Animals

Sprague-Dawley (SD) rats aged 1–3 days and male C57BL/6J mice aged 5 or 9 weeks were purchased from QingLongShan Animal Breeding Field (Jiangsu, China) and used in this study. The animals were housed in a specific pathogen-free environment under a 12-h light/dark cycle and could freely access food and water.

Myocardial hypertrophy model in vivo

The mice aged 9 weeks were randomly divided into myocardial hypertrophy or control group (n = 5 per group). Myocardial hypertrophy in mice was induced by chronic infusion of Ang-II (MCE) at a rate of 1000 ng/kg/min for 3 weeks using osmotic minipumps (Alzet Model 2004), and the control group mice were infused with saline at the same dose. The operation was performed as previously described [16]. In brief, the pump was prefilled with Ang-II or saline and then incubated in sterile saline at 37 °C for 48 h. After the mice were anesthetized with 3.0% isoflurane mixed with oxygen, an incision was made on the back skin of the mice, and the pump was implanted into the subcutaneous area, followed by suturing the incision. After the operation, the mice were given buprenorphine (0.1 mg/kg) to reach analgesia. Finally, the regaining consciousness mice were returned to cages and fed until the end of the experiment.

To explore the function of METTL3 in vivo, an AAV9 vector containing the short-hairpin RNA (shRNA) targeting METTL3 (shMETTL3) or negative control (shNC) was purchased from GeneChem and used to inhibit METTL3 expression in hearts. The sequence of shMETTL3 is listed in Additional file 1: Table S1. Briefly, 5-week-old mice were randomly divided into three groups: AAV9-shNC + saline (n = 7), AAV9-shNC + Ang-II (n = 8), AAV9-shMETTL3 + Ang-II (n = 7). Each mouse was injected with the AAV-9 vector containing 2.5 × 1011 viral genome particles via tail vein. After 4 weeks, the pumps containing saline or Ang-II were implanted according to the above procedure for 3 weeks to induce cardiac remodeling.

Echocardiography and histopathological examination

After 3 weeks of Ang-II infusion, the mice were anesthetized with 1.0% inhaled isoflurane and cardiac echocardiography was performed using an ultra-high-resolution small animal ultrasound imaging system (VisualSonics Vevo 3100) to evaluate the structural parameters and cardiac function. The data were analyzed from three consecutive cardiac cycles of each measurement. Several indicators, including left ventricular anterior wall thickness (LVAW), LV ejection fraction (EF, %), and LV fractional shortening (FS, %), were collected. Subsequently, the mice were euthanized to remove hearts, and body weight (BW) and heart weight (HW) were obtained to calculate the HW:BW ratio. Furthermore, the widest part of the middle ventricular tissue of hearts was collected and fixed in 4% paraformaldehyde overnight, embedded in paraffin, and cut into sections of 5 μm thickness. Then the sections were stained with hematoxylin and eosin (H&E, Sigma) to observe the general morphology. The sections were incubated with wheat germ agglutinin (WGA, Sigma) to evaluate the cross‐sectional areas of cardiomyocytes.

Cell culture and treatment

Primary neonatal rat cardiomyocytes (NRCMs) were isolated from the hearts of 1–3-day-old SD rats according to a previously described method [17]. In brief, the hearts were washed and minced in ice-cold Hank’s balanced salt solution (Gibco) and digested with a mixture of 0.03% trypsin (Hyclone) and 0.04% collagenase type II (Sigma) to isolate cardiomyocytes. Then, cardiac fibroblasts were removed using differential attachment technique, and NRCMs were seeded onto six‐well culture plates and cultured in Dulbecco’s modified Eagle medium (DMEM)/F12 medium (Hyclone) supplemented with 10% fetal bovine serum (FBS, Gibco) and 100 U/mL penicillin/streptomycin (Gibco) at 37 °C with 5% CO2 at a density of 2 × 105 cells per well. The myocardial hypertrophy model in vitro was induced by Ang-II (MCE) at a concentration of 400 nmol/L for 48 h.

HEK293T cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and cultured in DMEM medium (Hyclone) supplemented with 10% FBS (Gibco) and 100 U/mL penicillin/streptomycin (Gibco) at 37 °C with 5% CO2.

Cell immunostaining

The cells were plated on culture slides and fixed in 4% paraformaldehyde for 30 min, washed with PBS, and stained with an anti-α-actinin antibody (Santa Cruz) in immunofluorescence buffer at 4 °C overnight. Then, the sections were washed with PBS and incubated with the secondary antibody (Abways) for 90 min. The nuclei were stained with Hoechst. Images were obtained using a fluorescence inverted microscope (Olympus) and analyzed with ImageJ.

Cell transfection

The specific shRNA (Sangon Biotech)-targeting METTL3 and DKK2 were designed and cloned into the pLKO.1 vector respectively for the generation of shMETTL3 and shDKK2 to inhibit their expression in NRCMs. The genomic sequence encoding rat METTL3 was cloned into the pLVX vector for the generation of the pLVX-METTL3 vector to overexpress METTL3 in NRCMs. The empty lentivirus vector was set as the negative control. The detailed sequence information of shMETTL3 and shDKK2 are shown in Additional file 1: Table S2. The carrier vectors and helper plasmids were transfected into the HEK293T using Lipofectamine 2000 (Invitrogen) regents according to the manufacturer’s protocol. This process was provided and carried out by GenePharma. The lentivirus was transfected into the NRCMs at a multiplicity of infection (MOI) of 100 for 24 h, and the transfection mixture was replaced with normal complete medium for another 24 h before stimulation with Ang-II. Then, 100 nM mimics (GenePharma) and 100 nM inhibitor (GenePharma) of miR-221/222, as well as the same dose of corresponding negative control (GenePharma), were transfected into NRCMs with help of Lipofectamine 2000 (Invitrogen) kit according to the manufacturer’s instructions for 24 h prior to the addition of Ang-II. Detailed information on the sequence is also shown in Additional file 1: Table S2.

Real-time PCR analysis

Total RNA was extracted by TRIzol reagent (Invitrogen) from NRCMs and heart tissues following the manufacturer’s instructions. The concentrations of RNA were detected by NanoDrop ND-1000 spectrophotometer (Thermo Scientific). Then, RNA was reverse-transcribed into cDNA using the HiScript QRT Super Mix (Vazyme) and miRNA First-Strand Synthesis Kit (Vazyme) according to the manufacturer’s protocol. The real-time polymerase chain reaction was carried out using IQ SYBR Green Supermix (Bio-Rad) and the Prism 7500 SDS (Applied Biosystems). Finally, the relative expression value of genes was calculated using the 2−ΔΔCt method. U6 was used as the internal control of miR-221/222, and β-actin was used as the internal control for the other genes. The primer sequences (Sangon Biotech) of the genes are shown in Additional file 1: Tables S3 and S4.

Western blot

Total protein was extracted by Whole-Cell Lysis Assay (KeyGEN BioTECH) from NRCMs and heart tissues, and the concentration was defined by the BCA Protein Assay Kit (KeyGEN BioTECH). Protein extractions were boiled and denatured, separated by 10% SDS–PAGE and then transferred onto PVDF membranes (Millipore). After blocking with 5% skimmed milk powder, the PVDF membranes were incubated overnight at 4 °C with the following rabbit-sourced primary antibodies, including anti-METTL3 antibody (Abcam, ab195352, 1:1000), anti-ANP antibody (Abcam, ab189921, 1:1000), anti-BNP antibody (Abcam, ab239510, 1:1000), anti-DKK2 antibody (Abcam, ab95274, 1:1000), anti-β-catenin antibody (Abcam, ab32572, 1:1000), anti-c-Myc antibody (Abcam, ab32072, 1:1000), and anti-β-actin antibody (ABways, AB0035, 1:5000). Subsequently, the membranes were washed with TBST and incubated with HRP-conjugated secondary antibody (Abways, AB0101, 1:5000; AB0102, 1:2000) at room temperature for 1 h. Finally, bands were detected using ECL (KeyGEN BioTECH) with a chemiluminescence system (Bio-Rad).

RNA m6A quantification

Total RNA was isolated, and the m6A content was measured using an m6A RNA methylation quantification kit (Epigentek) following the manufacturer’s instruction. Briefly, 200 ng RNA was added to the wells of the assay plate. Then, the capture antibody solution and the detection antibody solution were added to the assay wells according to the manual. The m6A levels were quantified with colorimetry method by reading the absorbance of each well at 450 nm and calculating relative m6A abundance.

RNA immunoprecipitation (RIP) assay

NRCMs overexpressing METTL3 were UV-irradiated at 254 nm, 400 mJ/cm2 (Stratagene Stratalinker), and then lysed with RIP lysis buffer (Absin) at 4 °C through disruptive sonication. Immunoprecipitation of endogenous DGCR8 was performed by anti-DGCR8 antibody (Abcam) or control IgG antibody overnight at 4 °C. After washing, the immunoprecipitated protein–RNA complex was treated with proteinase K. RNAs were extracted by standard phenol/chloroform procedure. Then, the pri-miRNAs were detected with qRT-PCR, and %(IP/Input) and %(IgG/Input) were obtained. Finally, the fold enrichment of m6A level on pri-miRNAs was calculated by %(IP/Input)/%(IgG/Input).

For m6A RIP (meRIP) experiments, RNA extracted from NRCMs overexpressing METTL3 and control NRCMs was treated with deoxyribonuclease I (Sigma). The RNAs were fragmented by sonication for 10 s on the ice-water mixture. Immunoprecipitation was performed using an anti-m6A antibody (Abcam) or control IgG antibody previously bound to magnetic Dynabeads (Life Technologies) in the RIP immunoprecipitation buffer (Millipore) and incubated with DNA-free fragmented RNAs. Beads were then treated with proteinase K (20 mg/mL) for 1.5 h at 42 °C. RNAs were extracted by standard phenol/chloroform procedure. Then, the pri-miRNAs were detected with qRT-PCR, and %(IP/Input) and %(IgG/Input) were obtained. The fold enrichment of pri-miRNAs binding to DGCR8 was calculated by %(IP/Input)/%(IgG/Input).

Dual-luciferase assay

The target genes of miR-221/222 were predicted by the online webtool TargetScan (http://www.targetscan.org) [18] under default parameters. On the basis of the predicted intersection of target genes in three species of human, rat, and mouse, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed with another bioinformatic online tool, Enrichr (https://maayanlab.cloud/Enrichr/) [19].

The dual-luciferase assay was applied for validating the predicted connection between miR-221/222 and DKK2. Briefly, the psiCHECK2 plasmids (Promega) containing wild-type DKK2 (DKK2-wt) or DKK2 mutated (DKK2-mut) at the putative miR-221/222 binding sites were constructed. The psiCHECK2-DKK2-wt and psiCHECK2-DKK2-mut plasmids with either miR-221/222 mimic or negative control were delivered into HEK293T cells by Lipofectamine 2000 (Invitrogen) for 48 h. Subsequently, the HEK293T cell lysate was added to Renilla luciferase or firefly luciferase, and the luminescence was determined by SpectraMax M5 (Molecular Devices). The firefly luciferase was used as an internal control.

Statistical analysis

All data were analyzed by SPSS 22.0 software, and expressed in the form of mean ± standard deviation. Statistical comparisons were performed using unpaired two-tailed t-test when two groups were compared. Comparisons of data among multiple groups were carried out by one-way analysis of variance (ANOVA). P < 0.05 was regarded as statistically significant.