Rat cardiomyocyte cell line H9c2 and 293T cells were both purchased from the American Type Cult ure Collection (ATCC; Manassas, VA, USA). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) (both from Gibco, Rockville, MD, USA) and 1% penicillin-streptomycin solution (Sigma-Aldrich, St. Louis, MO, USA). Cells were routinely cultured in a humidified incubator containing 5% CO₂ at 37°C. For the induction of hypoxia, H9c2 cells were grown in a hypoxia chamber containing 94% N₂, 5% CO₂ and 1% O₂ at 37°C.

The miR-200c mimics, miR-200c inhibitor and negative controls (NCs) were all purchased from GenePharma (Shanghai, China) and transfected into cells using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA), following the manufacturer's instructions. The GATA-4 siRNA and NC siRNA were both purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) and transfected into cells according to the manufacturer's instructions.

Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies) and reverse transcribed into cDNA using M-MLV Reverse Transcriptase (BioTeke Co., Ltd., Beijing, China) or TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). The cDNA was used as the template for RT-qPCR with SYBR-Green PCR Master Mix and appropriate primers on 7900HT Fast Real-Time PCR system (both from Applied Biosystems). U6 was used as the internal control for normalization of miR-200c. β-actin was used as the internal control for normalization of GATA-4 and Bcl-2. Relative gene expression was calculated by the 2^−ΔΔCq method. The primer sequences used in the experiments were as follows: miR-200c forward, 5′-GTAATACTGCCGGGTAATGATGGA-3′ and reverse, 5′-GTGCAGGGTCCGAGGT-3′; U6 forward, 5′-GCGCGTCGTGAAGCGTTC-3′ and reverse, 5′-GTGCAGGGTCCGAGGT-3′; GATA-4 forward, 5′-GTGCCAACTGCCAGACTACC-3′ and reverse, 5′-AGCCTTGTGGGGACAGCTTC-3′; Bcl-2 forward, 5′-AGTTCGGTGGGGTCATGTGTG-3′ and reverse, 5′-CCAGGTATGCACCCAGAGTG-3′; β-actin forward, 5′-TCAGGTCATCACTATCGGCAAT-3′ and reverse, 5′-AAAGAAAGGGTGTAAAACGCA-3′.

Cell viability was assessed using the MTT colorimetric assay. In brief, the cells were seeded in 96-well plates at 2×10⁴ cells/well and cultured overnight. The cells were transfected with the miR-200c inhibitor or NC inhibitor for 24 h and then subjected to hypoxic conditions for 24 h. Afterwards, the cells were treated with 20 µl of 5 mg/ml MTT (Sigma-Aldrich) and cultured for 4 h. The purple-colored formazan crystals in living cells were solubilized by 200 µl of dimethyl sulfoxide (DMSO; Sigma-Aldrich). After 15 min, the absorbance of the solution at 490 nm was detected by a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Cell injury was measured using an LDH assay kit (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer's instructions. The cells were lysed by 0.2% Triton X-100 (Sigma-Aldrich) and the supernatants were collected after centrifugation at 10,000 × g for 10 min at 4°C. The supernatants were then incubated with pyruvate and nicotinamide adenine dinucleotide hydrogen for 30 min at 37°C. After the addition of 0.4 M NaOH, the absorbance at 530 nm was detected by a microplate reader (Bio-Rad Laboratories, Inc.).

Cell apoptosis was measured by using an Annexin V/PI apoptosis detection kit (Beyotime Institute of Biotechnology, Haimen, China) following the manufacturer's recommended instructions. In conclusion, the cells were digested with 2.5 g/l trypsin (Sigma-Aldrich) and then washed with phosphate-buffered saline (PBS). The cells were then re-suspended in binding buffer supplemented with 10 µl of Annexin V. After incubation for 30 min, 5 µl of PI solution was added and the cells were incubated for a further 5 min. Cells were analyzed by flow cytometry (BD Biosciences, San Jose, CA, USA).

Caspase-3 activity was measured by a caspase-3 activity assay kit (Roche Applied Science), according to the manufacturer's instructions. In brief, the cells were lysed and the supernatants were collected after centrifugation at 16,000 × g for 15 min at 4°C. The supernatants were then incubated with 10 µl Ac-DEVD-pNA (2 mM) for 2 h at 37°C. The absorbance at 405 nm was determined using a microplate reader (Bio-Rad Laboratories, Inc.).

Proteins were lysed in lysis buffer and protein concentrations were measured using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology). Equivalent amounts of proteins were loaded on 10% sodium dodecyl sulfate-polyacrylamide gels for separation, followed by protein transfer to a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% non-fat dry milk for 1 h at 37°C, followed by incubation with primary antibodies at 4°C overnight. The membrane was washed with Tris-buffered saline containing 0.1% Tween-20 (TBST) and then incubated with horseradish peroxidase-conjugated secondary antibodies (1:2,000; sc-2004; Santa Cruz Biotechnology, Inc.) for 1 h at 37°C. After being washed with TBST, the protein bands were developed using a Pierce ECL Western Blotting kit (Pierce, Rockford, IL, USA). Gray values of protein bands were detected by Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). The primary antibodies including anti-GATA-4 (1:500; sc-9053), anti-Bcl-2 (1:500; sc-783) and anti-β-actin (1:600; sc-130656) were all purchased from Santa Cruz Biotechnology, Inc.

The 3′-UTR of GATA-4 harboring either the miR-200c binding site (GATA-4 3′-UTR-WT) or a mutant (GATA-4 3′-UTR-MT) was cloned into the pmirGLO luciferase vector (Promega, Madison, WI, USA). The constructed vectors were co-transfected into 293T cells with the miR-200c inhibitor or NC inhibitor using Lipofectamine 2000 (Invitrogen Life Technologies,) and incubated for 48 h. The cells were harvested and luciferase activities were measured using a Dual-GLO Luciferase Assay system (Promega).

The results are presented as mean ± standard deviation. Statistical analyses were performed using SPSS package version 18.0 (SPSS, Inc., Chicago, IL, USA). Statistical significance was determined by one-way analysis of variance (ANOVA) followed by a Bonferroni correction. Differences were regarded as statistically significant at values of p<0.05.

To investigate the possible role of miR-200c in ischemic heart disease, we detected the expression of miR-200c in cardiomyocytes exposed to hypoxia using RT-qPCR. The results showed that miR-200c was significantly upregulated after exposure to hypoxia in comparison to the control (Fig. 1A), implying that miR-200c has an important role in the hypoxic injury of cardiomyocytes.

To investigate the biological effect of miR-200c on hypoxia-treated cardiomyocytes, we inhibited the expression of miR-200c in cells by transfecting them with an miR-200c inhibitor (Fig. 1B). We then detected the effect of miR-200c suppression on cell viability with the MTT assay. The results showed that cell viability was markedly impaired by hypoxia but was partially improved by miR-200c inhibition (Fig. 2A). We next evaluated the effect of miR-200c inhibition on hypoxia-induced cell injury with the LDH assay. We found that the hypoxia-induced cell injury was also significantly reversed by the downregulation of miR-200c (Fig. 2B). Taken together, these data suggest that downregulation of miR-200c improves cell survival under hypoxic conditions.

To verify the protective effect of miR-200c inhibition on cardiomyocytes under hypoxia, we further investigated the effect of miR-200c inhibition on hypoxia-induced apoptosis. The Annexin V/PI apoptosis assay showed that hypoxia-induced apoptosis was significantly suppressed by the downregulation of miR-200c (Fig. 3A and B). Furthermore, the increased activity of the pro-apoptotic protein, caspase-3, induced by hypoxia was also markedly decreased by miR-200c inhibition (Fig. 3C). Overall, these results indicate that the downregulation of miR-200c suppresses hypoxia-induced cardiomyocyte apoptosis.

To investigate the underlying mechanism by which the suppression of miR-200c provides a protective effect against hypoxia, we predicted the target genes of miR-200c with bioinformatics analysis. We found that GATA-4, an important transcription factor for cardiomyocyte survival (23,24), was a potential target gene of miR-200c. The 3′-UTR of GATA-4, harboring the complementary seed-matched binding sites of the miR-200c binding site (GATA-4 3′-UTR-WT) and a mutant (GATA-4 3′-UTR-MT), are shown in Fig. 4A. To confirm the presence of the interaction between miR-200c and GATA-4 3′-UTR, GATA-4 3′-UTR-WT or GATA-4 3′-UTR-MT was cloned into the pmirGLO vector that was used for the luciferase reporter assay. The results showed that the luciferase activity of pmirGLO-GATA-4 3′-UTR-WT was significantly decreased by miR-200c overexpression (Fig. 4B). However, the luciferase activity of pmirGLO-GATA-4 3′-UTR-MT was not obviously affected by miR-200c overexpression (Fig. 4B). These results suggest that miR-200c directly targets the 3′-UTR of GATA-4. We then examined the direct effect of miR-200c on GATA-4 expression by RT-qPCR and western blot analysis. The results showed that the downregulation of miR-200c significantly increased the mRNA (Fig. 5A) and protein (Fig. 5B) expression of GATA-4, which were decreased by hypoxia in the cardiomyocytes. Taken together, these results suggest that GATA-4 is a direct target gene of miR-200c in cardiomyocytes.

To further investigate the molecular basis of miR-200c in regulating cardiomyocyte survival, we detected the expression of the anti-apoptotic gene Bcl-2, a downstream gene of GATA-4 (23). The results showed that the downregulation of miR-200c significantly upregulated the mRNA (Fig. 6A) and protein (Fig. 6B) expression of Bcl-2 in response to hypoxia, implying that miR-200c regulates Bcl-2 expression.

To verify that GATA-4 contributes to the miR-200c inhibition-mediated protective effect against hypoxia, we silenced the expression of GATA-4 at the same time as suppressing miR-200c. The results showed that the promotive effect of miR-200c suppression on GATA-4 expression was significantly abolished by the knockdown of GATA-4 (Fig. 7A). Similarly, the increased expression of Bcl-2 induced by miR-200c suppression was eliminated by the knockdown of GATA-4 (Fig. 7B). As expected, the protective effect of miR-200c suppression against hypoxia was also markedly reversed by the knockdown of GATA-4 (Fig. 7C and D). Taken together, these results suggest that downregulation of miR-200c protects cardiomyocytes against hypoxia through the promotion of GATA-4 expression.