Ethics Statement Animal study were approved by Soochow University Scientific and Animal Ethics Committee (Approval Number: 20150086) and were in compliance with the Chinese national regulations on the use of experimental animals. Procedures for animal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (US National Institutes of Health; National Institutes of Health publication no. 85-23, revised 1996). We used SPF grade C57/BL6 male suckling mouse aged 1-3d in our experiment. All animals were purchased from the laboratory animal center of Soochow University. They were maintained on standard diet and water at a 12/12 light/dark cycle at the animal center of the first affiliated hospital of soochow university.

Mouse was sacrificed by euthanasia with carbon dioxide. Cardiomyocytes were isolated from hearts by adding 0.25% trypsin (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA)/0.02% EDTA (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for digestion and the digestive fluid was collected by centrifugation. After centrifugation, the cells were inoculated in 25 cm² culture flask and cultured in 37˚C, 5% CO₂ saturated humidity incubator (Thermo). For construction of hypoxia condition, cardiomyocytes were incubated for 24 h with 1% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and then transferred to hypoxia buffer (Gibco; Thermo Fisher Scientific, Inc.) and cultured in a saturated (95% N₂ + 5% CO₂) container at 37˚C.

Recombinant adenovirus vector containing microRNA-22 plus a recombinant GFP (Adeno-miR22) or GFP only (Adeno-null) were produced by GenScript Company (Nanjing, China). Cardiomyocytes were cultured to 70% confluency. The supernatant was reduced to 800 mL, and purified adenoviral vector was added to the medium at multiplicities of infection of 10 to 100. After incubation at 37˚C, 5% CO₂ for 2 h, 1200 mL complete medium was added and cells were cultured for 12 h. Next, virus-containing medium was removed and replaced with fresh medium. Transduction efficiency and expression of microRNA-22 were detected by Taqman RT-PCR. Primers were designed by Invitrogen company. RNA extraction and PCR reaction process were conducted according to the manufacturer’s construction. Gene expression was assessed as ΔΔCt = (sample Ct miR-22 − sample CtU6 mRNA) − (control Ct miR-22 − control Ct U6 mRNA). The fluorescence intensity of GFP was detected by flow cytometry (Thermo) and fluorescence microscopy (ZEISS). The expression of microRNA-22 and downstream PTEN protein expression were detected. The experimental cells were divided into two groups including Adeno-miR22 group and Adeno-null group.

Cell viability was assessed using a CCK-8 assay, based on the enzymatic reduction of WST-8 in living cells and the production of a proportional color change. Briefly, 1 × 10⁴ cells suspended in 150 µl complete medium were seeded in each well of a 96-well culture plate and incubated at 37˚C under 5% CO₂ for 24 h. The WST-8 cell proliferation reagent (50 µl) was added and incubated for 4 h under 5% CO₂ at 37˚C. The negative control comprised WST-8 reagent and complete medium with no cells. The absorbance was measured at 450 nm using a spectrophotometer (Huier Company, Hangzhou, China) and the optical density (OD) was calculated as OD = ODsample − ODcontrol. Each experiment was performed in triplicate wells and repeated three times.

DNA synthesis was measured using Promega’s Click-iT^Ò EdU kit. Briefly, the cells cultured in 6-well plates were incubated with EdU for 24 h. Following fixation and permeabilization, 0.5 ml Click-iT^Ò reaction cocktail was added and incubated for 30 min at room temperature in the dark. Hoechst 33342 (Invitrogen, USA) was used to stain the cell nuclei. The cells were observed using a fluorescence microscope, and nuclei undergoing DNA synthesis were stained red. The numbers of proliferating nuclei were counted in 10 randomly selected fields (magnification, ×100) and the average was calculated. The rate of DNA synthesis was calculated as the number of proliferating nuclei/100. Each experiment was performed in triplicate wells and repeated three times.

Cell apoptosis was assessed by caspase-3 activity. The activity of caspase-3 was determined using the caspase-3 activity kit (Beyotime Institute of Biotechnology, Shanghai, China). Cell lysates were prepared after their respective treatment with various designated treatments. Assays were performed on 96-well microtitre plates by incubating 10 μl protein of cell lysate per sample in 80 μl reaction buffer (1% NP-40, 20 mM Tris-HCl (PH 7.5), 137 mM Nad and 10% glycerol) containing 10 μl caspase-3 substrate (Ac-DEVD-pNA) (2 mM). Lysates were incubated at 37˚C for 4 h. Samples were measured with an ELISA reader at an absorbance of 405 nm. The detail analysis procedure was described in the manufaturer’s protocol. To further measure the extent of cell injury, the LDH activity was also tested. At the end of the incubation period, the culture supernatants were collected. The activity of LDH was detected at 490 nm according to the LDH assay kit (Beyotime Institute of Biotechnology, Shanghai, China).

The harvested cells were lysed with RIPA lysis buffer and protease inhibitor cocktail. Protein concentrations were quantified using a BCA protein assay kit. 30 ug proteins were separated using 10% SDS-polyacrylamide gel electrophoresis and transferred onto a PVDF membrane. The membrane was blocked with Tris-buffered saline-Tween 20 (TBS-T) and 5% nonfat dried milk for 2 h, and incubated with primary antibody (mouse monoclonal to PTEN, 1:1000, Cat No. AB32199, abcam, Cambridge, UK) overnight at 4˚C. Following washing twice with TBS-T and incubation with peroxidase-conjugated secondary antibodies (goat anti mouse 1:1000, A996702 Amyjet Scientific, China) for 1h at room temperature, the bands were detected using a chemiluminescence detection system (Invitrogen, USA). The band intensities were quantified using the Photo-Image System (Siemens, Germany).

Adenovirus mediated over-expression of microRNA-22 in cardiomyocytes co-expressed GFP. The ratio of GFP positive cells was detected by flow cytometry, indicating that more than 95% cells were successfully transfected with GFP (Figure 1). The expression of microRNA-22 was confirmed by qRT-PCR after adenovirus transfection. The results showed that the expression of microRNA-22 was significantly increased by 10.75 times compared with that of adeno-null group, *p < 0.05. This suggested that adenovirus transfection with high expression of microRNA-22 was successful (Figure 2). Western blot showed that the protein expression of PTEN was down-regulated after high expression of microRNA-22 (Figure 3).

After adenovirus transfection, EdU was used to detect the proliferation of nuclei in both groups, which represented nuclei proliferation. Red represented for positive nuclei and blue for all nuclei. Compared with adeno-null group, the nuclei proliferation in adeno-miR22 group was significantly increased, *p < 0.05 (Figure 4).

The cell growth curve recorded. Cell viability was measured by CCK-8 assay. Apoptosis was detected by Caspase-3/LDH assay. Compared with adeno-null group, the cells with high expression of microRNA-22 showed increased cell growth velocity and viability as well as decreased cell apoptosis, *p < 0.05 vs. adeno-null group (Figures 5(A)-(D)).