Exosomal Transfer Of MiR-30a Between Cardiomycytes Regulates Autophagy After Hypoxia

Part I Expression of Serum Exosomal MicroRNA-30a in Patients with AMIObjective:Recent studies have indicated a protective role of circulating microRNA transferred by exosomes in ischemic heart disease. However, whether the miR-30a which correlated with beclin-1(an identified indicators of autophagy) transferred by exosomes and the underlying mechanisms of miR-30a regulation in patients with AMI are poorly understood. In this study, we observed elevated miR-30a was highly enriched in exosomes from serum of acute myocardial infarction (AMI) patients.Method:145 patients with chest pain as the main symptom were enrolled in this study. The blood samples of patients with AMI were obtained at 4 h (±30 min),24 h (±30 min),72 h (±30 min) and 1 w (±60 min) after the onset of chest pain. The blood samples of patients in control and UA group were collected after admission in a fasting state on the following morning of the admission day. Real-time PCR was used to detect the expression level of miR-30a in serum and exosomes isolated form serum. The expression level of exosomes was measured through Western blot and flow cytometry analysis.Result:MiR-30a elevated in serum of AMI patients in a time-dependent manner. By FACS analysis, the fluorescence expression of CD63 (a commonly used marker of exosomes) isolated from patients with UA and AMI (24h) showed respectively 2.64 (±0.27) and 2.94 (±0.53) fold compared to control group (P<0.01). By western blotting, CD63 expression in exosomes isolated from serum of AMI patients elevated in a time-dependent manner, peaked at 4h and reduced distinctly at 72h. MiR-30a increased significantly at 4h (5.86±1.43 fold vs. control group, P<0.01) and 24h (4.17±1.24 fold vs. control group, P<0.01) after AMI onset in exosomes isolated from patients'serum. MiR-30a is also found to be up-regulated in exosomes from UA patients.Conclusion:MiR-30a is increased obviously and highly enriched in exosomes from serum of AMI patients.Part II Exosomal transfer of miR-30a between cardiomycytes regulates autophagy after hypoxiaObjective:Recent studies have indicated a protective role of physiological autophagy in ischemic heart disease. However, the underlying mechanisms of autophagy regulation after ischemia are poorly understood. Exosomes are nano-sized vesicles released from cells which have been recognized to play critical roles in mediating cell-to-cell communication through the transfer of microRNA. In this study, we observed elevated miR-30a was highly enriched in exosomes from culture medium of cardiomyocytes after hypoxic stimulation in vitro.Method:The cardiomyoblasts cell line H9c2 were cultured in DMEM supplemented with 10% FBS. To mimic the ischemic injury in vitro, cells were exposed to low-glucose DMEM and then placed in a hypoxic incubator containing 3% O2 for different times. Real-time PCR was used to detect the levels of miR-30a in hypoxic myocardial cells and exosomes isolated form conditioned medium. Real-time PCR was also used to detect the levels of miR-30a in hypoxic myocardial cells cardiac cells which transfected with HIF-la siRNA or co-cultured with 2ME (an inhibitor of HIF-la). The level of exosomes secreted from hypoxic myocardial cell was measured by Western blot and flow cytometry analysis. The exosomes secreted form hypoxic myocardial cells were co-cultured with normal cultured myocardial cells. Then the expression level of miR-30a in the co-cultured cells was detected by Real-time PCR. Exosomes isolated from culture medium of H9c2 cells after hypoxia were labeled by a PKH67 dye, the transfer of exosomes from cell to cell was observed by confocal microscopy. The H9C2 cells transfected by pCT-CD63-GFP were seeded into to the upper chambers of Transwell inserts in 24-well tissue culture plates, the non-transfected cardiomyocytes seeded into the lower well, the transfer of exosomes was observed by confocal microscopy. Confocal microscopy was used to observe the autophagy levels of the hypoxic cells which were transfected with pSELECT-GFP-mLC3 and miR-30a inhibitor or co-cultured with DMA (an exosomal pathway inhibitor). The autophagy levels of the hypoxic cells which were transfected with miR-30a inhibitor or co-cultured with DMA were measured by real-time PCR and western blot. Phase images and TUNEL signals of H9c2 cells were observed through fluorescent microscopy.Result:Hypoxia induced miR-30a elevation in cardiomyocytes, inhibition of hypoxia inducible factor (HIF)-1a inhibited miR-30a up-regulation in response to hypoxia. MiR-30a was transferred between cardiomyocytes via exosomes after hypoxia. Hypoxia induced autophagy of cardiomyocytes, and inhibition of miR-30a or exosome releasing contributed to maintaining of autophagic response in cardiomyocytes after hypoxia. Inhibition of miR-30a or exosome releasing attenuated apoptosis of cardiomyocytes induced by hypoxia.Conclusion:The present study shows that exosomes from hypoxic cardiomyocytes regulate autophagy through transferring miR-30a in a paracrine manner which reveals a new pathway of autophagy regulation and may comprise a promising strategy to ischemic heart disease.