Cell culture and treatment
Rat cardiomyocytes (H9c2) were purchased from ATCC (VA, USA). All cells were cultured in DMEM (Gibco, MD, USA) containing 10% FBS (Gibco) at 37°C with 5% CO2. To construct an in vitro model of MIRI, H9c2 cells were subjected to OGD/R treatment as previously described (24). In brief, H9c2 cells were cultured in serum and glucose-free DMEM (Gibco) and cultured under the hypoxic conditions (2% O2, 93% N2 and 5% CO2) for 2 h, 4 h, 6 h, 8 h or 12 h, followed by reoxygenation (37oC and 5% CO2) for 4 h. The control cells were cultured under normal condition.
Cell transfection
The short hairpin RNA of Smad7 (sh-Smad7), sh- Smurf2, the overexpression plasmid of Smad7 (oe-Smad7), oe-Smurf2, miR-322 mimics/inhibitor and their negative controls were transfected into cells with Lipofectamine™ 3000 (Invitrogen, CA, USA). The above plasmids were all obtained from GenePharma (Shanghai, China).
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay
H9c2 cells were seed into 96-well plate (3000 cells/per well) and cultured for 24 h followed by incubation with 20 µL MTS reagent (500 µg/mL) ((Promega, WI, USA) for 2.5 h at 37℃. After incubation, the absorbance at 492 nm was detected by a microplate reader (Bioteke, Beijing, China).
Cell apoptosis assay
Cells were incubated with 500 µl of 1X Annexin-binding buffer containing 10 µl Annexin V-FITC and 5 µl PI stain (Beyotime, Shanghai, China) followed by flow cytometry analysis (Becton,Dickinson and Company).
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining
Cells were fixed, permeabilized and washed with PBS. The procedure was conducted by using TUNEL staining kit purchased from Beyotime (Shanghai, China), and the nucleus was stained with DAPI (Beyotime).
Dual luciferase reporter gene assay
The fragment of SMAD7 or Smurf2 was amplified by PCR. Site-directed mutagenesis was performed using a site-directed mutagenesis kit (Stratagene, CA, USA). Wt and mut reporter plasmid of SMAD7 or Smurf2 sequence was cloned into the pmiRGLO vector (Promega, WI, USA). Then, cells were co-transfected with SMAD7-wt or Smurf2-wt, SMAD7-mut or Smurf2-mut plasmids and miR-322 mimics or mimics NC. The Luciferase activity was examined using a dual-luciferase reporter assay system (Promega). The same method was employed to verify the interaction between HIF-1α and miR-322 and the interaction between β-catenin and HIF-1α.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted with TRIzol (Beyotime). For mRNA and miRNA, the cDNA was synthesized using the cDNA synthesis kit (Toyobo, Tokyo, Japan) and the first-strand cDNA synthesis kit (Sangon, Shanghai, China). Then, SYBR (Thermo Fisher Scientific, MA, USA) was employed for qRT-PCR assay. GAPDH and U6 were used as the reference gene for mRNA and miRNA, respectively. Data was analyzed with 2−ΔΔCT method. The primers used in the study were listed as follows (5’-3’):
miR-322 (F): ACTGGATACGACTCCAAA
miR-322 (RT): GTCGTATCCAGTGCAGGGTCCGAGGTATTCGC
Smad7 (F): CCCGGCGGCGAGGACGAGGAG
Smad7 (R): GGATGGTGGTGACCTTTGGCAC
Smurf2 (F): CAGTGGTTTTCCGTGCAGTG
Smurf2 (R): AGAGCGACTGGAGGAAGGAT
HIF-1α (F): AGCAATTCTCCAAGCCCTCC
HIF-1α (R): TTCATCAGTGGTGGCAGTTG
GAPDH (F): ATGACTCTACCCACGGCAAG
GAPDH (R): GGAAGATGGTGATGGGTTTC
U6 (F): CTCGCTTCGGCAGCACA
U6 (R): AACGCTTCACGAATTTGCGT
Western blot
Total proteins were extracted by using RIPA (Thermo Fisher Scientific), which were then transferred to a PVDF membrane (Millipore, MA, USA). The membranes were subsequently incubated overnight with antibodies against Smad7 (Abcam, Cambridge, UK, 1:1000, ab216428), Smurf2 (Abcam, 1:1000, ab53316), Smad3 (Abcam, 1:1000, ab40854), β-catenin (Abcam, 1:1000, ab32572), HIF-1α (Abcam, 1:1000, ab179483) and β-actin antibody (Abcam, 1:1000, ab8226). Membranes were washed with PBS-T followed by incubation with the corresponding secondary antibody (Abcam, 1:10000, ab7090; 1:2000, ab6728) for 60 min. Protein bands were analyzed by an ECL detection kit (Beyotime).
Data analysis
All our data were obtained from three independent experiments. Statistical data was analyzed by GraphPad Prism 8 and expressed as means ± SD. The differences among two groups were analyzed by Student’s t-tests. One-way ANOVA was performed to assess the differences among multiple groups. The p values less than 0.05 were considered significant.