones, growth factors, and RNAs have been determined to be the regulators of CH (Braunwald, 2015; Lyon, Zanella, Omens, & Sheikh, 2015), the potential molecular mechanisms about CH are still largely unknown. The aim of this study is to explore novel molecular regulators in CH.

To our knowledge, the majority of mammalian genome is transcribed to noncoding RNAs (ncRNAs; 2012). ncRNAs are crucial factors in regulating the physiological functions of mammalian cells (Esteller, 2011; Jandura & Krause, 2017; Klingenberg, Matsuda, Diederichs, & Patel, 2017). On the basis of length, ncRNAs are classified into two groups. One group is long noncoding RNAs (lncRNAs) that are longer than 200 nucleotides (nt). The other group is microRNAs (miRNAs) whose length between 20 and 24 nt. Both lncRNAs and miRNAs have been demonstrated to be crucial regulators in the progression of CH (Care et al., 2007; Micheletti et al., 2017; Uchid, 2017a; Wang et al., 2014, 2016). Dysregulation of lncRNAs has been demonstrated to be one of the leading causes of CH (Lv et al., 2018; Zhou, Li, Feng, Zhang, & Fang, 2018). In this study, we chose 10 lncRNAs, which is possibly involved in CH progression from GeneCard. To identify the potential correlation between these 10 lncRNAs and CH progression, we examined the fold change of these 10 lncRNAs in the sham group and transverse aortic constriction (TAC) group. The results showed that three lncRNAs were differentially expressed in the TAC group. Angiotensin II (Ang‐II) is known as a drug which can induce CH (Lai et al., 2017; L. Wang et al., 2018). Next, we examined the expression condition of those three lncRNAs in Ang‐II‐induced cardiomyocytes. As a result, only the expression of lncRNA SYNE1 antisense RNA 1 (SYNE1‐AS1) was increased in Ang‐II‐induced cardiomyocytes. Further experiments demonstrated that SYNE1‐AS1 facilitated CH.

According to previous studies, lncRNAs can act as competing endogenous RNAs) by sponging miRNAs to upregulate messenger RNAs (mRNAs; Y. Li, Wang, Sun, & Zhu, 2018; Wo, Guo, Li, Yang, & Wo, 2018). Here, we investigated whether SYNE1‐AS1 can regulate CH in the same manner. Mechanism investigation revealed the potential interaction between SYNE1‐AS1 and microRNA‐525‐5p (miR‐525‐5p). The previous study has reported the potential role of miR‐525‐5p in ischemia/reperfusion injury‐induced neuronal cell death (Zhao, Hua, Li, Sun, & Wu, 2015). However, the role of miR‐525‐5p in CH is almost unknown. Thus, we further determined the effect of miR‐525‐5p on the hypertrophic response. Further mechanism analysis revealed the downstream target of miR‐525‐5p. Sp1 transcription factor (SP1) has been demonstrated to be a transcription activator of lncRNAs (Chen, Zeng et al., 2018; H. Dong et al., 2018). Bioinformatics analysis and mechanism experiments certified that SP1 contributed to the transcription of SYNE1‐AS1. Rescue assays were conducted to demonstrate the involvement of SP1 in SYNE1‐AS1‐mediated hypertrophic responses. In conclusion, our study indicated that SP1‐induced upregulation of lncRNA SYNE1‐AS1 facilitated Ang‐II‐induced CH by regulating miR‐525‐5p/SP1 axis.

2. MATERIALS AND METHODS

2.1. Animal specimens

The 8‐week male C57BL6 mice (SPF, 20–25 g) obtained from Vital River Laboratory Animal Company (Beijing, China) were used to construct mouse model. The processes involved in this assay were carried out in accordance with the guidance of the Care and Use of Laboratory Animals published by the Ethics Committee of First Affiliated Hospital of Harbin Medical University. The TAC was applied to induce CH of the mouse model. In summary, after the anesthetization by using intraperitoneal ketamine (100 mg/kg) and xylazine (10 mg/kg), the transverse thoracic aorta of the mice was dissected. Later, the mice were placed on a ventilator for recovery with the core temperature at 37°C. The mice with a pressure gradient of less than 60 mmHg on the basis of the echocardiography in a week after TAC were excluded in this experiment. Anatomic M‐mode echocardiography from mice with matched echocardiographic images of the aortic arch was used to detect the stenosis of the transverse aorta. The sham operation for the corresponding matched mice experienced the same process, without the seam for the aorta.

2.2. Cardiomyocyte culture

Cardiomyocytes were isolated from the neonatal mice, followed by the relevant preparation like before (Wang et al., 2012). The isolated cardiomyocytes were divided into small pieces. Subsequently, the tissues were put into the digestion solution containing 0.1% of collagenase type IV, 0.1% of trypsin, 15 μg/ml DNase I, and 1% of chicken serum in HEPES‐buffered saline at 37°C. Next, the trypsin was neutralized with the adding of 10% of calf serum. After the centrifugation, the resolved cells were resuspended in Dulbecco's modified Eagle's medium/F12 with supplements, followed by the plate on the collagen‐coated silicone sheet in preparation for the cardiomyocytes extraction. The serum‐free medium was used to replace the cultivation medium at 24 hr after the seeding and the cultivation continued for another 24 hr before the assay. All the cells were treated with Ang‐II ahead of the assay.

2.3. Plasmid construction and transfection

The short hairpin RNA (shRNA) targeting SYNE1‐AS1 (sh‐SYNE1‐AS1) and control shRNA (sh‐NC) was purchased from Santa Cruz (Santa Cruz Biotechnology, San Diego, CA). For SYNE1‐AS1 and SP1 overexpression, the whole sequences of them were separately cloned into a pcDNA3.1 vector (Invitrogen, Carlsbad, CA). MiR‐525‐5p mimics and control mimics (NC) were purchased from GenePharma (Shanghai, China). Based on the manufacturers' protocol, Lipofectamine 2000 (Invitrogen) was used for transfection.

2.4. Quantitative reverse transcription‐polymerase chain reaction

RNA isolation was conducted by using TRIZOL reagent (Invitrogen). Reverse transcription was finished using 5XAll‐In‐One RT MasterMix (abmGood, Vancouver, Canada). Real‐time qPCR was carried out on an ABI 7500 fast real‐time PCR system (Applied Biosystems, Foster City, CA) using the EvaGreen qPCR Mastermix Kit (abmGood). GAPDH and U6 were used for normalization, respectively. The 2−ΔΔCt method was used to evaluate the fold change of all genes. The primers are listed in Supporting Information Table S1.

2.5. Northern blot

The level of SYNE1‐AS1 was measured by northern blot with an Ambion Northern Max‐Gly Kit (Austin, TX). Total RNA was isolated using TRIZOL reagent (Invitrogen). Probes for SYNE1‐AS1 were obtained using Biotin RNA labeling mix (Roche, Basel, Switzerland). RNA samples were segregated by electrophoresis and transferred onto nitrocellulose membrane. The membranes were then incubated with hydration buffer with probes. At last, Chemiluminescent Nucleic Acid Detection Module (Thermo Fisher Scientific, Waltham, MA) was used to detect RNA signals.

2.6. Western blot

The cultivated cells were lysed on ice by using mammalian PRO‐PREP solution (Thermo Fisher Scientific) and protease inhibitors (Sigma‐Aldrich, St. Louis, MO). After the centrifugation, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was used to split cell lysate protein (40 μg), which was then transferred onto polyvinylidene fluoride membranes (Bio‐Rad, Hercules, CA). Five percentage of milk powder was used to seal membranes in TBST, and then the immunoblotting was performed with primary antibodies such as an atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and β‐myosin heavy chain (β‐MHC) (dilution of 1:1,000; Abcam, Cambridge, UK). GAPDH was applied to be a loading control. The protein bands were determined by using HRP‐binate secondary antibodies (Abcam) and improved chemiluminescence (Pierce, Rockford, ILA). Digital images of luminescence were detected by Alpha Imager (FluorChem5500TM, Alpha Innotech, San Leandro, CA).

2.7. Cell surface area measurement

The cells were fixed and permeabilized with 4% formaldehyde and 0.1% Triton X‐100 in phosphate buffered saline, respectively. Afterward, cells were stained with α‐actin (Abcam, Cambridge, MA) at 4°C overnight. A daylight 594 goat antimouse antibody was used to incubate cells for 1 hr at room temperature. Before the immunofluorescence capture, cells were incubated with 4′,6‐diamidino‐2‐phenylindole for 10 min. A fluorescence microscope(37081; Carl Zeiss Meditec, Dublin, CA) was utilized for visualizing the immunofluorescence. Cell surface area was quantified by measuring 50 random cells from three independent experiments. The average value was recorded for analysis. Finally, the Image‐Pro Plus 6.0 (Media Cybernetics, Silver Spring, MD) software was used to calculate the surface areas.

2.8. Luciferase reporter assay

The gene for luciferase reporter analysis was cloned by using the specific plasmid. The luciferase reporter plasmid containing SYNE1‐AS1 sequence was constructed by Ribo Company (Guangzhou, Guangdong, China). Then, the Renilla luciferase plasmid was utilized to transfect the plasmids into the cells. Briefly, the cells were cotransfected with 50 or 100 nM NC or Mimics and 0.4 μg of the luciferase reporter vector on the 12‐well plates. After 48 hr of transfection, the cell lysates were ready. Subsequently, a Monolight 3010 luminometer (Pharmingen, San Diego, California) was used to detect the luciferase activity. The transfections were performed in triplicate.

The two binding sequences between SP1 and SYNE1‐AS1 promoter were obtained from JASPAR. To analyze the binding sequence that was responsible for the interaction between SP1 and SYNE1‐AS1 promoter. The two sequences and the mutant binding site 1 (MUT1) or mutant binding site 2 (MUT2) were synthesized and inserted into a pGL3‐basic vector, and then cotransfected with SP1 expression vector into cardiomyocytes. Relative luciferase activities were assessed using the Dual Luciferase Assay Kit (Promega Corporation Madison, WI).

2.9. Chromatin immunoprecipitation assay

Chromatin immunoprecipitation (ChIP) assay was carried out as previously described (Guo et al., 2016). A SP1‐specific antibody (Millipore) and the EZ‐Magna ChIP A/G Kit (Millipore, Billerica, MA) were used to conduct ChIP in cardiomyocytes.

2.10. Statistical analysis

The data from three independent experiments were displayed as the mean ± SD. Student's t test was applied to detect the statistical significance between the two groups. One‐way analysis of variance and Tukey's multiple comparison tests were performed to calculate data from assays. p < 0.05 was identified to be statistically significant. Statistical analysis software GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA) was applied to analyze the data.

3. RESULTS

3.1. LncRNA SYNE1‐AS1 was upregulated in CH in vivo and in cardiomyocytes treated with Ang‐II

At first, we operated TAC surgery for C57BL/6 mice. The mice undergone sham operation was taken as the control group (sham group). Atrial natriuretic factor (ANF), BNP, and β‐MHC are three hypertrophic markers (Chien, Knowlton, Zhu, & Chien, 1991; Jiang, Zhou, & Huang, 2016). In this study, the expression of these three markers was higher in TAC group (n = 12) than that in the sham group (n = 12) (Figure 1a), indicating the successful construction of TAC and sham group. Ten lncRNAs associated with CH were chosen from GeneCard (https://www.genecards.org/) and subjected to quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis in the TAC group and sham group. The results showed that three lncRNAs were significantly upregulated in the TAC group (Figure 1b). Ang‐II is known as an inducer of CH (Wang et al., 2014). Thus, we treated cardiomyocytes with different concentrations of Ang‐II (0, 0.5, and 1.0 mmol/L). The expression level of lncRNA SYNE1‐AS1 was significantly increased in response to the treatment of Ang‐II (Figure 1c). Therefore, we chose SYNE1‐AS1 to do subsequent experiments. The expression level of SYNE1‐AS1 in the sham group and TAC group was examined and shown in Figure 1d. All these results suggested the potential association of SYNE1‐AS1 with hypertrophic responses.

Figure 1.

Figure 1

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LncRNA SYNE1‐AS1 was upregulated in CH in vivo and in cardiomyocytes treated with Ang‐II. (a) The relative higher expression of ANF, BNP, and β‐MHC was found in the TAC group (n = 12). (b) The fold change of ten lncRNAs chosen from GeneCard was examined in the TAC group and sham group. (c) The expression levels of three lncRNAs (SYNE1‐AS1, LINC01312, and LINC00850) were tested in cardiomyocytes treated with different concentrations of Ang‐II (0, 0.5, and 1.0 mmol/L). (d) The expression pattern of SYNE1‐AS1 in the TAC group and sham group was examined with qRT‐PCR. *p < 0.05, **p < 0.01 versus control group. ANF: atrial natriuretic factor; Ang‐II: angiotensin II; BNP: brain natriuretic peptide; CH: cardiac hypertrophy; lncRNA: long noncoding RNA; qRT‐PCR: quantitative reverse transcription‐polymerase chain reaction; SYNE1‐AS1: SYNE1 antisense RNA 1; TAC: transverse aortic constriction; β‐MHC: β‐myosin heavy chain [Color figure can be viewed at wileyonlinelibrary.com]

3.2. Knockdown of SYNE1‐AS1 attenuated CH

To investigate the correlation between SYNE1‐AS1 expression and CH progression, we silenced SYNE1‐AS1 in Ang‐II‐induced cardiomyocytes by stably transfecting with sh‐SYNE1‐AS1 (Figure 2a). Immunofluorescence indicated that SYNE1‐AS1 knockdown attenuated the effect of Ang‐II on cell surface area (Figure 2b). Furthermore, knockdown of SYNE1‐AS1 efficiently decreased levels of hypertrophic markers (ANF, BNP, and β‐MHC) in Ang‐II‐induced cardiomyocytes (Figure 2c,d). We also overexpressed SYNE1‐AS1 in Ang‐II‐induced cardiomyocytes (Supporting Information Figure S1a). According to the result of immunofluorescence, overexpression of SYNE1‐AS1 led to the increased size of the cell surface area (Supporting Information Figure S1b). These data revealed the role of SYNE1‐AS1 in Ang‐II‐induced hypertrophic responses.

Figure 2.

Figure 2

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Knockdown of SYNE1‐AS1 attenuated cardiac hypertrophy. (a) SYNE1‐AS1 was silenced in Ang‐II‐induced cardiomyocytes by stably transfecting with sh‐SYNE1‐AS1. (b) The effect of SYNE1‐AS1 knockdown on the cell surface area of Ang‐II‐induced cardiomyocytes was assessed by immunofluorescence staining. (c,d) qRT‐PCR and western blot assays were conducted to examine the mRNA and protein levels of ANF, BNP, and β‐MHC in Ang‐II‐induced cardiomyocytes transfected with sh‐SYNE1‐AS1 or sh‐NC. *p < 0.05, **p < 0.01 versus control group. ANF: atrial natriuretic factor; Ang‐II: angiotensin II; BNP: brain natriuretic peptide; mRNA: messenger RNA; qRT‐PCR: quantitative reverse transcription‐polymerase chain reaction; SYNE1‐AS1: SYNE1 antisense RNA 1; sh‐NC: control shRNA; β‐MHC: β‐myosin heavy chain [Color figure can be viewed at wileyonlinelibrary.com]

3.3. SYNE1‐AS1 acted as a molecular sponge of miR‐525‐5p

LncRNAs can regulate CH by sponging miRNAs (Z. Li et al., 2018; Lv et al., 2018). In this study, we explored the downstream miRNAs of SYNE1‐AS1 by using bioinformatics analysis. We found top 17 miRNAs (binding score >0.8) that can bind with SYNE1‐AS1 from DIANA (http://carolina.imis.athena‐innovation.gr/diana_tools/web/index.php). To investigate whether these miRNAs could be regulated by SYNE1‐AS1, we separately overexpressed and silenced SYNE1‐AS1 in cardiomyocytes (Figure 3a) to examine the expression changes of these miRNAs. It was found that the expressions of miR‐525‐5p and miR‐885‐5p were negatively regulated by SYNE1‐AS1 (Figure 3b). In addition, miR‐525‐5p was found to be expressed lower in the TAC group (Figure 3c), which is opposite with SYNE1‐AS1 (Figure 3d). Therefore, we further demonstrated the interaction between SYNE1‐AS1 and miR‐525‐5p. Moreover, the binding sequence between SYNE1‐AS1 and miR‐525‐5p was predicted (Figure 3e). A dual luciferase reporter assay was conducted to demonstrate the interaction between SYNE1‐AS1 and miR‐525‐5p. The experimental results indicated that miR‐525‐5p overexpression reduced the luciferase activity of wild‐type SYNE1‐AS1 (SYNE1‐AS1‐WT) but not that of mutant type SYNE1‐AS1 (SYNE1‐AS1‐MUT; Figure 3f), indicating the interaction between SYNE1‐AS1 and miR‐525‐5p.

Figure 3.

Figure 3

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SYNE1‐AS1 acted as a molecular sponge of miR‐525‐5p. (a) SYNE1‐AS1 was silenced or overexpressed in cardiomyocytes by transfecting with sh‐SYNE1‐AS1 or SYNE1‐AS1 expression vector. (b) The expression changes of all 17 miRNAs were tested in cardiomyocytes transfected with sh‐SYNE1‐AS1 or SYNE1‐AS1 expression vector. (c) The expression levels of miR‐885‐5p and miR‐525‐5p were detected in the TAC group and sham group. (d) The negative expression association between SYNE1‐AS1 and miR‐525‐5p in the TAC group was analyzed. (e) The binding sequence between SYNE1‐AS1 and miR‐525‐5p was predicted. (f) A luciferase reporter assay was conducted to demonstrate the interaction between SYNE1‐AS1 and miR‐525‐5p. *p < 0.05, **p < 0.01 vs control group. N.S: no significance. miRNAs: microRNAs; SYNE1‐AS1: SYNE1 antisense RNA 1; TAC: transverse aortic constriction [Color figure can be viewed at wileyonlinelibrary.com]

3.4. miR‐525‐5p attenuated the hypertrophic responses

To identify the effect of miR‐525‐5p on hypertrophic responses of cardiomyocytes, we overexpressed miR‐525‐5p in Ang‐II‐induced cardiomyocytes by transfecting with miR‐525‐5p mimics (Figure 4a). Immunofluorescence suggested that miR‐525‐5p mimics attenuated the effect of Ang‐II on cell surface area (Figure 4b). Moreover, the increased levels of ANF, BNP, and β‐MHC in Ang‐II‐induced cardiomyocytes were efficiently decreased by miR‐525‐5p mimics (Figure 4c,d). Therefore, we confirmed that miR‐525‐5p attenuated the hypertrophic responses in cardiomyocytes treated with Ang‐II.

Figure 4.

Figure 4

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miR‐525‐5p attenuated the hypertrophic responses. (a) miR‐525‐5p was overexpressed in Ang‐II‐induced cardiomyocytes by transfecting with miR‐525‐5p mimics. (b) Immunofluorescence staining suggested that attenuated miR‐525‐5p mimics attenuated the effect of Ang‐II on the cell surface area. (c,d) The increased levels of ANF, BNP, and β‐MHC in Ang‐II‐induced cardiomyocytes were efficiently decreased by miR‐525‐5p mimics. *p < 0.05, **p < 0.01 vs control group. ANF: atrial natriuretic factor; Ang‐II: angiotensin II; BNP: brain natriuretic peptide; β‐MHC: β‐myosin heavy chain [Color figure can be viewed at wileyonlinelibrary.com]

3.5. SYNE1‐AS1 enhanced the expression of SP1 by sponging miR‐525‐5p

To our knowledge, lncRNAs can regulate gene expression by sponging miRNAs (Ling et al., 2013; Shan et al., 2016). In the present study, we found 23 potential targets of miR‐525‐5p by using two bioinformatics prediction tools (PITA and RNA22; Figure 5a). Among which, SP1 has been reported both in CH and miRNA‐mRNA axis (Domínguez, 2013; Ji et al., 2017; Sack, Disch, Rockman, & Kelly, 1997; Xia et al., 2017). Therefore, we chose SP1 to do further study. The relative high expression of SP1 was examined in the TAC group (Figure 5b), which was opposite to miR‐525‐5p (Figure 5c) but was consistent with SYNE1‐AS1 (Figure 5d). The putative binding sequence between miR‐525‐5p and SP1 was predicted and illustrated (Figure 5e). Similarly, a luciferase reporter assay was conducted to demonstrate the interaction between miR‐525‐5p and SP1. As shown in Figure 5f, overexpression of miR‐525‐5p led to the decreased luciferase activity of SP1 3′UTR (SP1‐WT). Whereas, the luciferase activity of SP‐MUT was not significantly changed. The same results were observed when cardiomyocytes were transfected with sh‐SYNE1‐AS1 (Figure 5g). Finally, both mRNA and protein levels of SP1 were decreased in response to the upregulation of miR‐525‐5p or downregulation of SYNE1‐AS1 (Figure 5h). On the basis of the above, we confirmed that SYNE1‐AS1 positively regulated SP1 by sponging miR‐525‐5p.

Figure 5.

Figure 5

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SYNE1‐AS1 enhanced the expression of SP1 by sponging miR‐525‐5p. (a) 23 potential targets of miR‐525‐5p were predicted by using two bioinformatics prediction tools (PITA and RNA22). (b) SP1 was found to be highly expressed in the TAC group. (c) The negative correlation between miR‐525‐5p and SP1 in the TAC group was analyzed. (d) The positive correlation between SYNE1‐AS1 and SP1 in the TAC group was analyzed. (e) The putative binding sequence between miR‐525‐5p and SP1 was predicted. (f) Luciferase reporter assay was conducted to demonstrate the interaction between miR‐525‐5p and SP1. (g) The luciferase activity of SP1‐WT or SP1‐MUT was evaluated in cardiomyocytes treated with sh‐SYNE1‐AS1 or sh‐NC. (h) The mRNA and protein levels of SP1 were detected in cardiomyocytes transfected with sh‐SYNE1‐AS1 or SYNE1‐AS1 expression vector. *p < 0.05, **p < 0.01 vs control group. SYNE1‐AS1: SYNE1 antisense RNA 1; TAC: transverse aortic constriction [Color figure can be viewed at wileyonlinelibrary.com]

3.6. Transcription factor SP1 contributed to SYNE1‐AS1 transcription

Furthermore, we explored the molecular mechanism that contributed to the upregulation of SYNE1‐AS1. According to the ChIP‐seq result of UCSC (http://genome.ucsc.edu/), SP1 is a potential transcription factor of SYNE1‐AS1 (Figure 6a). To determine the regulatory effect of SP1 on the SNYE1‐AS1 expression, SP1 was separately silenced or overexpressed in cardiomyocytes (Figure 6b). qRT‐PCR and northern blot assays showed that SP1 positively regulated SYNE1‐AS1 in cardiomyocytes (Figure 6c,d). The binding motif of SP1 obtained from JASPAR (http://jaspar.genereg.net/) was illustrated (Figure 6e). Next, we chose the top two binding sites of SP1 to SYNE1‐AS1 promoter to do further study. Luciferase activity analysis showed that the luciferase activity of SYNE1‐AS1 promoter containing two putative binding sites (WT) with SP1 was efficiently increased by SP1. In addition, a mutation in the putative binding sites 1 (from −139 to − 148 bp) did not affect the luciferase activity of SNYE1‐AS1. Effect of SP1 on the luciferase activity of SNYE1‐AS1 promoter was abolished after mutation of binding site 2 (from −898 to − 907 bp; Figure 6f). Therefore, we confirmed that site 2 was responsible for the interaction between SP1 and SYNE1‐AS1 promoter. Furthermore, ChIP assay certified the affinity of SP1 to SNYE1‐AS1 promoter (Figure 6g). Taken together, lncRNA SNYE1‐AS1 was activated SP1 and facilitated CH by regulating the miR‐525‐5p/SP1 axis.

Figure 6.

Figure 6

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Transcription factor SP1 contributed to SYNE1‐AS1 transcription. (a) The ChIP‐seq result of UCSC showed that SP1 is a potential transcription factor of SYNE1‐AS1. (b) SP1 was silenced or overexpressed in cardiomyocytes by transfecting with sh‐SP1 or SP1 expression vector. (c,d) The expression level of SYNE1‐AS1 was examined in cardiomyocytes transfected with sh‐SP1 or SP1 expression vector. (e) The binding motif of SP1 obtained from JASPAR was illustrated. (f) Luciferase activity analysis showed that site 2 was responsible for the interaction between SP1 and SYNE1‐AS1 promoter. (g) ChIP assay certified the affinity of SP1 to SNYE1‐AS1 promoter. **p < 0.01 vs control group. SYNE1‐AS1: SYNE1 antisense RNA 1 [Color figure can be viewed at wileyonlinelibrary.com]

3.7. SP1 involved in SYNE1‐AS1‐mediated hypertrophic responses in Ang‐II‐induced hypertrophic responses

We conducted rescue assays to demonstrate the function of the SP1‐SYNE1‐AS1 axis in CH. At first, both qRT‐PCR and northern blot assays revealed that SP1 overexpression partially rescued the decreased expression level of SYNE1‐AS1 in Ang‐II‐induced‐cardiomyocytes transfected with sh‐SYNE1‐AS1 (Figure 7a,b). Moreover, the inhibitory effect of SYNE1‐AS1 on Ang‐II‐induced hypertrophic responses was partially reversed by cotransfecting with pcDNA‐SP1 (Figure 7c–f). These data indicated that SP1‐induced upregulation of lncRNA SYNE1‐AS1 facilitated CH.

Figure 7.

Figure 7

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SP1 involved in SYNE1‐AS1‐mediated hypertrophic responses in Ang‐II‐induced hypertrophic responses. (a,b) Both qRT‐PCR and northern blot assays revealed that SP1 overexpression partially rescued the decreased expression of SYNE1‐AS1 caused by sh‐SYNE1‐AS1 in Ang‐II‐induced‐cardiomyocytes. (c–f) The inhibitory effect of SYNE1‐AS1 on Ang‐II‐induced hypertrophic responses was partially reversed by co‐transfecting with pcDNA‐SP1. *p < 0.05, **p < 0.01 vs control group. Ang‐II: angiotensin II; qRT‐PCR: quantitative reverse transcription‐polymerase chain reaction; SYNE1‐AS1: SYNE1 antisense RNA 1 [Color figure can be viewed at wileyonlinelibrary.com]

4. DISCUSSION

During the process of cardiac reprogramming, cardiomyocytes are the most primary cell type, and gene expression is the basis of pathological hypertrophic cardiomyocytes (Rohini, Agrawal, Koyani, & Singh, 2010). In addition, the dysregulation of the fetal genes like ANP, BNP, and β‐MHC were vital factors for the molecular and cellular events engaged in the development of myocytes (Bishopric & Kedes, 1991; Gardner, 2003).

Recently, accumulating studies have demonstrated that lncRNAs also play critical functions in heart development and diseases (Grote et al., 2013; Haddad et al., 2006; Klattenhoff et al., 2013; Visel et al., 2010), among which only certain lncRNAs are acknowledged to be regulators for CH (Shao et al., 2017; Song et al., 2016; Uchida, 2017a). In this study, we examined the expression of ten lncRNAs which were chosen from GeneCard. Among which, only three lncRNAs were differentially expressed in TAC group. Subsequently, the expression of these three lncRNAs was assessed in cardiomyocytes treated with different concentrations of Ang‐II. qRT‐PCR examination revealed that the expression level of lncRNA SYNE1‐AS1 was increased in cardiomyocytes that were treated with increasing concentrations of Ang‐II. Considering the upregulation of SYNE1‐AS1 in both TAC group and Ang‐II‐induced cardiomyocytes, we silenced SYNE1‐AS1 to examine the hypertrophic responses. Interestingly, SYNE1‐AS1 knockdown led to the smaller cell size, indirectly reflecting the inhibitory effect of SYNE1‐AS1 knockdown on CH. Moreover, Ang‐II‐induced upregulations of ANF, BNP, and β‐MHC were partially rescued by SYNE1‐AS1 knockdown. Based on these findings, we confirmed that upregulation of SYNE1‐AS1 contributed to the CH.

It has been documented that lncRNAs can regulate CH by sponging miRNAs (Wang et al., 2014). Based on the bioinformatics analysis, we predicted some miRNAs that can bind with SYNE1‐AS1. Among which, miR‐525‐5p was negatively regulated by SYNE1‐AS1. Moreover, the expression of miR‐525‐5p was opposite with SYNE1‐AS1 in the TAC group. Further mechanism experiments suggested that miR‐525‐5p could bind with SYNE1‐AS1 in cardiomyocytes. To demonstrate that miR‐525‐5p can affect SYNE1‐AS1‐medited CH, we conducted experiments to identify the function of miR‐525‐5p. As a result, we found that miR‐525‐5p suppressed the hypertrophic progression.

Previous reports revealed that lncRNAs can regulate gene expression in CH by sponging miRNAs (Z. Li et al., 2018; Wei, Chen, Gao, & Li, 2017). In this study, we conducted mechanism investigation to detect the target mRNA of miR‐525‐5p. Based on the bioinformatics analysis, 23 potential targets of miR‐525‐5p were screened out. Among which, SP1 has been reported as a potential biomarker in CH and a target of miRNAs (Z. Dong et al., 2017; Hu et al., 2011; F. Wang et al., 2013). Therefore, we chose SP1 to do further study. Intriguingly, the results of mechanism experiments demonstrated that SYNE1‐AS1 positively regulated SP1 by sponging miR‐525‐5p. According to previous studies, SP1 is a transcription factor that can promote the transcription of lncRNAs (Chen, Xie et al., 2018; W. Wang et al., 2018). According to the ChIP‐seq result of UCSC, SP1 is a potential transcription factor of SYNE1‐AS1. Therefore, we hypothesized that SYNE1‐AS1 might be activated by SP1 to form a feedback loop. At first, we found that the SYNE1‐AS1 expression was positively regulated by SP1. Furthermore, ChIP assay and luciferase reporter assay validated the binding of SP1 to SYNE1‐AS1 promoter. Collectively, we confirmed that SP1‐SYNE1‐AS1‐miR‐525‐5p feedback loop regulated CH. Finally, rescue assays were conducted to demonstrate the function of SP1‐SYNE1‐AS1 axis in cardiac hypertrophy. The results showed that SP1 partially reversed the effect of SYNE1‐AS1 knockdown on hypertrophic responses, indicating that SYNE1‐AS1 and SP1 synergistically regulated CH. All our findings revealed that SP1‐activated lncRNA SYNE1‐AS1 facilitated CH by regulating the miR‐525‐5p/SP1 axis (Supporting Information Figure S2). Our research findings may shed light on finding a novel therapeutic target for CH.

CONFLICTS OF INTEREST

The authors declare that they have no conflicts of interest.

Supporting information

Supporting Information

JCP-234-14319-s001.tif (391.5KB, tif)

Supporting Information

JCP-234-14319-s003.tif (200KB, tif)

Supporting Information

JCP-234-14319-s002.xls (28.5KB, xls)

ACKNOWLEDGEMENT

The authors sincerely appreciate all lab members.

Contributor Information

Ye Wang, Email: wangye_cry@163.com.

Bangruo Qi, Email: qibangruo@163.com.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Information

JCP-234-14319-s001.tif (391.5KB, tif)

Supporting Information

JCP-234-14319-s003.tif (200KB, tif)

Supporting Information

JCP-234-14319-s002.xls (28.5KB, xls)

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