Bone marrow mesenchymal stem cell-secreted exosomes carrying microRNA-125b protect against myocardial ischemia reperfusion injury via targeting SIRT7

Abstract

MicroRNA-125b (miR-125b) reduces myocardial infarct area and restrains myocardial ischemia reperfusion injury (I/R). In this study, we aimed to investigate the effect of bone marrow mesenchymal stem cell (BMSC)-derived exosomes carrying miR-125b on I/R rats. The myocardial I/R model in rats was constructed by ligation of the left anterior descending coronary artery (LAD). Rats were randomly divided into I/R and Sham group. Lv-cel-miR-67 (control) or Lv-miR-125b was transfected into BMSCs. Exosomes were extracted from transfected BMSCs, and separately named BMSC-Exo-67, BMSC-Exo-125b, and BMSC-Exo. MTT assay and flow cytometry were used to detect the viability and apoptosis of I/R myocardium cells, respectively. The expression of cell apoptosis proteins and the levels of inflammatory factors were examined by Western blot and ELISA assay, respectively. The target relationship between miR-125b and SIRT7 was predicted by using StarBase3.0, and was confirmed by using dual-luciferase reporter gene assay. qRT-PCR, immunohistochemistry staining, and Western blot were used to evaluate the expression of SIRT7 in myocardium tissues in I/R rats. BMSC-derived exosomes were successfully isolated and identified by TEM and positive expression of CD9 and CD63. The expression of miR-125b was down-regulated in I/R myocardium tissues and cells. BMSC-Exo-125b significantly up-regulated miR-125b in I/R myocardium cells. The intervention of BMSC-Exo-125b significantly increased the cell viability, decreased the apoptotic ratio, down-regulated Bax and caspase-3, up-regulated Bcl-2, and decreased the levels of IL-1β, IL-6, and TNF-α in I/R myocardium cells. SIRT7 was a target of miR-125b, and BMSC-Exo-125b significantly down-regulated SIRT7 in myocardium cells. In addition, the injection of BMSC-Exo-125b alleviated the pathological damages and down-regulated SIRT7 in myocardium tissues of I/R rats. BMSC-derived exosomes carrying miR-125b protected against myocardial I/R by targeting SIRT7.

Keywords: Apoptosis; Exosomes; Inflammatory factor; Ischemia reperfusion; miR-125b.

Fig. 1

Characterization of exosomes derived from bone marrow mesenchymal stem cells (BMSCs). a Cellular morphology (P1, P3) of BMSCs observed under an inverted fluorescence microscope. Scale bar: 100 μm. b Flow cytometry was used to analyze the surface antigens (CD44, CD105, CD31) in BMSCs. c, d The morphology of FBS-derived exosomes and BMSC-derived exosomes was observed under transmission electron microscopy (TEM). Scale bar: 50 μm. e Western blot was used to examine the expression of CD9, CD63 in BMSCs, and BMSC-derived exosomes

Fig. 2

Myocardial ischemia reperfusion injury (I/R) model in rats. a Hemodynamic test of I/R rats. b Hematoxylin–eosin (HE) staining was performed to detect the pathological changes of myocardium in I/R rats (× 400). c Triphenyltetrazolium chloride (TTC) assay was used to evaluate the infarct size in I/R rats

Fig. 3

Bone marrow mesenchymal stem cells (BMSCs)-derived exosomes increased the expression of miR-125b in I/R myocardium cells. a, b The relative expression of miR-125b was examined by qRT-PCR in myocardium tissues and cells. c, d The relative expression of miR-125b was detected by qRT-PCR in BMSC cells and BMSC-derived exosomes, respectively. e BMSC-Exo-125b was traced by labeling PKH26 (red). f The relative expression of miR-125b was examined by qRT-PCR in I/R myocardium cells. (Color figure online)

Fig. 4

BMSC-Exo-125b inhibited the apoptosis of ischemia reperfusion injury (I/R) myocardium cells. a The viability of myocardium cells was examined by MTT. b Apoptotic ratio (%, cell ratio in LR + UR) of myocardium cells was examined by flow cytometry. Lower left quadrant (LL), viable cells; upper left quadrant (UL), necrotic cells; lower right quadrant (LR), early apoptotic cells; upper right quadrant (UR), late apoptotic cells. c Western blot was used to detect the expression of apoptotic proteins in myocardium cells. d ELISA was performed to examine the levels of inflammatory factors

Fig. 5

MiR-125b was negatively correlated with SIRT7. a A binding site at 3′-UTR of SIRT7 was predicted on miR-125b by StarBase3.0. b The luciferase activity was measured by dual-luciferase reporter gene assay. c Relative mRNA expression of SIRT7 in ischemia reperfusion injury (I/R) myocardium tissues was examined by qRT-PCR. d Spearman’s correlation analysis was used to detect the correlation between miR-125b and SIRT7 expression. e, f qRT-PCR and Western blot were used to detect the mRNA and protein expression of SIRT7 in myocardium cells

Fig. 6

BMSC-Exo-125b restored the cardiac function of myocardial ischemia reperfusion injury (I/R) rats. a Hemodynamic test of I/R rats. b HE staining was performed to detect the pathological changes of myocardium in I/R rats (× 400). c Triphenyltetrazolium chloride (TTC) assay was used to evaluate the infarct size in I/R rats. d qRT-PCR was used to examine the relative mRNA expression of SIRT7 in myocardium tissues. e Relative density of SIRT7 was detected by immunohistochemistry in myocardium tissues (× 400). f Western blot was used to examine the relative protein expression of SIRT7 in myocardium tissues