Epigenetic regulation mechanism of DNA methylation and miRNAs on the expression of the ALOX5AP gene in patients with ischemic stroke

Xiaoshuai Bie; Huiling Zhao; Zhaojing Zhang; Xiaoou Wang; Yingying Luan; Yuanli Wang; Shangdong Yang; Liyan Xu; Xuran Zhang; Baixue Zhou; Hui Dong; Yan Xu; Dongzhi Yang; Hong Zheng; Ying He

doi:10.3892/etm.2021.10919

Epigenetic regulation mechanism of DNA methylation and miRNAs on the expression of the ALOX5AP gene in patients with ischemic stroke

Exp Ther Med. 2021 Dec;22(6):1484. doi: 10.3892/etm.2021.10919. Epub 2021 Oct 26.

Authors

Xiaoshuai Bie¹, Huiling Zhao², Zhaojing Zhang¹, Xiaoou Wang¹, Yingying Luan¹, Yuanli Wang¹, Shangdong Yang¹, Liyan Xu³, Xuran Zhang⁴, Baixue Zhou¹, Hui Dong¹, Yan Xu¹, Dongzhi Yang⁵, Hong Zheng¹, Ying He¹

Affiliations

¹ Department of Medical Genetics and Cell Biology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Henan 450052, P.R. China.
² Department of Reproductive Genetics of Anyang Maternal and Child Health Care Hospital, Anyang, Henan 455000, P.R. China.
³ Henan Eye Institute, Henan Eye Hospital, Henan Provincial People's Hospital, Zhengzhou, Henan 450003, P.R. China.
⁴ Medical Laboratory of The First Affiliated Hospital of Henan University of CM, Zhengzhou, Henan 450000, P.R. China.
⁵ School of Life Sciences of Zhengzhou University, Zhengzhou, Henan 450052, P.R. China.

Abstract

5-lipoxygenase-activating protein (FLAP), encoded by the arachidonate 5-lipoxygenase-activating protein (ALOX5AP) gene, can adjust the biogenesis of proinflammatory leukotrienes to increase the adhesion and permeability of the vascular internal wall. Moreover, it participates in the process of atherosclerosis and is closely associated with ischemic stroke (IS). Accumulating evidence has shown that the expression levels of the ALOX5AP gene are upregulated in patients with IS. However, the mechanism of ALOX5AP action in IS remain elusive. The present study hypothesized that epigenetic regulation, including DNA methylation and microRNA (miR/miRNA) regulation, affects the expression levels of the ALOX5AP gene. Therefore, 200 patients with a first diagnosis of acute IS and 200 healthy control subjects were enrolled in the present study. Initially, the mRNA expression levels of the ALOX5AP gene were examined by reverse transcription-quantitative PCR. It was found that the mRNA levels of ALOX5AP gene in the IS group were significantly higher compared with controls (P<0.05). Subsequently, the methylation status of 17 CpG sites located in the promoter region of ALOX5AP was assessed by MethyTarget sequencing. However, the levels of methylation exhibited no significant differences between IS and control groups (P>0.05). Moreover, the expression levels of miR-335 and miR-495 were examined as two potential miRNAs targeting the ALOX5AP gene. The expression levels of miR-335 and miR-495 in the IS group were significantly lower compared with the control group (P<0.05). Finally, the luciferase assay results indicated that the luciferase activity of the experimental group following co-transfection of miRNA mimic and wild-type reporter gene plasmid was significantly lower compared with the other experimental groups (P<0.05), suggesting that miR-335 and miR-495 could specifically bind to the 3'-untranslated region of the ALOX5AP gene, thereby downregulating its expression. The present study provided preliminary evidence demonstrating that epigenetic regulation affects the expression of the ALOX5AP gene in patients with IS.

Keywords: DNA methylation; arachidonate 5-lipoxygenase-activating protein; epigenetics; ischemic stroke; microRNAs.

Grants and funding

Funding: The research reported in this publication was supported by a grant from the National Science Foundation of China-Henan United fund (grant no.U2004114).