Objectives To examine the hypothesis that microRNAs (miRs) contributes to the survival of MSCs under hypoxic conditions through regulating autophagy, thus enhancing the therapeutic effect of cells transplantation on myocardial infarction (MI).
Methods The miRNAs transcriptome was analysed by microarray and verified by real-time PCR in human MSCs cultured in hypoxic or normal condition. The gain- and loss-of-function methods through lentivirus infection were administrated to evaluate the effect of miRs on autophagy in human MSCs under hypoxia. Cells viability was measured by MTT and cells autophagy was quantified by immunoblot, immunofluoresence, and transmission electron microscopy analyses with or without respective incubation of bafilomycin A1 or rapamycin. Meanwhile, a luciferase reporter assay was performed to confirm associations between miRNAs and their putative targets. Moreover, the expression of sry gene was checked to confirm the impact of miRNAs on the survival of male MSCs in the infarcted myocardium of female rats on day four after the transplantation; meanwhile, echocardiology was applied to assess the cardiac remodelling and myocardial function in a four-week follow-up.
Results MiR-210 was up-regulated significantly in hypoxic MSCs, and forced expression of miR-210 in MSCs, compared with control group, was evidenced to increase cell viability under hypoxic conditions (3.8 ± 0.6 vs. 1.0 ± 0.7, P<0.05), and to enhance autophagy by the increase in the conversion of LC3I to LC3II (3.6 ± 0.5 vs. 2.2 ± 0.3, P<0.05), up-regulation of the expression of Beclin-1 (2.8 ± 0.6 vs. 1.2 ± 0.7, P<0.05), promotion of the formation of autophagosomes and autophagosome-fused lysosomes (4.8 ± 1.1 vs. 1.3 ± 0.5, P<0.05), reduction in the signs of broken or swollen mitochondria (2.8 ± 0.7 vs. 6.3 ± 1.4, P<0.05), as well as activation of the signalling pathway of protein kinase-1-Akt-mammalian target of rapamycin (P<0.05). Administration of bafilomycin A1 or rapamycin were respectively observed to attenuate or strengthen the above effects of miR-210 (P all<0.05). Computational prediction indentified Rubicon, generally regarded as an inhibitive factor of autophagy, was the suppressed target for miR-210. In in vivo study, the survival rate of MSCs-miR-210 in infarcted heart was discovered to remarkably higher than that of MSCs-null (6.3 ± 1.5 folds vs. 2.6 ± 0.8 folds, P<0.05). Meanwhile, transplantation of MSCs-miR-210 induced statistical improvement of cardiac function [LVEF: (50.2 ± 3.9)% vs. (42.9 ± 2.7)%, P<0.05] and obvious reduction of left ventricular end-diastolic volume (4.6 ± 0.7 vs.6. 4 ± 0.5, P<0.05) compared with that of MSCs-null.
Conclusions MiR-210 promotes autophagy through decreasing the expression of Rubincon, and enhances the viability of MSCs under hypoxic conditions both in culture and in a model of MI rats, suggesting that miR-210 might be developed as a novel target to improve the cells survival after transplantation in infarcted myocardium.