Experiments were in compliance with the council of China on Animal Care and were approved by the Animal Ethics Committee of the Medical College of He Xi University. Sprague-Dawley (SD) male rats (180–250 g) were randomly divided into two groups including, the myocardial infarction group (n=3) and sham operation group (n=3). Rats were anesthetized with pentobarbital sodium (0.1%) and were assisted breathing with small animal ventilator. Their electrocardiograms were recorded with II leads and the third, fourth rib gap was open to expose the left atrial appendage. Anterior descending artery was ligated with 7–0 ligation line 2 mm at the lower edge of the right in the left atrial appendage. Two weeks later, rats were anesthetized, and the heart was quickly removed. After the residual blood was washed with normal saline, the samples were stored at −80°C.

Rats of both groups were anesthetized, and their heart was taken out. Saline was used to wash residual blood. Following 24 h of fixation with 4% poly-formaldehyde, tissues were embedded with paraffin, sliced and stained using H&E and Masson procedures.

The epicardium of heart of SD neonatal rats (1–3 days) was tore with pincett. The residual blood was washed with 1X PBS. The sample was cut with a pair of scissors (about 1 mm³), and double enzymes were added (0.25% pancreatin + 0.1% collagenase B) to digest at 37°C using shock for a total of 10 times, initially for 10 min, and then 6 min each time. The supernatant of each collection was terminated digestion with DMEM culture medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Gibco). After centrifugation of cell suspension at 800 × g for 10 min, the cells were cultured in 50 ml culture flask after resuspension with 10% FBS, and then stored at 37°C in a 5% CO₂ incubator. CFb was obtained after differential for 75 min and discarding the supernatant. Cells were cultured with 10% FBS DMEM and 5% CO₂ at 37°C. Cell passage occurred in 1–2 days, and cells between passages 2–3 were used for experiments. Serum-free DMEM was used to starve cells for 24 h.

After washing with serum-free DMEM, the cells were cultured with 2 ml serum-free DMEM for 4–6 h (6-well plate). miR-370 mimics (5′-CAGGUCACGUCUCUGCAGUUACAC-3′), miR-370 antisense inhibitor (5′-GTCCAGTGCAGAGACGTCAATGTG-3′), NC (5′-CACAUUGTGCUCUCUGCACUGCTC-3′) (Guangzhou RioBio Co., Ltd., Guangzhou, China) and transfection reagent lipofectmine^®2000 (Invitrogen Life Technologies, Carlsbad, CA, USA) and 500 µl Opti-MEM (Gibco) were mixed, with a standing time of 5 min. Two kinds of mixture mixed and stood for 20 min, and then transfected cells. 6 h later, culture medium was replaced and corresponding stimulation was added.

After treatment with the Sircol collagen assay kit (Biocolor), cell samples were stored at 4°C for 24 h. After centrifugation, 100 µl supernatant was added with 1 ml Sircol dye, mixing, 4°C, 30 min. After centrifugation, the supernatant was removed and 700 µl Sircol alkali reaction fluid was resuspended. Data were obtained using a spectrophotometer (540 nm; Hitachi, Tokyo, Japan).

Total RNA in cardiac tissue and CFbs was extracted with TRIzol (Invitrogen Life Technologies). Levels of GAPDH, ColIa1, ColIIIa1, TGFβ1, TGFβRII and miR-370 were detected with qRT-PCR and SYBR-Green (Takara Bio, Dalian, China) (Table I).

Total protein in cardiac tissue and CFbs was extracted with protein lysis buffer (Beyotime Biotech, Jiangsu, China). Proteins were isolated using 60 µg protein system and 10% SDS polyacrylamide gel, and transferred to the polyvinylidene fluoride membrane. After three hours of blocking with 5% skim milk, TGFβ1, TGFβRII, a-SMA (a-smooth muscle actin, Gene Tex) and internal reference APDH (Anti Gene) primary antibody diluent was used to seal the membrane for 16 h. 1X TBST washing membrane for 15 min each time for a total of three times. The secondary antibody (Anti Gene) dilution was incubated for 2 h and then membranes were washed in 1X TBST for 15 min each time for a total of three times. Thereafter, ECL (Thermo Fisher Scientific, Waltham, MA, USA) luminescence was applied. The Bio-Rad Biology system was used to capture images and the results were analyzed.

We used two databases, TargetScan6.2 (http://www.targetscan.org/index.html) and PicTar (http://pictar.mdc-berlin.de), to predict the combination of miR-370 and TGFβII mRNA.

3′-UTR in TGFβRII was sub-cloned based on PCR, and the miRNA binding site in reporter gene vector was constructed. The construct was inserted into multiple cloning sites. After miRNA reporter vector (Ambion Life Technologies, Carlsbad, CA, USA) expressed by pMIR-REPORT™ luciferase (HindIII and SpeI sites) in the serum-free medium for 24 h, human embryonic kidney cell (HEK-293) (1≤10⁵/well) 1ug PGL3 target DNA (firefly luciferase vector) and 0.1 µg pRL-TK (Ranilla luciferase vector driven by TK) were transfected using liposome (Lipofectamine^® 2000, Invitrogen Life Technologies). Forty-eight hours after transfection of dual luciferase, double luciferase activity was detected, strictly according to the method provided by Promega (8).

Statistical analysis was performed using SPSS 13.0 (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism (version 6.0; GraphPad Software, Inc, La Jolla, CA, USA). All data were expressed as mean ± standard error. Analysis of variance and Dunnett's test were conducted in comparisons among multiple groups. P<0.05 was considered to indicate a statistically significant difference.

MASSON staining and qRT-PCR results showed that 2 weeks after myocardial infarction, the collagen deposition in the infarct border area was significantly increased in the myocardial infarction group when compared with the sham operation group (Fig. 1). The expression of TGFβ1 and TGFβRII was significantly increased and miR-370 expression decreased in the border area of the heart infarction. Western blotting results showed that the expression of α-SMA was also significantly increased in the border area, which suggested that CFbs in the infarct border area partially differentiated into myofibroblasts (Figs. 2 and 3).

When SD rat CFbs were passaged for the third generation and cells were fused to 85%, the cells were synchronized with serum-free DMEM for 24 h and stimulated with AngII for 24 h, and then the indicators were detected. It showed that TGFβ1, and TGFβRII mRNA and protein levels were increased in fibroblasts under the induction of AngII. The increase of α-SMA promoted the transformation of fibroblasts into myofibroblasts and increased the expression of collagen. The results also showed that AngII significantly inhibited the expression of CFb miR-370, which indicated that the decrease of miR-370 may be related to the increase of TGFβRII.

Dual luciferase reporter gene assay showed that luciferase activity was significantly decreased in the plasmid group co-transfected with miR-370 mimics and contained the 3′UTR gene sequence of TGFβRII when compared with the control group. miR-370 mimics and inhibitor co-transfection significantly inhibited decreased luciferase activity induced by miR-370 mimics, while there were no effects in the NC transfection group (Fig. 4).

qRT-PCR results showed that the levels of TGFβRII, ColI1a and ColIII1a mRNAs in the miR-370 mimics + AngII group were significantly decreased compared with the AngII group. There were no significant differences in the levels of the above indexes in the miR-370 inhibitor + AngII, NC + AngII and AngII groups. Level of TGFβ1 mRNA did not differ in the above four groups. Western blot analysis revealed that, compared with the other three groups, TGFβRII and α-SMA protein levels and collagen secretion decreased in the miR-370 mimics + AngII group, however, miR-370 mimics did not inhibit the increase in TGFβ1 induced by AngII (Figs. 5 and 6).