Introduction

The heart initially develops cardiac hypertrophy under excessive stress as an adaptive response to reduce wall stress and prevent cardiac dysfunction [1, 2]. However, sustained overload causes cardiac dysfunction, eventually leading to heart failure and even sudden death [3,4,5]. Therefore, it is of great importance to explore and uncover the molecular mechanisms of cardiac hypertrophy.

Long non-coding RNAs (lncRNAs) are a group of transcribed RNA molecules with more than 200 nucleotides, which have no obvious protein-coding potential [6]. Previous studies have indicated that the aberrant expression of lncRNAs could be critical in multiple cardiovascular diseases [7]. For instance, Lv et al. reported that lncRNAPlscr4 controlled cardiac hypertrophy by regulating miR-214 [8]. LncRNAs have various pivotal roles with multiple mechanisms, including chromatin remodeling and RNA stability, etc [9,10,11,29, 30]. LncRNAA02Rik, a novel member of lncRNAs, was increased in the cardiac hypertrophy model-a finding that triggered the present study. Through gain- and loss-of-function experiments, artificial lncRNAA02Rik overexpression in normal cardiomyocytes significantly enlarged cell area and enhanced the protein and mRNA levels of hypertrophic markers (BNP and β-MHC). In contrast, silencing lncRNAA02Rik in hypertrophic neonatal mouse ventricular myocytes and TAC-induced hypertrophic mouse hearts markedly ameliorated hypertrophic heart function, decreased cell surface area upregulation and markedly reduced the mRNA and protein levels of BNP and β-MHC. These results indicated that lncRNAA02Rik might have a pro-hypertrophy role, and further detailed studies were warranted to address this phenomenon.

Accumulating evidence has shown that lncRNAs could function as ceRNA to indirectly exert biological functions in various diseases, including cardiac hypertrophy [31, 32]. For example, the lncRNAH19 functioned as a ceRNA to mediate cardiac fibrosis [33]. To explore whether lncRNAA02Rik mediated cardiac hypertrophy by acting as ceRNA, we first conducted bioinformatics analysis using the miRanda software and determined that miR-135a contained potential binding sequences for lncRNAA02Rik. After the prediction, RNA FISH was performed to detect the subcellular localization of lncRNAA02Rik, and the results showed that most lncRNAA02Rik was located in the cytoplasm. In particular, forcing the overexpression or silence of lncRNAA02Rik could weaken or promote miR-135a expression, respectively. Additionally, co-transfection of lncRNAA02Rik and miR-135a could reverse the pro-hypertrophic effect of lncRNAA02Rik. At the same time, luciferase results indicated that lncRNAA02Rik could directly bind to miR-135a. Therefore, we concluded that lncRNAA02Rik promoted cardiac hypertrophy by competitively sponging miR-135a. Next, we investigated the effect of miR-135a on cardiac hypertrophy.

In general, miRNA-mediated gene expression and the regulation of further downstream signaling events exerted an appreciable influence on cardiac hypertrophy progression [34]. However, the role of miR-135a in cardiac hypertrophy has not been reported. In the present study, miR-135a was revealed to be significantly inhibited, both in the TAC-induced mouse heart and cultured mouse myocardial cells following 24 h of Ang-II treatment. We then found that forcing miR-135a overexpression could significantly repress the myocardial cell area and the expression of BNP and β-MHC proteins in vitro. On the contrary, the anti-miR-135a significantly enhanced their expressions in cardiac hypertrophy. Furthermore, to identify the direct target for miR-135a, TargetScan software was used to predict the downstream genes of miR-135a. We found that TCF7 was the most likely target gene for the miR-135a. The luciferase activity and TCF7 protein expression results confirmed that TCF7, a central component of the Wnt signaling pathway whose downstream target was c-Myc, was a direct target for miR-135a. In this study, TCF7 and c-Myc levels were significantly increased in TAC-induced mouse hearts and Ang-II-treated cultured mouse myocardial cells. Furthermore, silencing TCF7 could significantly diminish the cardiomyocyte area and decrease BNP and β-MHC protein levels and mRNA expression. Taken together, the data suggested that the miR-135a protected against cardiac hypertrophy by inhibiting TCF7 expression and then blocking the Wnt signaling pathway. Moreover, the luciferase experiment demonstrated that lncRNAA02Rik sponging miR-135a could abolish the derogation of the target gene TCF7 by miR-135a. Furthermore, co-transfection of lncRNAA02Rik and miR-135a could ameliorate the pro-hypertrophic effect of lncRNAA02Rik. Therefore, lncRNAA02Rik promoted cardiac hypertrophy through the miR-135a/TCF7 signaling pathway.

Taken together, our study revealed that lncRNAA02Rik, as a critical pro-hypertrophic lncRNA, could promote cardiac hypertrophy via the miR-135a/TCF7 signaling pathway, implying that lncRNAA02Rik could be considered as a novel therapeutic target. Accordingly, siA02Rik or other forms of the lncRNAA02Rik inhibitor could be developed into novel therapeutic agents for treating cardiac hypertrophy.

Materials and methods

Bioinformatics analysis of lncRNAA02Rik

To observe the expression patterns of lncRNAA02Rik in mouse hearts and other tissues, we first downloaded the expression profile of lncRNAA02Rik from the MGI database (http://www.informatics.jax.org/). Then, we classified the expression data into “expressed” or “non-expressed” groups based on the expression thresholds of the MGI database and exhibited these in a heatmap. We also observed the transcriptional activity of lncRNAA02Rik in the heart. Briefly, we downloaded ChIP-seq data (histone: H3K4me3 and H3K27ac; Co-factor: P300 and Pol2) of the heart from the UCSC table browsers (http://genome.ucsc.edu/), and data visualization was conducted using the IGV browser with mm9 as the reference genome. In addition, we also downloaded heart development expression data from the ENCODE database (https://www.encodeproject.org/).

Cardiac hypertrophy induced by pressure overload in vivo

All the animal experimental procedures were performed following the Guide for the Care and Use of Laboratory Animals published by the US NIH (publication, 8th Edition, 2011), and approved by the Experimental Animal Ethics Committee of Harbin Medical University-Daqing. Briefly, C57BL/6 mice of 22–24 g (8–10 weeks old) were anesthetized by intraperitoneal injection with sodium pentobarbital (30 mg/kg, i.p.). To visualize the aortic arch, under aseptic conditions, a 2–3 mm longitudinal incision was performed in the proximal sternum. An overlaying blunted 27-gauge needle and a 6-0 silk suture were then used for transverse aorta constriction; the needle was quickly removed to make the constriction. Mice received meloxicam (1.6 mg/kg, s.c.) immediately after surgery and again 12 h postoperatively. Mice were kept warm under a heat lamp and monitored until they were awake and ambulatory, at which time they were returned to their home cages. After 3 weeks, echocardiography analysis was conducted to evaluate cardiac function, including left ventricular posterior wall end-diastolic diameter (LVPWd), left ventricular anterior wall end-diastolic diameter (LVAWd), left ventricular ejection fraction (LVEF), and fractional shortening (FS). Finally, the mice were sacrificed to examine the heart weight (HW), body weight (BW), left ventricular weight (LVW), and tibial length (TL) [8].

Neonatal mouse cardiomyocyte culture

Neonatal mouse cardiomyocytes were harvested as previous study [35,36,37]. Briefly, hearts were obtained from C57BL/6 mice (1 to 3 days old) and repeatedly rinsed with ice-cold phosphate-buffered saline solution. Thereafter, ventricular tissues were minced with scissors and enzymatically dissociated using 0.25% trypsin at 37 °C, and pooled cell suspensions were centrifuged and then resuspended in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. The suspension was coated on the culture bottle for 90 min, allowing the fibroblasts to attach preferentially to the bottom of the bottle. Weak-adherent and non-adherent cells were regarded as cardiomyocytes and transferred to a new culture bottle for further experiments, and 0.01 nmol/L 5-Bromo-2-deoxydriuine was added to exclude the cardiac fibroblasts. Finally, to induce hypertrophy, angiotensin-II (Ang-II) was added to the cardiomyocyte at a concentration of 100 nmol/L for 24 h.

Western blotting

Myocardial cells and heart tissue were lysed in cold radioimmunoprecipitation assay buffer. A bicinchoninic acid protein assay was used to quantify protein samples. Identical quantities of protein samples were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. Thereafter, the membrane was blocked with 5% skim milk at room temperature for 2 h, followed by incubation with the specific primary antibody at 4 °C overnight: anti-BNP antibody (1:1,000 dilution, sc-271185, Santa Cruz), anti-β-MHC antibody (1:2,000 dilution, SAB2106550, Sigma), anti-TCF7 antibody (1:300 dilution, A01315-2, Boster), anti-c-Myc antibody (1:300 dilution, BM4042, Boster), and anti-β-actin antibody (1:2000 dilution, TA-09, ZSGB-BIO). β-actin was used as a loading control. After washing thrice for 10 min each time in Tris-buffered saline with Tween (TBST), the membrane was incubated with the secondary antibody at room temperature for 1 h and washed again in TBST. Subsequently, the bands were scanned using an Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE, USA).

Hematoxylin-eosin (HE) staining

After anesthesia, the hearts were quickly removed and immersed in 4% paraformaldehyde solution for 24 h. Next, the tissue was cut into 5-μm-thick cross-sectional slices. These slices were stained with HE to evaluate histopathology. Photographs were captured using an Olympus BX60 microscope (Olympus Optical, Tokyo, Japan), and cell areas were calculated using image analysis software (Image-Pro Plus 6.0 software).

Immunofluorescence staining

The cells on coverslips were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.2% Triton X-100 for 15 min. Thereafter, goat serum was used to block cells for 30 min at room temperature. α-Smooth muscle actin (α-SMA) antibody (1:200 dilution, #19245, Cell Signaling) and cardiac troponin T antibody (1:100 dilution, A4914, ABclonal) were added, followed by incubation at 4 °C overnight [38, 39]. The second antibody was then added, followed by incubation in the dark for 1 h. The slides were observed under a fluorescence microscope (Leica, Heidelberg, Germany), and cell areas were calculated using image analysis software (Image-Pro Plus 6.0 software).

Quantitative real-time polymerase chain reaction (qPCR)

TRIzol was used to extract the total RNA from the heart tissues of mice or cultured cells following the manufacturer’s protocol. qPCR was performed using a LightCycler® 480 system (Roche, Basel, Switzerland) with SYBR Green I Master Mix (Roche). Each reaction system consisted of 20 ng cDNA. After a 40-cycle reaction, amplification was used to calculate the CT value (ΔCT) of target genes and the difference between the ΔCT of those genes and the 18 s, U6, or GAPDH gene. In addition, the 2−ΔΔCT equation was used to determine the relative amount of lncRNA, miRNA, or mRNA in specific target genes.

The sequences of primers were as follows:

lncRNAA02Rik:

Forward: TGTCCATTCTGAGGGTTCTG

Reverse: ACGATTGAGGACTTCTGGC

miR-135a-5p reverse transcriptase primer:

CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTCACATAG

miR-135a-5p:

Forward: CTGGTAGGTATGGCTTTTTATTC

Reverse: TCAACTGGTGTCGTGGAGTC

miR-22-3p reverse transcriptase primer:

CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACAGTTCT

miR-22-3p:

Forward: ACACTCCAGCTGGGAAGCTGCCAGTTGAAG

Reverse: GGTGTCGTGGAGTCGGCAA

miR-135b-5p reverse transcriptase primer:

GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACTCACAT

miR-135b-5p:

Forward: GGTATGGCTTTTCATTCCT

Reverse: CAGTGCGTGTCGTGGAGT

miR-451a reverse transcriptase primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACGCAA

miR-451a:

Forward: ACACTCCAGCTGGGAAACCGTTACCATTACT

Reverse: CTGGTGTCGTGGAGTCGGCAA

miR-873-5p reverse transcriptase primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGTCAAA

miR-873-5p:

Forward: GCAGGAACTTGTGAG

Reverse: GTGCAGGGTCCGAGGT

β-MHC:

Forward: TATCGATGACCTGGAGCTGA

Reverse: AGTATTGACCTTGTCTTCCTC

BNP:

Forward: ACAGAAGCTGCTGGAGCTGA

Reverse: CCGATCCGGTCTATCTTGTG

GAPDH:

Forward: ACAGCAACAGGGTGGTGGAC

Reverse: TTTGAGGGTGCAGCGAACTT

TCF7:

Forward: TCGGGTGTGGAGAAGACTGGCAT

Reverse: CTGGCTGATGTCCGCTGGTG

c-Myc:

Forward: CGTTGGAAACCCCGCAGACA

Reverse: GATATCCTCACTGGGCGCGG

U6:

Forward: CGCTTCACGAATTTGCGTGTCAT

Reverse: GCTTCGGCAGCACATATACTAAAAT

18s:

Forward: TAGAGGGACAAGTGGCGTTC

Reverse: CGCTGAGCCAGTCAGTGT

Knockdown of lncRNAA02Rik by siRNA and shRNA

LncRNAA02Rik-specific siRNA (siA02Rik) and shRNA (shA02Rik) were commercially manufactured by GenePharma (Shanghai, China) as well as IBSBIO Tech. According to the manufacturer’s protocol, the cells were transfected with siA02Rik/shA02Rik (1 µg/mL) and X-tremeGENE Transfection Reagent (Roche, Penzberg, Germany) with 300 μl Serum-free Medium for 5 min. Subsequently, the two reagents were mixed and combined for 18 min at room temperature, after which the mixture was put into cells for 6 h at 37 °C. Thereafter, cardiomyocytes were maintained in the culture medium for 48 h until subsequent experiments.

The sequences of siA02Rik-1 were Forward: CAGAGACUGUCAAGAGUCAGA, Reverse: UGACUCUUGACAGUCUCUGUA; siA02Rik-2 sequences were Forward: GAAUGAUCACUCUGUUAAAUU, Reverse: UUUAACAGAGUGAUCAUUCCA; siA02Rik-3 sequences were Forward: GGACUGUGCUCAAGGCACAGA, Reverse: UGUGCCUUGAGCACAGUCCUG; the sequences of siRNA negative control (siN.C) were Forward: UUCUCCCAACGUGUCACGUTT, Reverse: ACGUGACACGUUCGGAGAATT; shA02Rik sequences were GGACTGTGCTCAAGGCACAGA; the sequences of shRNA negative control (shN.C) were CCTAAGGTTAAGTCGCCCTCG.

Cells were separated into four groups: (1) Control group; (2) Ang-II group: cells were treated with 100 nmol/L Ang-II for 24 h; (3) Ang-II + siA02Rik/Ang-II + shA02Rik group: cells were transfected with siA02Rik/shA02Rik for 24 h then removed it and treated with 100 nmol/L Ang-II for 24 h; (4) N.C group: cells were transfected with siN.C/shN.C for 24 h then removed it and treated with 100 nmol/L Ang-II for 24 h.

Overexpression of lncRNAA02Rik by plasmid

LncRNAA02Rik cDNA was combined with the pcDNA3.1 vector. An empty vector was regarded as the negative control. Similar to siRNA transfection, plasmid vectors were transfected into cells at a 2.5 mg/L concentration.

Cells were separated into four groups: (1) Control group; (2) lncRANAA02Rik group: lncRNAA02Rik plasmid was transfected into cells for 24 h; (3) Vector group: pcDNA3.1 empty vector was transfected into cells for 24 h; (4) Ang-II group.

miRNA transfection

The mimics and inhibitors of miR-135a-5p were synthesized by GenePharma and the protocol was the same as that for siRNA transfection.

The sequences of miR-135a-5p mimics were Forward: UAUGGCUUUUUAUUCCUAUGUGA, Reverse: ACAUAGGAAUAAAAAGCCAUAUU; miR-135a-5p inhibitor (AMO-miR-135a-5p) sequences were UCACAUAGGAAUAAAAAGCCAUA; negative control (N.C) sequences were Forward: UUCUCCGAACGUGUCACGUTT, Reverse: ACGUGACACGUUCGGAGAATT; negative control inhibitor (AMO-N.C) sequences were CAGUACUUUUGUGUAGUACAA.

Cells were separated into six groups: (1) Control group; (2) Ang-II group; (3) Ang-II + miR-135a group: cells were treated with Ang-II for 24 h then removed and transfected with miR-135a-5p mimics for 24 h; (4) Ang-II + miR-135a+AMO-135a group: cells were treated with Ang-II for 24 h then removed it and transfected with miR-135a-5p mimics and AMO-miR-135a-5p for 24 h; (5) Ang-II + N.C group: cells were treated with Ang-II for 24 h then removed it and transfected with the negative control for 24 h; (6) Ang-II + AMO-N.C group: cells were treated with Ang-II for 24 h then removed it and transfected with the negative control inhibitor for 24 h.

Knockdown of TCF7 by siRNA

siRNA targeting TCF7 (siTCF7), and non-targeting control were synthesized by GenePharma. The transfection of siTCF7 was similar to that of siA02Rik/shA02Rik.

The sequences of siTCF7 were Forward: GGAAGAGAGGACAAGGAAUTT, Reverse: AUUCCUUGUCCUCUCUUCCTT.

The sequences of negative control (N.C) were Forward: UGGAGCAAGUUUGGCAGGAGCUAUU, Reverse: AAUAGCUCCUGCCAAACUUGCUCCA.

Cells were separated into four groups: (1) Control group; (2) Ang-II group; (3) Ang-II + siTCF7 group: cells were treated with Ang-II for 24 h then removed it and transfected with siTCF7 for 24 h; (4) Ang-II + N.C group: cells were treated with Ang-II for 24 h then removed it and transfected with the negative control for 24 h.

Luciferase reporter assays

A lncRNAA02Rik fragment with miR-135a binding sites and TCF7 3′-UTRs was magnified by PCR. Briefly, HEK293 cells were incubated in 48-well culture plates. Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) was used to transfect miR-135a-5p mimics at 10 pmol and luciferase reporter vectors at 40 ng/well. A dual-luciferase reporter assay kit (Promega) was used to detect firefly and renilla luciferase activity. Luciferase activity was normalized using firefly luciferase against renilla luciferase.

Adeno-associated virus-9-shA02Rik (AAV9-shA02Rik) infection in vivo

AAV9-shA02Rik and AAV9-shN.C were injected into mice via the tail vein (units: 1.2 × 1012 vg/mL, 100 μl, IBSBIO Tech). Sham group mice were treated similarly to TAC group mice but with 100 μl saline.

Mice were separated into four groups: (1) Sham group; (2) TAC + N.C group: saline was injected into mice on the second day after TAC for 3 weeks; (3) TAC + shA02Rik group: AAV9-shA02Rik was injected into mice on the second day after TAC for 3 weeks; (4) TAC + shN.C group: AAV9-shN.C was injected into mice on the second day after TAC for 3 weeks.

Statistical analysis

All statistical analysis was performed using SPSSv19.0 (SPSS Inc, Chicago, IL, USA), and data were presented as mean ± SD. The Student’s t-test was used to compare differences between two groups. A one-way ANOVA was used to compare differences among groups. If the ANOVA was significant, SNK-q was used to evaluate the statistical significance of differences between the two groups. P < 0.05 was considered to be statistically significant.