1. Ethical statement
Animal experiments were conducted according to the Guidelines for the Care and Use of Laboratory Animals and were approved by Ethics Committee of Xuzhou Central Hospital. Surgical operations are performed under anesthesia by sodium pentobarbital (50 mg/kg) injecting intraperitoneally.
2. Cell culture and identification
Human umbilical mesenchymal stem cells (HuMSCs) were gained from Clinical Center of Reproductive Medicine in Nanjing. HuMSCs were cultured with Eagle’s Minimum Essential Medium (EMEM, Gibco, USA) containing 10% fetal bovine serum(FBS, Gibco, USA), 5ng/ml epidermal growth factor and 5ng/ml alkaline fibroblast growth factor. Human umbilical vein endothelial cells were cultured in Dulbecco modified eagle medium (DMEM, Gibco, USA) containing fetal bovine serum (10%), 100 μg/ml streptomycin, 100 U/ml penicillin, and 110 mg/ml sodium acetone. All those cells were cultured in an incubator which contains 5% CO2 at 37℃. HuMSCs used for experiments were between passages 3 and 6. Oil red staining or alcian blue were used to examine differentiation capacity of HuMSCs for adipogenesis and chondrogenesis.
3. Neonatal rat cardiomyocytes (NRCMs) isolation and culture
Myocardial cells were extracted from those 1-3-days old suckling rats. Ventricles were cleaned and shredded using 1× ADS (NaCl 68g/L, HEPES 47.6g/L, Na2HPO4 1.38g/L, Glucose 6g/L, KCl 4g/L and MgSO4 1g/L) buffer solution with pH 7.35‐7.45. After that, tissues were digested by 0.6 mg/ml pancreatin and 0.4 mg/ml collagenase II. Collect the supernatant and add one-fifth horse serum of the volume of supernatant after each digestion. Finally, the cells were purified and cultured in DMEM consisting of 10% horse serum, 5% fetal bovine serum, 100μg/ml streptomycin and 100 U/ml penicillin, and placed in an incubator with a CO2 content of 5% and a temperature of 37°C.
4. Extraction and identification of exosomes
Isolation of exosome was proceed as described previously9. Briefly, huMSCs with 80% confluence were washed with PBS and then cocultured in exosome free medium for 48 h. The supernatant were centrifuged at 1500g for 30 minutes and incubated with Exosome Isolation Reagent (C10130-2, RiboBio, China) for 12 hours at a temperature of 4°C, and centrifuged at 2000 g for half an hour. The precipitates were suspended with PBS and stored at 80 ℃. Bicinchoninic acid assay (BCA, Thermo Fisher Scientific, Waltham, MA) was used to detect exosomes protein concentration by measuring absorbance at 562 nm. Transmission electron microscopy (TEM; Hitachi H-7650; Japan) was used to observe morphology of exosomes and western blot was carried out to detect surface markers CD81, CD63 and TSG101. Nanoparticle Tracking Analysis (NTA) was used to analyze the size and concentration of exosomes.
5. Lentiviral constructions and infection
The lentivirus was gained from Shanghai Genechem Co., LTD. There are two types of lentiviral recombinant vectors. One is Ubi-MCS-SV40-EGFP-IRES-puromycin, used as hsa-miR-214-3pOE (overexpression of hsa-miR-214-3p) virus. Another is H1-MCS-CMV-EGFP, used as hsa-miR-214-3pNC (Control) virus. HuMSCs were incubated in 24-well plates, grow to 50% confluence, and infected with hsa-miR-214-3pOE virus or hsa-miR-214-3pNC virus, respectively. The fluorescence signals were observed and the expression of hsa-miR-214-3p was detected by qRT-PCR.
6. Exosome uptake assay
Exosomes were labelled by 1 μmol Dil (ThermoFisher, USA) and were cultured with cells for 6 or 24 h in vitro. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, Shanghai, China). Zeiss laser-scanning confocal microscope (LSM5 Live, Carl Zeiss, Germany) were used to evaluate the absorbance of exosomes.
7. Migration assay
HUVECs were planted on 6-well plate with 2 × 105 cells/well. Cells were treated with 1 mL test medium for 24 hours. After that, cells were rinsed and scratched by P200 pipette tip. Cell migration was evaluated 24 hours later. All samples were observed with three replicates.
8. Tube formation of HUVECs assay
Capillary tube formation assay was used to observe angiogenesis of HUVECs. HUVECs were incubated in 96-well plate coated with growth factor reduced Matrigel (356,230; BD Biosciences, SanJose, CA, USA). Six hours later, formation of capillary-like tubes was observed and the tube length was analyzed by Image J software. All experiments were carried out with three replicates.
9. Immunofluorescence and TdT‐mediated dUTP nick‐end labeling staining
Cardiomyocytes were fixed with 4% paraformaldehyde for 15 min, and then permeabilized with 0.5% Triton X‐100 for 15 min. After that, cells were incubated with 10% goat serum for 2 hours, α‐actinin primary antibody (1:150; A7811; Sigma, Santa Clara, CA) overnight at 4℃ and second antibody for 1 hour at room temperature. TdT‐mediated dUTP nick‐end labeling (TUNEL) staining was used to examine apoptotic cells following manufacturer's instructions (Roche, Mannheim, Germany).
10. MI model establishing and exosomes injection
Sprague Dawley rats (Male, 6-8 weeks) were provided by animal center of Nanjing Medical University (Nanjing, China). Rats were injected 50 mg/kg sodium pentobarbital and mechanical ventilated via orotracheal intubation. The left anterior descending coronary artery was ligated 1.5 mm at the lower edge of the left auricle by thoracotomy. Exosomes (50ug) were injected into 4 portions at the border of the infarction area. All surgeries and analyses were blind interventions.
11. Cardiac function assessment
The cardiac function was measured by transthoracic echocardiography 28 days after exosome injection with VEVO 2000 high-resolution micro-imaging system. A 30 MHz transducer was used to measure the left ventricular short-axis in M-mode. The left ventricular ejection fraction (LVEF) and left ventricular shortening rate (LVFS) were calculated by Vevo2000 workstation software.
12. Masson trichrome staining and Hematoxylin-Eosin staining
Heart tissues were collected, fixed and sliced to 5μm. Mason's trichrome staining as described above9. The proportion of infarct area is the sum of infarct area of each section/sum of LV area of each section × 100%. Hematoxylin-Eosin (HE) stain was used to measure the level of inflammatory cells.
13. Fluorescence in situ hybridization (FISH) of miR-214
MiR-214OE exosomes (50ug) were injected at the edge of the infarction area. After 6 hours, the rats were sacrificed. The miR-214 oligonucleotide probes (Sangon Biotech, China, 8 ng/μL) was incubated with Cy3 or FAM in pre-hybridization buffer at 37°C for 1 h, and then hybridized overnight at 37°C. With sections 2 × SSC, 1 × SSC, 0.5 × SSC rinsing and DAPI staining, the internalization of miR-214 in recipient cells or myocardium was observed by confocal microscope.
14. Immunofluorescence
Immunofluorescence was performed as reported previously24. Briefly, heart tissue were collected and then fixed, embedded, and sectioned. After that, the section was stained with primary antibody anti-actin and CD31 (1:200, ab7388, Abcam, Cambridge). The nuclei was stained with DAPI.
15. Western blot analysis
Western blotting was performed with a standard protocol as previously described9. Antibodies were used as follows: GAPDH (1:1000; 5174S; Cell Signaling Technology), Bcl-2 (1:1000; 2870S; Cell Signaling Technology),, AKT (1:1000; 4961S; Cell Signaling Technology), p-AKT (1:1000; 4060S; Cell Signaling Technology), BAX (1:1000; 5023, Cell Signaling Technology), PTEN (1:1000; ab31392, Abcam).
16. Quantitative real‐time polymerase chain reaction analysis
Total RNAs were extracted by Trizol and treated with RNase‐free DNase I (1/20 μL, Promega Corp, Madison, WI). Reverse transcription of cDNAs was performed using Reverse Transcription Kit (Takara, Dalian, China). Polymerase chain reaction analysis (PCR) analysis was performed with SYBR green PCR Master Mix (Applied Biosystems, Foster, CA, USA) on ABI‐7900 Real‐Time PCR Detection System (7900HT; Applied Biosystems). The exosomal level of miR-214-3p was normalized to that of cel-miR-39 (C39). The cellular miR-214-3p expression was normalized to U6. The cellular expression of PTEN mRNA was normalized to that of glyceraldehyde3-phosphate dehydrogenase (GAPDH). The related gene expression was normalized to that of GAPDH. The primer sequences are listed in Additional file: Table S1.
17. Flow cytometry analysis
Apoptosis of HUVECs were examined by flow cytometry using Annexin V Alexa Fluor647/PI/Apoptosis detection kit (Fcmacs Biotech, Nanjing, China). Briefly, cells were washed, digested and resuspended in a binding buffer with double distilled water at a ratio of 1:3, incubated by 5 μl Annexin V and 10 μl propidium iodide (PI) for 15 min at room temperature in the dark. After that, 400 μl PBS was added and flow cytometry analysis was performed on FAC Scan. Data were analyzed by FlowJo software.
18. Luciferase assay
The PTEN 3'UTR cDNA sequences containing mir-214-3p binding site was inserted into pmir-GLO-promoter vector (Promega, Madison, USA). HEK-293T cells were transfected with miR-NC or miR-214-3p mimics and seeded into 96-well plates. The sequences of miRNAs transfected were shown in Additional file: Table S2. After that, cells were co-transfected with 100 ng pmiR-GLO-PTEN-WT or pmiR-GLO-PTEN-MUT. The luciferase activity was detected by EnSpire® 2300 Multimode Plate Reader (Perkin Elmer Singapore Pte. Ltd., Singapore) 24 hours after transfection. luciferase activities of firefly were normalized to Renilla luciferase activity.
19. Statistical Analysis
All data are expressed in the way of mean±standard deviation. The two-tailed test was used to compare the means between the two groups, and one‐way analysis of variance was used for multiple experimental groups. SPSS statistical software (version 17.0, SPSS Inc., Chicago, IL) was used for statistical analysis. P value of less than 0.05 was considered statistically significant.