Li Jiaxin, Chen Jieshan, Zhao Mingyi, Li Zhetao, Liu Nanbo, Fang Heng, Fang Miaoxian, Zhu Ping, Lei Liming, Chen Chunbo
The Second School of Clinical Medicine, Southern Medical University, Guangzhou, China.
Department of Intensive Care Unit of Cardiac Surgery, Guangdong Cardiovascular Institute, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou, China.
Ann Transl Med. 2022 Apr;10(7):417. doi: 10.21037/atm-22-1289.
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.
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.
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.
This study demonstrated that the downregulated might contribute to MIRI process by increasing the mA modification of Trio mRNA and downregulating Trio.
N - 甲基腺苷（mA）修饰是一个动态且可逆的过程，可能在心血管疾病中发挥作用。然而，mA修饰在心肌缺血/再灌注损伤（MIRI）中的机制仍不清楚。
采用MIRI小鼠模型和氧糖剥夺/再灌注（OGD/R）HL - 1细胞模型。在一项研究中，通过斑点印迹法测定总RNA的mA修饰水平，并通过实时定量聚合酶链反应（RT - qPCR）和蛋白质免疫印迹法筛选与mA修饰相关的关键基因。在另一项研究中，通过3 -（4,5 - 二甲基噻唑 - 2 - 基）- 5 -（3 - 羧甲氧基苯基）- 2 -（4 - 磺基苯基）- 2H - 四氮唑（MTS）法、乳酸脱氢酶（LDH）和心肌肌钙蛋白I（cTnI）水平以及流式细胞术检测RNA去甲基化酶AlkB同源物5（ALKBH5）对细胞增殖、细胞损伤和凋亡的影响。此外，通过甲基化RNA免疫沉淀测序（MeRIP - seq）和RNA测序（RNA - seq）在过表达ALKBH5的HL - 1细胞中测定mA修饰改变和差异表达的信使RNA（mRNA）。最后，通过RT - qPCR检测有前景的靶向基因的mRNA水平，并通过MeRIP - qPCR检测其mA修饰水平。
我们的结果表明RNA的mA修饰参与了MIRI，其中（此处原文缺失相关内容）被下调。在功能上，通过在实验细胞中过表达或沉默ALKBH5，我们验证了其在MIRI过程中对细胞增殖、细胞损伤和凋亡的保护作用。此外，我们在过表达ALKBH5的HL - 1细胞中提供了大量具有mA修饰变化的潜在差异mRNA。在机制上，我们进一步筛选出最具潜力的靶向mRNA，并表明三重功能域（Trio）mRNA可通过降低Trio的mA水平被ALKBH5上调。
本研究表明，（此处原文缺失相关内容）下调可能通过增加Trio mRNA的mA修饰并下调Trio来促进MIRI进程。
需注意，原文中存在部分缺失信息，已在译文中以括号标注。
