Downregulated ALKBH5 contributes to myocardial ischemia/reperfusion injury by increasing mA modification of Trio mRNA.

BACKGROUND

The modification of N-methyladenosine (mA) is a dynamic and reversible course that might play a role in cardiovascular disease. However, the mechanisms of mA modification in myocardial ischemia/reperfusion injury (MIRI) remain unclear.

METHODS

A mouse model of MIRI and a cell model of oxygen-glucose deprivation/reperfusion (OGD/R) HL-1 cells were employed. In an study, the total RNA mA modification levels were determined by dot blot, and the key genes related to mA modification were screened by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot. In an 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 mA modification-changed and differentially expressed messenger RNA (mRNA) were determined by methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) in -overexpressed HL-1 cells. Finally, the mRNA levels of the promising targeted gene were examined by RT-qPCR and its mA modification levels were examined by MeRIP-qPCR.

RESULTS

Our results showed that RNA mA modification was involved in MIRI, in which 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 mA 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 mA level of Trio.

CONCLUSIONS

This study demonstrated that the downregulated might contribute to MIRI process by increasing the mA modification of Trio mRNA and downregulating Trio.