In the present study, we examined the expression and role of endogenous microRNA-146a in MIRI, and we explored the possible target genes and pathways. Our results indicated that microRNA-146a was upregulated at an early stage of MIR, and endogenic microRNA-146a deficiency increased MIRI, demonstrating a protective role in this process. The role of endogenic microRNA-146a was consistent with that of exogenous microRNA-146a mimics previously reported, which also proved that the increase of microRNA-146a in the first hour after reperfusion was a compensatory protection initiated by the body itself. However, this compensatory protective effect slowly disappeared after that. Therefore, upregulation of microRNA-146a occurs as early as the reperfusion occurs and as long as the injury duration would be beneficial for ischaemic myocardium. Thus, interventions that can increase microRNA-146a, such as drugs, surgery or direct exogenous addition of microRNA-146a mimics at a suitable time, can be used to protect ischaemic myocardium after reperfusion.
MicroRNA is a class of non-coding single-stranded RNA molecules encoded by endogenous genes of approximately 22 nucleotides [3, 12, 13]. They are capable of completely or incompletely binding to the target genes and then degrading them or modulating their functions [3, 12, 13]. It has been demonstrated that microRNA-146a can target many genes, such as IRAK1, IRAK2, TRAF6, RIG-I, IRF-5, STAT-1, PTC1, Numb, and WASF2, to play a variety of roles in human diseases, including cancers and inflammatory immune diseases [14,15,16,17,18]. IRAK1 and TRAF6 are the downstream molecules of the TLR-induced NF-κB signalling pathway, and they have often been identified as the target genes of microRNA-146a in inflammatory immune diseases containing MIRI [11, 19,20,21]. However, a few other target genes of microRNA-146a in MIRI are known. In this study, we found that mediator complex subunit 1 (Med1) was one target gene of microRNA-146a, and TRAP220 protein encoded by Med1 was upregulated when microRNA-146a was deficient.
The multi-subunit mediator is an evolutionarily conserved transcription co-regulatory nuclear complex in eukaryotes. It is needed for the transcription regulation of gene expression in general, as well as in a gene specific manner. Mediator complex subunits interact with different transcription factors and as components of the RNA Pol II transcription initiation complex; in doing so, they act as a bridge between gene specific transcription factors and general Pol II transcription machinery . Med1 is also known as TRAP220 in mice and RB18A in humans. Researchers have reported that cardiac Med1 was necessary for survival in mice because it regulated cardiac metabolic and mitochondrial genes. Genetic deletion of Med1 resulted in embryonic lethality, largely due to impaired cardiac development [23, 24]. Other studies have also shown that cardiomyocyte-specific ablation of the Med1 subunit of the mediator complex might cause lethal dilated cardiomyopathy . However, no data detailing whether Med1 can influence MIRI are available. In our study, we found that the upregulation of TRAP220 in microRNA-146a deficient mice that experienced myocardial ischaemia reperfusion was accompanied by an amplified ratio of Bax/Bcl2 and increased cleaved caspase-3. The rescue study verified that Med1 was indeed one target gene of microRNA-146a in MIRI.
Apoptosis is a sophisticated process that is the main cause of MIRI . There are two main pathways involved in apoptosis: the extrinsic (death receptor) pathway and the intrinsic (mitochondrial) pathway [27, 28]. However, the last stage of both pathways is initiated by the cleavage of caspase-3 and results in DNA fragmentation, degradation of cytoskeletal and nuclear proteins, cross-linking of protein, formation of apoptotic bodies, expression of ligands for phagocytic cell receptors and finally, uptake by phagocytic cells. In the intrinsic pathway, stimuli, including radiation, toxins, viral infections, hyperthermia, hypoxia and free radicals, are able to cause changes in the inner mitochondrial membrane, which may result in an opening of the mitochondrial permeability transition pore and loss of the mitochondrial transmembrane potential. Then, two main groups of normally sequestered pro-apoptotic proteins are released from the intermembrane space into the cytosol, consisting of cytochrome c, Smac/DIABLO, and the serine protease HtrA2/Omi, which will activate the caspase-dependent mitochondrial pathway. Cytochrome c initiates activation of a series of kinases, including caspase-3, which is the ultimate executor of apoptosis [27, 28]. These apoptotic mitochondrial events can be regulated by the Bcl-2 family proteins, such as anti-apoptotic proteins Bcl-2 and pro-apoptotic proteins Bax, which control the release of cytochrome c from the mitochondria via alteration of mitochondrial membrane permeability [27, 28]. p53, a tumour suppressor protein, has a critical role in regulation of the Bcl-2 family [27,28,29,30,31]. The p53 tumour suppressor gene is a transcription factor that regulates the cell cycle. It can activate DNA repair proteins when DNA has sustained damage, hold the cell cycle at the G1/S regulation point on DNA damage recognition, and initiate apoptosis if the DNA damage proves to be irreparable. p53 is one regulator of Bcl2 and Bax [27,28,29,30,31]. Researchers have revealed that RB18A, a member of the Med1 family, self-oligomerized and interacted with the p53 protein in vitro when K562 (an erythroleukaemia cell line) and H1299 (pulmonary embryo carcinoma) cells, two human p53-null cell lines, were used to transfect with p53wt cDNA in the presence or absence of RB18A cDNA [29, 30]. In addition, with immunoprecipitation, they demonstrated that in vivo, RB18A directly interacted (through its C-terminal domain) with wild type p53 (p53 wt), acting as a cofactor of transcription by directly regulating p53-transactivating activity on the promoters of Bax, p21Waf1 and IGF-BP3 genes [29, 30], while RB18A alone did not affect cell apoptosis. In this process, RB18A activated Bax promoter and inhibited p21Waf1 and IGF-BP3 promoters [29, 30, 32]. TRAP220, a 220 KDa thyroid hormone receptor-associated protein, which shared 99% sequence identity within the RB18A coding sequence (with only minor sequence variations), was also demonstrated to interact with p53 wt as RB18A [29, 30, 33]. In our study, we inferred that the increased TRAP220 protein in the KO mice that experienced myocardial ischaemia reperfusion might interact with p53 protein, activate the promoter of Bax, and then lead to the cleavage of caspase 3, which mediated apoptosis.
Our study is the first to clarify the expression and role of endogenous microRNA-146a after MIRI. We also found one new target gene of microRNA-146a, Med1, which might mediate apoptosis through regulating the p53 related signal pathway in MIRI. However, more studies are still needed to verify the apoptosis signal pathway in this process.