Therapeutic delivery of microRNA-125a-5p oligonucleotides improves recovery from myocardial ischemia/reperfusion injury in mice and swine

Rationale: Clinical application of mesenchymal stem cells (MSCs) and MSC-derived exosomes (MSC-Exos) to alleviate myocardial ischemia/reperfusion (I/R) injury is compromised by the low cell engraftment rate and uncontrolled exosomal content. As one of their active ingredients, single-component microRNA therapy may have more inherent advantages. We sought to find an ideal microRNA candidate and determine whether it could reproduce the cardioprotective effects of MSCs and MSC-Exos. Methods: Cardiac function and myocardial remodeling in MSC, MSC-Exo, or microRNA oligonucleotide-treated mouse hearts were investigated after I/R injury. The effects of microRNA oligonucleotides on cardiac cells (macrophages, cardiomyocytes, fibroblasts, and endothelial cells) and their downstream mechanisms were confirmed. Large animals were also employed to investigate the safety of microRNA therapy. Results: The results showed that microRNA-125a-5p (miR-125a-5p) is enriched in MSC-Exos, and intramyocardial delivery of their modified oligonucleotides (agomir) in mouse I/R myocardium, as well as MSCs or MSC-Exos, exerted obvious cardioprotection by increasing cardiac function and limiting adverse remodeling. In addition, miR-125a-5p agomir treatment increased M2 macrophage polarization, promoted angiogenesis, and attenuated fibroblast proliferation and activation, which subsequently contributed to the improvements in cardiomyocyte apoptosis and inflammation. Mechanistically, Klf13, Tgfbr1, and Daam1 are considered the targets of miR-125a-5p for regulating the function of macrophages, fibroblasts, and endothelial cells, respectively. Similar results were observed following miR-125a-5p agomir treatment in a porcine model, with no increase in the risk of arrhythmia or hepatic, renal, or cardiac toxicity. Conclusions: This targeted microRNA delivery presents an effective and safe strategy as a stem cell and exosomal therapy in I/R cardiac repair.

The medium was then collected and centrifuged at 2,000 rcf for 10 min and at 10,000 rcf for 30 min. After passing through a 0.22-μm filter (Millipore, SLGP033RB) to remove the cell debris, the supernatant was centrifuged at 100,000 rcf for 70 min, and the pellet was resuspended in PBS. The mixture was centrifuged again at 100,000 rcf for 70 min, and the pellet containing isolated exosomes was resuspended in PBS and stored at -80°C for further use. The exosome concentration was determined by BCA assay (Beyotime Biotech, P0012). Exosomes from cardiac fibroblasts were collected as a control.
For MSC-Exo characterization, exosomes were dissolved in 1 mL PBS, and the particle concentration and size distribution were evaluated via a NanoSight LM10 apparatus (Malvern). The expression of exosomal marker proteins (CD63, CD9, TSG101, and Alix) was determined by immunoblotting. The exosomal ultrastructure was assessed via transmission electron microscopy (TEM) as described previously [3]. Briefly, the resuspended exosomes were fixed with 2.5% glutaraldehyde, post-fixed in buffered 1% OsO4 with 1.5% K4Fe(CN)6, embedded in 1% agar, and processed according to the standard Epon812 embedding procedure. Exosomes were visualized on thin sections (60 nm) via a Tecnai G2 T12 transmission electron microscope (FEI).

Next Generation Sequencing (NGS)
microRNA sequencing and NGS data analysis of exosome samples were performed by LC Sciences [4]. The total exosomal RNA was purified via a miRNeasy kit (QIAGEN, 217684) according to the manufacturer's protocols, and the quality and quantity of RNA were analyzed by a 2100 Bioanalyzer (Agilent). Sequencing libraries were generated through the TruSeq Small RNA Sample Preparation protocol, which includes the ligation of specific RNA adapters to both the 3′ and 5′ ends for each sample, followed by reverse transcription, amplification, and purification of small-RNA libraries (size range 22 to 30 nucleotides). Sequencing was performed on an Illumina HiSeq 2500 platform, and adapter dimers, junk, low complexity, common RNA families (rRNA, tRNA, snRNA, snoRNA), and repeats were removed from the raw reads with an in-house program, ACGT101-miR (LC Sciences). Unique sequences with lengths of 18 to 26 nucleotides were mapped to specific species precursors in miRBase 22.0 by BLAST search to identify known microRNAs and novel 3p-and 5p-derived microRNAs; length variations at both the 3′ and 5′ ends and one internal sequence mismatch were allowed in the alignment. The unique sequences mapping to specific species of mature microRNAs in hairpin arms were identified as known microRNAs. Unique sequences mapping to the other arm of known specific species precursor hairpin opposite to the annotated mature microRNA-containing arm were deemed novel 3p-or 5p-derived microRNA candidates. The remaining sequences were mapped to other selected species precursors (except specific species) in miRBase 22.0 by BLAST search, and the mapped pre-microRNAs were further BLASTed against the specific species genomes to determine their genomic locations. Unmapped sequences were BLASTed against the specific genomes, and the hairpin RNA structures containing sequences were predicated from the flank 80 nucleotide sequences using RNAfold software (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi). The relative expression of the 14 upregulated miRNAs and 5 downregulated microRNAs in MSC-Exos compared to FB-Exos were displayed in a heat-map, and data were expressed as fold change (log2) of exosomal microRNAs normalized read counts ( Table S2). The microRNA sequencing data have been deposited in the Genome Sequence Archive in the National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (project number: PRJCA007694, GSA accession number: CRA005702), and are publicly accessible at https://ngdc.cncb.ac.cn/gsa.

MiR-125a-5p Inhibition in MSCs and MSC-Exos
Synthetic mmu-miR-125a-5p antagomir or negative control (NC) antagomir was mixed with culture medium to form a final antagomir concentration of 200 nM. Next, antagomir was transfected directly into MSCs for 24 h, and cells and exosomes depleted with miR-125a-5p were isolated for further experiments.

Animals
All animal procedures and protocols (mice and swine) were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (8th Edition, 2011) and approved by the Institutional Animal Care and Use Committee of Tongji University. Female C57BL/6 mice (20 g, 6-8 weeks old) were obtained from Shanghai Jiesijie Laboratory Animal Co., Ltd, and Bama swine (female, 20 kg, 16-20 weeks old) were purchased from Shanghai Jiagan Laboratory Animal Co., Ltd. The animals were fed a standard laboratory diet and maintained in a 12-h light/12-h dark cycle.

Myocardial Ischemia/Reperfusion (I/R) Surgery and Treatment in Mice
The mouse model with myocardial I/R was established surgically by ligation of the left anterior descending (LAD) coronary artery as previously described [5]. Briefly, mice were placed in an induction chamber and then connected to a small rodent ventilator with 2% isoflurane. Subsequently, the chest was opened to expose the heart, left auricle, and LAD. The LAD was ligated with an 8-0 silk suture approximately 2 mm below the edge of the left auricle for 60 min, followed by reperfusion. Ligation was considered successful when the anterior wall of the left ventricle (LV) turned pale. The experimental treatments were administered at the onset of reperfusion. Animals in groups with different doses of miR-125a-5p agomir and MSC were intramyocardially injected with a concentration gradient of miR-125a-5p agomir (10, 20, and 40 nmol) or MSC (1 × 10 5 , 3 × 10 5 , and 5 × 10 5 ) in 15 μL PBS (similarly hereafter), respectively; animals in the I/R control, MSC, and MSC-Exo groups were injected with 15 μL PBS, 5 × 10 5 MSCs, and 10 μg MSC-Exos, respectively; animals in the miR-125a-5p and NC agomir groups were administered with 20 nmol (~8 mg/kg of 20 g) mmu-miR-125a-5p agomir, and 20 nmol NC agomir, respectively; animals in the MSC 125a-anta and MSC NC-anta groups were treated with 5 × 10 5 MSCs transfected with mmu-miR-125a-5p antagomir and NC antagomir, respectively; and animals in the MSC 125a-anta -Exo and MSC NC-anta -Exo groups were treated with 10 μg exosomes isolated from MSC 125a-anta and MSC NC-anta , respectively. Next, the thoracotomy site was closed in layers with 6-0 sutures. After reinstallation of spontaneous respiration, the mice were extubated and allowed to recover from anesthesia. Animals were maintained on a 37°C heating pad during the myocardial I/R surgery until recovery. All agents were directly injected into the border zone of the I/R hearts at the onset of reperfusion, and sham-operated mice underwent the same procedure, excluding LAD ligation. At the end of the experiment, in addition to the hearts used for flow cytometry (FCM) analysis, the remaining hearts were cut in half; half was used for histological studies, and the other half was used for RNA and protein extraction and enzyme-linked immunosorbent assay (ELISA).

FCM Analysis
Non-cardiomyocytes were collected from mouse hearts as described previously [7]. Mononuclear cells, including macrophages, were then isolated from non-cardiomyocytes using a centrifugal-based method (500 rcf for 30 min) with percoll gradients (Cytiva, 17089102) [8]. Cells were blocked with mouse Fc block (BD Biosciences, 553141) for 5 min at 4°C, incubated with primary and isotype control antibodies for 30 min at 4°C, resuspended in 200 μL staining buffer, and evaluated using a CytoFLEX flow cytometer and FlowJo software.

Luciferase Reporter Assay
The Klf13 sequences containing 233 to 239 bp, Tgfbr1 sequences containing 2243 to 2249 bp, and Daam1 sequences containing 1146 to 1153 bp were amplified and cloned between the XhoI and SalI sites of the pmirGLO dual-luciferase microRNA target expression vector (Promega, E1330). The mutant Klf13, Tgfbr1, or Daam1 3¢UTR pmirGLO vector was generated using a QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene, 200522) according to the manufacturer's instructions. RAW 264.7 cells, cardiac fibroblasts, and ECs were cultured until 60% confluency and then co-transfected with miR-125a-5p agomir and either the Klf13, Tgfbr1, or Daam1 3¢UTR pmirGLO vector or the mutant Klf13, Tgfbr1 or Daam1 3¢UTR pmirGLO vector, respectively. Cells transfected with the no-insert pmirGLO vector were used as a control. Twenty-four hours after transfection, the cells were analyzed in a luciferase activity using a Dual-Glo Luciferase Assay System (Promega, E2920). The relative luciferase activity was determined by the ratio of firefly luminescence/Renilla luminescence.

Myocardial I/R Surgery and Treatment in Swine
Porcine myocardial I/R surgery was performed as previously described [4]. Briefly, animals were anesthetized with inhaled 2% isoflurane, intubated, and ventilated with a respirator and supplemental oxygen. Body temperature, electrocardiogram (ECG), blood pressure, and arterial oxygen saturation were monitored throughout the surgical procedure. After left thoracotomy, the pericardial sac was opened to expose the LAD and its diagonal branches, and the LAD was occluded for 60 min at a site between the 1st and 2nd diagonal branches and then reperfused. Ligation was considered successful when the anterior wall of the LV turned pale and ST elevation was observed on ECG. The experimental treatments were administered at the onset of reperfusion. Animals in the miR-125a-5p group received 1.2 μmol (~0.5 mg/kg of 20 kg) hsa-miR-125a-5p agomir (in 1 mL PBS, similarly hereafter); NC agomir animals received 1.2 μmol NC agomir; animals in the I/R control group received 1 mL PBS; and animals in the sham group underwent all surgical procedures except occlusion and recovered without any experimental treatment. After closing the chest in layers, a Reveal LINQ insertable cardiac monitor (ICM) device (Medtronic) was implanted subcutaneously to monitor the electrical activity. Animals in all treatment groups received standard postoperative care, including analgesia and antibiotic administrations, until the animals ate normally and became active.

Telemetric Monitoring
The electrical activity from conscious swine was continuously monitored by a Reveal LINQ ICM device (Medtronic), which was implanted subcutaneously after the surgery and treatment. The arrhythmic events and heart rates were recorded. A tachy episode was defined as a rate faster than 100 beats/min; heart pause was defined as a complete disappearance of electrical activity; a brady episode was defined as a rate slower than 60 beats/min; paroxysmal supraventricular tachycardia (ST) was defined as narrow QRS complexes (< 120 ms) with regular tachycardia rhythm (160-220 beats/min), and hidden or inverted P waves [10,11]; atrial tachycardia (AT) and atrial fibrillation (AF) were automatically recognized by the ICM device; paroxysmal ventricular tachycardia (VT) was defined as ≥ 3 consecutive ventricular beats that occurred at a rate faster than 100 beats/min, or continuous ventricular beats that terminated spontaneously within 30 s; ventricular fibrillation (VF) was defined as a chaotic tachycardia without consistently identifiable QRS complexes; and sustained ventricular tachycardia was defined as sustained ventricular beats lasting longer than 30 s [12].

Cardiac Multi-Detector Computed Tomography (MDCT)
MDCT images were obtained 28 days after myocardial I/R surgery and treatments. Animals were anesthetized with 2% isoflurane, positioned in a supine position, and scanned with ECG monitoring by an Aquilion ONE TSX-301C MDCT scanner (Canon Medical). A 35-mL bolus of Iopamidol (Bracco Sine, Iopamiro 370) was injected intravenously opacifiying the LV chamber at a rate of 3.5 mL/s during the first phase acquisitions. An additional 25 mL of Iopamidol was subsequently administered at a rate of 1.0 mL/s, and delayed enhancement (de)-MDCT images were acquired with a coronary CT angiography model 7 min after contrast delivery (second phase acquisitions). Images were acquired using the following parameters: gantry rotation time = 275 ms, detector collimation = 0.5 mm × 320 (isotropic voxels = 0.5 × 0.5 × 0.5 mm 3 ), tube voltage = 100 kV, and tube current = 400 mA.
All raw data were reconstructed in 5% steps from 0%-95% throughout the R-R interval, and images in optimal phase were reformatted separately for myocardial perfusion (first-pass images) and scar assessment (de-MDCT images). Cardiac functional parameters, including LVEF, stroke volume, LVESV, LVEDV, wall motion, and myocardial perfusion, were evaluated on a Vitrea 2 computer workstation (Vital Images). The infarct size on day 28 post-cardiac I/R was determined by six cross-sections of the de-MDCT images from the apex to base by dividing the infarct area by the total LV area.

Hemodynamic Measurements
Invasive monitoring of aortic and LV pressure, and LV function of the anesthetized animals was acquired via a transvascular (through the left external carotid artery) polyvinyl chloride catheter introduced into the LV lumen as previously described [4]. Aortic and LV pressures were continuously monitored with a PowerLab system (AD Instrument), and LV end-diastolic pressure (LVEDP), peak contraction velocity (dP/dtmax), and peak relaxation velocity (dP/dtmin) measurements were made on day 28 after myocardial I/R. After the measurement, blood samples were drawn and blood tests were performed by the Department of Laboratory Medicine, Shanghai East Hospital, Tongji University School of Medicine.

RNA Isolation and Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR)
Total RNA and microRNA were extracted by TRIzol reagent (Thermo Fisher Scientific, 15596018) following the manufacturer's protocols. The concentrations of RNA were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific), and samples with an A260/A280 ratio from 1.8 to 2.0 were applied for reverse transcription. mRNAs were reverse transcribed using a PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, RR047A). Then, 20-μL reactions, including 1 μg mRNA and primers, were detected by a QuantStudio 7 Flex system (ABI) using PowerTrack SYBR Green Master Mix (ABI, A46109). microRNAs were reverse transcribed with a TaqMan microRNA Reverse Transcription Kit (ABI, 4366596) following a quantitative analysis of microRNAs by TaqMan Advanced microRNA Assays (ABI, 4440887). The PCR thermocycling conditions were 95°C for 1 min, followed by 40 cycles of 95°C for 10 s and 60°C for 32 s. The relative expression of microRNAs was normalized to U6 or cel-miR-39, and the relative mRNA expression was normalized to Gapdh and calculated using the standard 2 -(ΔCtsample-ΔCtcontrol) method. The primers used for PCR are listed in Table S3.

Histological Studies 2,3,5-triphenyltetrazolium Chloride (TTC) Staining
Mouse hearts were collected 3 days after I/R injury and cut into five slices perpendicular to the LAD from the apex to base, and slices were immediately immersed in 1% TTC (Sigma-Aldrich, T8877) at 37°C in the dark for 10 min to distinguish infarcted tissue from viable myocardium.

Tissue Sample Preparation
Mouse heart tissues were harvested and perfused with PBS, followed by embedding with Tissue-Tek O.C.T. Compound (SAKURA, 4583) on liquid nitrogen, and subsequently subjected to serial cryosectioning (8 μm-thickness). For porcine hearts, the LV wall was cut vertically into six rings (R1 to R6) from the apex to base, and each ring was cut into eight samples (S1 to S8) in sequence. Then, the tissues from S1 to S5 of R2 to R4 (i.e., from the site of agomir injection) were embedded and cryosectioned.

Masson's Trichrome Staining
Tissue sections were stained using a Masson's trichrome Stain Kit (Solarbio, G1340) according to the manufacturer's instructions and observed under an optical microscope. For mouse tissues, histological evaluation of infarct size (at day 3 post-myocardial I/R) and fibrosis size (at day 28) were calculated as a percentage of the infarct area (or fibrosis area)/total LV area. The severity of fibrosis was classified as follows: > 45% fibrotic area, ''severe;'' 25%-45% as ''moderate;'' or < 20% as ''mild'' for I/R hearts [13]. For porcine tissues, interstitial fibrosis was determined by the ratio of fibrosis area/each random high-power field of view.

Hematoxylin and Eosin (H&E) Staining
Tissue sections were stained using a H&E Staining Kit (Beyotime Biotech, C0105) following the manufacturer's protocols before being observed under an optical microscope. The infiltration of inflammatory cells was evaluated by the ratio of inflammatory cell area (white dotted)/each random high-power field of view.

Immunostaining
Heart sections or cell slides were fixed with 4% paraformaldehyde, permeabilized in 0.25% triton X-100, blocked in Ultra-V Block buffer for 10 min at room temperature, incubated overnight at 4°C with primary antibodies, and then incubated for 2 h at room temperature with the corresponding secondary antibodies. For wheat germ agglutinin (WGA) staining, sections were stained for 1 h at room temperature using a FITC-labeled WGA dye (Sigma-Aldrich, L4895). For terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, sections were stained with an In Situ Cell Death Detection Kit (Roche, 12156792910) according to the manufacturer's instructions. Nuclei were counterstained with DAPI (Beyotime Biotech, P0131), and images were taken using a TCS SP8 STED 3X confocal microscope (Leica). To analyze the capillary density, sections were immunofluorescently stained for CD31 and cardiac troponin I (cTnI), and images were analyzed by ImageJ software (v1.8.0, NIH) to determine the number of capillaries surrounding each cardiomyocyte. The positively stained cells or areas were counted in 3 to 5 sections per heart, and 4-10 high-power fields per section.

ELISA
Three days and 7 days (in mice) after the myocardial I/R procedure, the heart tissue and whole blood samples from mice and swine were collected. Blood samples were centrifuged at 1,000 rcf for 10 min to obtain serum, and the heart tissue was homogenized using an electric homogenizer. The protein levels of IL-6 and IL-10 in mouse serum and heart tissue were determined using IL-6 (Biolegend, 431315) and IL-10 (Biolegend, 431425) ELISA kits. The concentrations of IL-6 and IL-10 in swine were detected by IL-6 (Abcam, ab100755) and IL-10 (Bertin Bioreagent, A05414) ELISA kits, followed by the manufacturer's protocols.

Western Blotting
Heart tissue proteins were isolated using an electric homogenizer and RIPA lysis buffer (Beyotime, P0013) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) (Beyotime, ST506). Protein extracts (20 μg) in each lane were separated by gel electrophoresis, transferred to a polyvinylidene fluoride membrane (Millipore, IPVH00010), blocked by 5% skimmed milk for 2 h, and then incubated with primary antibodies overnight at 4°C. The membranes were incubated with HRP-conjugated secondary antibodies for 2 h at room temperature and exposed through enhanced chemiluminescence.

Statistical Analysis
Data are presented as the mean ± standard error of mean (SEM). Quantitative analysis of experimental images was performed using ImageJ software. Differences between two mean values were evaluated by Student's t-test, and multiple comparisons at a single time point were determined by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. To evaluate differences between groups at multiple time points, data were analyzed using two-way ANOVA followed by Tukey's post hoc test. For survival analysis, a Kaplan-Meier survival curve was generated, and the log-rank statistics test was rendered. Statistical analyses were performed using commercially available Prism 8 software (GraphPad), and P < 0.05 was considered statistically significant.

Figure S4. Effects of miR-125a-5p agomir or MSC administration at different doses on myocardial function recovery in myocardial ischemia/reperfusion (I/R) mice. (A-C) On day 28 post-myocardial I/R, cardiac function, including (A) left ventricular (LV) ejection fractions (LVEF), (B) LV fractional shortening (LVFS)
, and (C) LV internal diameter at the end-systole (LVIDs), was determined in mice treated with 10, 20, or 40 nmol miR-125a-5p agomir via echocardiography (n = 6 mice per group). (D-F) On day 28 post-myocardial I/R, the (D) LVEF, (E) LVFS, and (F) LVIDs were also measured in mice treated with 1 × 10 5 , 3 × 10 5 , or 5 × 10 5 MSCs (n = 6 mice per group). (G) Three days after myocardial I/R, the infarct size (%) of the mouse hearts was calculated by dividing the infarct area (blue dotted) by the total LV area in TTC-stained slices from the apex to base (n = 6 mice per group). Statistical analysis was performed by one-way ANOVA followed by Tukey's post hoc test. * P < 0.05 and ** P < 0.01, NS: No significance.   Quantification of mRNA expression levels of (A) collagen genes (Col1a1,Clo1a2,Col3a1,Col4a1,Col5a1,and Col5a2) and (B) myocyte hypertrophy-related genes (Nppa, Nppb, and Myh7) on day 28 post-myocardial I/R. (C and D) On day 3 and 7 after cardiac I/R, (C) IL-6, and (D) IL-10 protein concentrations in heart tissue and serum were determined using the corresponding ELISA kits (n = 6 mice per group). Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test in (A and B) and two-way ANOVA followed by Tukey's post hoc test in (C and D). * P < 0.05 and ** P < 0.01.   A and B) and two-way ANOVA followed by Tukey's post hoc test in (C). * P < 0.05 and ** P < 0.01, NS: No significance.

Figure S10
Figure S10. miR-125a-5p regulates macrophage polarization by inhibiting ERK1/2 signaling pathway activation in the I/R myocardium of mice. (A) On day 3 post-myocardial I/R, heart tissue was collected for western blotting. Representative immunoblots (left) and quantification (right) for Akt signaling pathway and MAPK-associated signaling cascades, including ERK1/2, P38, and JNK pathways. (B) ERK1/2 activator, LY2828360 or DMSO were injected intraperitoneally into the miR-125a-5p mice, and the representative immunoblots (left) and quantification (right) for iNOS and CD206 protein levels on day 3 post-myocardial I/R are shown (n = 4 mice per group). Statistical analysis was performed by one-way ANOVA followed by Tukey's post hoc test in (A) and Student's t-test in (B). ** P < 0.01, NS: No significance.

Figure S11
Figure S11. miR-125a-5p agomir regulates fibroblast proliferation and activation under the TGF-β1 condition via acting on Tgfbr1. (A) The 167 miR-125a-5p target genes predicted from online databases and 297 cardiac fibrosis-related genes predicted from Disgenet were intersected to obtain four candidate genes. (B) The mRNA expression levels of Rit1, Nlrc5, Tgfbr1, and Sirt7 were detected by RT-qPCR in cardiac fibroblasts transfected with miR-125a-5p agomir or NC agomir. (C) The mRNA expression level of Tgfbr1 was detected in normal fibroblasts and in fibroblasts transfected with miR-125a-5p antagomir or NC antagomir (n = 5 independent experiments). (D) A WT or mutant dual-luciferase reporter plasmid was constructed according to the predicted binding sequence in 3′ UTR of Tgfbr1 (blue) or mutant sequence (red), respectively. (E) Luciferase activity was determined in cardiac fibroblasts transfected with WT or mutant reporter plasmids and 100 nM miR-125a-5p agomir or NC agomir (n = 3 independent experiments). (F) The protein expression of TGF-β receptor-1 (TGFBR1) was assessed by western blotting in normal, normal + NC agomir, normal + miR-125a-5p agomir, TGF-β1 + NC agomir, and TGF-β1 + miR-125a-5p agomir groups. (G and H) To overexpress TGFBR1 in cardiac fibroblasts, cells were co-cultured with TGFBR1-OE lentiviruses (1 × 10 8 TU/mL) for 24 h, and TGFBR1-NC lentiviruses were used as a control. Cardiac fibroblasts were then treated with 100 nM miR-125a-5p agomir and some were treated with 20 ng/mL TGF-β1 for 24 h. Representative images and quantitative analysis of (G) Ki67 and (H) α-SMA positive cardiac fibroblasts. Scale bar: 50 μm (n = 4 independent experiments). (I) Quantitative assessment and representative images of Masson's trichrome staining in the border zone of TGFBR1-NC or TGFBR1-OE lentivirus-treated I/R myocardium. Scale bar: 100 μm (n = 4 mice per group). Statistical analysis was performed by one-way ANOVA followed by Tukey's post hoc test in (B-E) and Student's t-test in (F-I). * P < 0.05 and ** P < 0.01, NS: No significance.

Figure S12
Figure S12. miR-125a-5p agomir improves injured endothelial cell (EC) function by targeting Daam1. (A) The 167 miR-125a-5p target genes and 24 angiogenesis inhibitory genes were intersected to obtain three candidate genes. (B) The mRNA expression levels of Txnrd1, Dll4, and Daam1 were assessed in ECs transfected with miR-125a-5p agomir or NC agomir. (C) The level of Daam1 was investigated in normal ECs and ECs transfected with miR-125a-5p antagomir or NC antagomir (n = 5 independent experiments). (D) A WT or mutant dual-luciferase reporter plasmid was constructed according to the predicted binding sequence in the 3′ UTR of Daam1 (blue) or mutant sequence (red), respectively. (E) Luciferase activity was detected in ECs transfected with WT or mutant reporter plasmids and 100 nM miR-125a-5p agomir or NC agomir (n = 3 independent experiments). (F) The protein expression of dishevelled associated activator of morphogenesis 1 (DAAM1) was determined via western blotting in the normal, normal + NC agomir, normal + miR-125a-5p agomir, hypoxia + NC agomir, and hypoxia + miR-125a-5p agomir groups. (G) To overexpress DAAM1 in ECs, cells were co-cultured with DAAM1-OE lentiviruses (1 × 10 8 TU/mL) for 24 h, and DAAM1-NC lentiviruses served as a control. Cells were then treated with 100 nM miR-125a-5p agomir, and some were subjected to hypoxia. Representative images and quantitative assessment of EC tube formation. Scale bar: 75 μm (n = 4 independent experiments). (H) Representative images and quantitative analysis of CD31 staining for the border zone of myocardial I/R mice on day 28. Scale bars: 30 μm (n = 4 mice per group). Statistical analysis was performed by one-way ANOVA followed by Tukey's post hoc test in (B-E) and Student's t-test in (F-H). ** P < 0.01, NS: No significance.   ) and myocyte hypertrophy-associated genes (Nppa and Nppb) in hearts of the sham, I/R control, NC agomir, and miR-125a-5p agomir swine on day 28 post-myocardial I/R (n = 4 swine per group). (B) Representative images and quantitative assessment of Ki67 staining (proliferation-related marker; frames showing higher-magnification images of the area outlined by white dashed lines) in the border zone on day 28. Scale bars: 30 μm (n = 5-6 swine per group). Statistical analysis was performed by one-way ANOVA followed by Tukey's post hoc test. * P < 0.05 and ** P < 0.01, NS: No significance.