Background
The modification of N6-methyladenosine (m6A) is a dynamic and reversible course that might play a role in cardiovascular disease. However, the mechanisms of m6A modification in myocardial ischemia/reperfusion injury (MIRI) remain unclear.
Conclusions
This study demonstrated that the downregulated ALKBH5 might contribute to MIRI process by increasing the m6A modification of Trio mRNA and downregulating Trio.
Methods
A mouse model of MIRI and a cell model of oxygen-glucose deprivation/reperfusion (OGD/R) HL-1 cells were employed. In an in vivo study, the total RNA m6A modification levels were determined by dot blot, and the key genes related to m6A modification were screened by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot. In an in vitro study, the effects of AlkB homolog 5 (ALKBH5), an RNA demethylase, on cell proliferation, cell injury, and apoptosis were detected by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, lactate dehydrogenase (LDH) and cardiac troponin-I (cTnI) levels, and flow cytometry. Besides, the m6A modification-changed and differentially expressed messenger RNA (mRNA) were determined by methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) in ALKBH5-overexpressed HL-1 cells. Finally, the mRNA levels of the promising targeted gene were examined by RT-qPCR and its m6A modification levels were examined by MeRIP-qPCR.
Results
Our results showed that RNA m6A modification was involved in MIRI, in which ALKBH5 was downregulated. Functionally, by overexpressing or silencing ALKBH5 in experimental cells, we verified its protective properties on cell proliferation, cell injury, and apoptosis in the process of MIRI. Besides, we provided a mass of latent different mRNAs with m6A modification variation in ALKBH5-overexpressed HL-1 cells. Mechanistically, we further screened the most potential targeted mRNAs and suggested that triple functional domain (Trio) mRNA could be upregulated by ALKBH5 by reducing m6A level of Trio. Conclusions: This study demonstrated that the downregulated ALKBH5 might contribute to MIRI process by increasing the m6A modification of Trio mRNA and downregulating Trio.