Animals
Male C57/Bl6 mice were maintained in accordance with the guidelines published by the US National Institutes of Health. All study procedures were approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University. This study was conducted in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Academy Press (NIH, revised in 1996).
Treatment using Dox and exosome MIF in vivo
Eight-week-old male C57/Bl6 mice were injected three times with Dox (4 mg/kg body weight) on alternative days in a time span of 1 week (Monday, Wednesday, and Friday) for a cumulative dose of 12 mg/kg. For exosomeMIF treatment groups, exosomeMIF were injected on alternative days between Dox treatments (Tuesday, Thursday, and Saturday), as previously reported [13].
Echocardiographic Evaluation
The mice inhaled anesthetic isoflurane 14 days after treatment with Dox, and vevo 2100 was used to observe the echocardiographic parameters of mice and assess the cardiac functions (Vevo 2100, Visual Sonics, Canada).
Quantitative Reverse Transcription-polymerase Chain Reaction (qRT-PCR)
RNA was isolated using TRIzol reagent (Invitrogen, CA, USA), and cDNA was synthesized using an ImProm-II reverse transcription kit (Promega, WI, USA) following the manufacturer’s instructions. Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) was performed with SYBR Green to detect mRNA levels. The mRNA levels were calculated relative to the control GAPDH or U6 using the 2−ΔΔCq method. The sequences of all qRT-PCR primers are shown in Table 1.
Microarray Analysis
Exosomes derived from MSCs, or MSCs treated with MIF, were lysed immediately in 500 µL of TRIzol reagent (ThermoFisher Scientific, MA, USA) and stored at − 80 °C before purification using a standard phenol–chloroform extraction protocol with an RNAqueous Micro Kit (ThermoFisher Scientific). The transcriptome was subjected to microarray analysis using an Affymetrix human array (ThermoFisher Scientific) and normalized based on quantiles.
Western Blot Analysis
Western Blot analysis was conducted as previously described [25]. Primary antibodies, including p27kip (ab62364), p16INK4a (ab211542), Sirt2 (ab211033), and β-actin (ab179467), were purchased from Abcam.
Cardiomyocyte Isolation And Culture
Mouse ventricular myocytes were isolated from C57BL/6 mice. The animals were euthanized by cervical dislocation. The hearts were obtained and digested in a digestion solution containing 0.25% trypsin and collagenase I. Myocytes were separated after 3 h of differential sedimentation and adhesion. They were then cultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS).
Senescence-associated β-galactosidase Assay (SA-β-gal Assay)
The cellular senescence was measured using the SA-β-gal assay (Cell Signaling Technology, MA, USA). Briefly, the cells at the density of 2 × 104 were fixed with 2% paraformaldehyde and incubated with the SA-β-gal staining solution as previously described [26].
Agomir Studies
Chemically modified oligonucleotides were designed to mimic miR-221-3p. Eight-week-old C57/Bl6 mice were injected (intraperitoneally) with agomir-221-3p (80 mg/kg) or control agomir (80 mg /kg) for three consecutive days as previously reported [27].
Cell Culture And Cell Treatment
BM‑MSCs were isolated using a standard protocol, as described previously [28]. Briefly, bone marrow was isolated from the femur and tibia of mice by flushing with PBS. Adherent MSCs were propagated and maintained at 37˚C and in the presence of 5% CO2 in high-glucose DMEM supplemented with 10% FBS and 1% penicillin/streptomycin.
For MIF treatment, the cells were cultured with a medium containing 100 ng/mL of recombinant MIF and incubated at 37 °C for various periods as previously reported [29].
HL-1 murine cardiomyocytes were a kind gift of Dr. William C. Claycomb. The cells were maintained in fibronectin-coated flasks, supplemented with 10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, and 2 mM L-glutamine, and kept semi-confluent at all times.
Isolation Of Exosomes
Exosomes were isolated and purified from the supernatants of MSCs and MIF-treated MSCs. The supernatants were collected after 48-h culture. The exosome quick extraction solution was added to the filtered solution at a 1:5 ratio and stored at 4 °C for at least 12 h. The precipitation (exosomes) was dissolved in PBS and stored at − 70 °C. The characterization of exosomes was carried out as previously reported [20].
Cell Cycle Assay
Cold anhydrous ethanol (70%) was employed to fix the cells. Then, the cells were treated with propidium iodide (Sigma, MO, USA) and RNase A. A flow cytometer equipped with CellQuest software was used to detect the cell cycle distribution.
Relative Telomere Length Measurement
Relative telomere length quantification in HL-1 cells was performed using a qPCR approach based on a previously established method [30], using GAPDH as the normalizing gene. The primer pairs used to detect the telomere length are listed in Table 1.
Relative Telomerase Activity Measurement
The telomerase activity of HL-1 cells was examined using a Telo TAGGG Telomerase PCR Enzyme-linked immunosorbent assayPlus kit according to the manufacturer’s instructions as described previously [31].
Fluorescence tracing of exosomes in vitro
Exosomes were labeled with DiI incubated with the dye (1 mM) at the volume ratio of 500:1 for 30 min, followed by exosome isolation. For the in vitro tracing of exosomes, cardiomyocytes were incubated with DiI-labeled exosomes for 3 h. The cell nuclei were stained with DAPI (1:1000, Invitrogen) for 10 min at 37 °C. Fluorescence was detected under a microscope.
Small Interfering RNA Transfection
LncRNA–NEAT1 expression in MSCs was knocked down using small interfering (si)RNAs, with a nontargeting siRNA as a negative control (Invitrogen). Sirt2 expression in cardiomyocytes was also knocked down using siRNAs. The procedures were conducted as described previously [31]. The transfection efficiency was detected using qRT-PCR and western blot analysis.
MiR-221-3p Overexpression
Cardiomyocytes were seeded into six-well plates at a density of 1 × 105 cells per well. The cells were transfected with miR-221-3p mimic or negative control (NC) mimic (Pre-miR miRNA Precursors, Life Technologies, Karlsruhe, Germany) using X-treme transfection reagent (Roche Applied Science, Penzberg, Germany), according to the manufacturer’s protocol, to induce the overexpression of miR-221-3p. The cells were harvested for further analysis 48 h after transfection, and the transfection efficiency was analyzed using qRT-PCR.
Luciferase Reporter Assay
The 3′-untranslated regions (UTRs) of LncRNA–NEAT1 and Sirt2 were synthesized, annealed, and inserted into the SacI and HindIII sites of the pmiR-reporter luciferase vector (Ambion), downstream of the luciferase stop codon to induce the mutagenesis of LncRNA–NEAT1 and Sirt2. The constructs were validated by sequencing. Cardiomyocytes were seeded into a 24-well plate for luciferase assay. After overnight culture, the cells were co-transfected with a wild-type (WT) or mutant plasmid and an equal amount of miR-221-3p mimic or miR-NC mimic. Luciferase assays were performed using a dual-luciferase reporter assay system (Promega) 24 h after transfection.
Statistical analysis
Data were expressed as the mean ± standard deviation (SD). Differences between groups were tested by one-way analysis of variance, and comparisons between the two groups were evaluated using the Student’s t test. Analyses were performed using SPSS package v19.0 (SPSS Inc., IL, USA). A P value less than 0.05 was considered statistically significant.