Sprague-Dawley rats (1–3 days, SPF grade, Certification No. 11400700368598) were obtained from the Beijing Huafukang Biological Technology Co. Ltd. (Beijing, China). The experimental protocols were approved by the Ethics Committee of the Ethics of Animal Experiments of Chengde Medical College (Approval ID: CDMULAC-20191031-009). The animal experiments were approved by the Research Ethics Committee of Chengde Medical College, and were in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996).

Cardiomyocytes were obtained from 1 to 3-day-old neonatal rats as previously described, with a few minor modifications (Cai et al., 2018). Briefly, after being digested in 0.25% trypsin solution, myocytes were isolated by selective adhesion of myocytes at a 2 h pre-plating interval. Cardiomyocytes were cultured in DMEM supplemented with 1% penicillin and streptomycin, 10% fetal bovine serum and 0.1 mM 5-bromo-2-deoxyuridine in a 37°C incubator with 5% CO₂. MiR-27a-3p-mimics (5′-UUC​ACA​GUG​GCU​AAG​UUC​CGC-3′), miR-27a-3p-inhibitor (5′-GCG​GAA​CUU​AGC​CAC​UGU​GAA-3′), Hoxa10-overexpress and corresponding NC were synthesized by Shanghai Sangon Biotechnology, and transiently transfected with liposome3000 transfection reagent (Invitrogen, Carlsbad, CA, United States) and serum-free medium. Eight hours after transfection, a new serum-free medium with or without Ang II (10 μM) was used instead. After 48 h of culture, the cells were collected for protein/total RNA extraction (Ma et al., 2018).

After treatments, the cardiomyocytes were washed with PBS for three times, then the protein lysate was added and stored at −80°C. Place cells on the ice to dissolve, scrape off the cells with a protein scraper and centrifuge at 13,500 rpm/min × 15 min, 4°C. Collect the supernatant protein solution. Determination of protein concentration by BCA method, proteins were separated by SDS-PAGE and transferred to a PVDF membrane. After blocking with 5% fat-free milk at room temperature for 120 min, the membranes were incubated overnight with the corresponding primary antibody at 4°C, including anti-Kv4.3 (1:1,000, CST), anti-Hoxa10 (1:500, Sigma), anti-ANP (1:1,000, SANTA), anti-BNP (1:1,000, SANTA), anti-MHC (1:1,000, SANTA) and anti-GAPDH (1:10,000, ABclonal). After being washed with TBST by three times, each time 10 min, the membranes were incubated with secondary antibodies (1:8,000, ABclonal) for 2 h at room temperature and washed with TBST again. The proteins were scanned using Odyssey version 1.2 (LI-COR Biosciences, Lincoln, NE, United States) and Image Studio software.

Total RNA samples from different groups of cardiomyocytes were extracted and treated with DNA enzyme 1, and then routine quantitative RT-PCR (qRT-PCR) was used for detection. Real-time quantitative RT-PCR was determined by Taqman probe method (ABI 7500 fast). In the determination of mRNA, GAPDH was used as the internal reference, and U6 was used as the internal reference for microRNAs. Compared with the control group, the related expressions of microRNAs and mRNAs under different treatments were calculated.

Cardiomyocytes in different treatment groups were washed with PBS for three times, and 15 min was fixed with 4% paraformaldehyde at 37°C. Get rid of paraformaldehyde, wash the cells with PBS, then add a mixed solution (0.4% Triton + 10 mg BSA +1 ml PBS) and place 90 min at room temperature. After that, goat serum blocked 30 min at 37°C, then α-actinin was added and incubated at 4°C for 48 h. Finally, after incubated with fluorescent second antibody for 1 h, the cells were washed by PBS for three times, and the morphological changes of cells were observed by fluorescence microscope.

GraphPad Prism software was used for statistical analysis. Values were expressed as the mean ± SEM. Differences between two groups were analyzed using t-test. The significance of the difference between groups in review were analyzed by one-way analysis of variance (ANOVA). A value of *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 were considered statistically significant difference for all analyses.

Firstly, the cardiomyocytes from rats were extracted and exposed to Ang II (10 μM) for 48 h to established cardiac hypertrophy cell model in vitro. Then the expressions of myocardial hypertrophy-related genes and miRNAs in cardiomyocytes were detected. Results showed that the expressions of myocardial hypertrophy-related genes and proteins including ANP, BNP and β-MHC were markedly up-regulated in Ang II-treated cardiomyocytes compared with control group (Figures 1A,B,C,E). Immunofluorescence results showed that the area of cardiomyocytes in Ang II treatment group was significantly larger than the control group (Figure 1F), indicating that Ang II induced cardiac hypertrophy cell model in vitro was successfully established. At the same time, we also found that the expression of miR-27a-3p was up-regulated in cardiomyocytes after Ang II treatment compared with control group (Figure 1D). This result suggested that miR-27a-3p might be involved in myocardial hypertrophy induced by Ang II.

To clarify whether miR-27a-3p was involved in cardiac hypertrophy, miR-27a-3p-inhibitor, a specific antagonist of miR-27a-3p, was used in the following experiments. Four hours after the transfection of miR-27a-3p-inhibitor, the serum-free culture medium was replaced and co-cultured with Ang II (10 μM) for 48 h. PCR results showed that the expressions of NPPA, NPPB, and MHY7 in miR-27a-3p-inhibitor + Ang II group was significantly lower than Ang II group, while KCND3 was higher than Ang II group (Figures 2A–D), and the protein expressions showed similar tendencies (Figure 2F). The α-actinin staining showed that the area of cardiomyocytes in miR-27a-3p-inhibitor + Ang II group was significantly smaller than Ang II group (Figure 2E). These results indicated that the inhibition of miR-27a-3p mitigated the process of myocardial hypertrophy and structural remodeling induced by Ang II.

In addition, we detected the effects of miR-27a-3p-mimics and inhibitor on cultured rat ventricular cardiomyocytes. PCR results showed that the expression of miR-27a-3p in miR-27a-3p-mimics group was significantly increased compared with control group, while the expression of miR-27a-3p in miR-27a-3p-inhibitor group was significantly decreased compared with miR-27a-3p-mimics group and the control group (Figure 3A). Furthermore, the expressions of NPPA, NPPB, and MHY7 were significantly up-regulated after miR-27a-3p-mimics treatment, which were effectively alleviated by miR-27a-3p-mimics and miR-27a-3p-inhibitor co-treatment (Figures 3B–D). These results suggested that the overexpression of miR-27a-3p owned the potential to promote cardiomyocyte hypertrophy.

The results above proved that miR-27-3p was involved in hypertrophic growth of cardiomyocytes, but its mechanism still needs to be elucidated. Recent study revealed that potassium channel gene KCND3 acts as an important mediator in cardiomyocyte hypertrophy (Wang et al., 2019). Consistently, we found that the expressions of Kv4.3 protein and KCND3 gene were down-regulated in Ang II-induced cardiomyocyte hypertrophy (Figures 4A,B). Furthermore, miR-27-3p-mimics transfection induced a reduction expression of Kv4.3 channel protein in cardiomyocytes. On the contrary, miR-27-3p-mimics and miR-27-3p-inhibitor co-treatment attenuated the reduction of Kv4.3 protein expression in hypertrophic cardiomyocytes, and PCR results showed the similar tendencies (Figures 4C,D). These results indicated that miR-27-3p regulated cardiomyocyte hypertrophy by affecting the expression of Kv4.3 channel protein. However, no binding sites of miR-27-3p in the 3′UTR region of KCND3 gene were found.

Next, we further searched the downstream targets of miR-27a-3p in the pathogenesis of cardiac hypertrophy and electrical remodeling. The TargetScan prediction showed that there was a binding sequence between miR-27a-3p and Hoxa10-mRNA 3′UTR region (Figure 5A). As shown in Figure 5B, we designed wild and mutant types of plasmids of Hoxa10 3′UTR region and transfected into HEK293T cells. Luciferase assay showed that the fluorescence intensity of miR-27a-3p group was significantly lower than the NC group in wild type, but there was no change in mutant type (Figure 5C). In addition, compared with NC group, the mRNA expression of Hoxa10 was significantly inhibited by miR-27a-3p-mimics in cardiomyocytes (Figure 5D). These results confirmed that there was a binding between miR-27a-3p and Hoxa10-mRNA 3′UTR region. Furthermore, miR-27a-3p-mimics significantly inhibited the expression of Hoxa10, while this process could be alleviated by miR-27a-3p-mimics and miR-27a-3p-inhibitor co-treatment. Besides, miR-27a-3p-inhibitor can also alleviate the decreased expression of Hoxa10 induced by Ang II treatment (Figure 5E). These results suggested that miR-27a-3p could negatively regulate the expression of Hoxa10.

In order to confirm whether Hoxa10 was involved in myocardial hypertrophy process. The cardiomyocytes were cultured with Hoxa10-overexpress for 8 h, then the serum-free medium was replaced, and the cells were co-cultured with Ang II for 48 h. PCR results showed that the mRNA expression of Hoxa10 in Hoxa10-overexpress group was significantly higher than that in NC group (Figure 6A). Compared with NC group, the expression of cardiac hypertrophy related genes including NPPA, NPPB, and MHY7 in NC + Ang II group was significantly up-regulated, while KCND3 was down-regulated (Figures 6B–E). As shown in Figure 6F, the protein expressions showed similar tendencies. Moreover, the protein expression of Hoxa10 was also down-regulated synchronously in NC + Ang II group. While this process could be inhibited by Hoxa10-overexpress (Figure 6B–F). The α-actinin staining showed that the area of cardiomyocytes in Ang II + Hoxa10-overexpress group was significantly smaller in NC + Ang II group, indicating that Hoxa10-overexpress reversed the process of myocardial hypertrophy induced by Ang II (Figure 6G). To sum up, these results suggested that by targeting the miR-27a-3p/Hoxa10/Kv4.3 axis could effectively inhibit the process of myocardial hypertrophy and cardiomyocyte hypertrophy, which provided us a new therapeutic strategy for myocardial hypertrophy and electrical remodeling.