Abstract
Atherosclerosis (AS) is a life-threatening vascular disease. RNA N6-methyladenosine (m6A) modification level is dysregulated in multiple pathophysiologic processes including AS. In this text, the roles and molecular mechanisms of m6A writer METTL3 in AS progression were explored in vitro and in vivo. In the present study, cell proliferative, migratory, and tube formation capacities were assessed through CCK-8, Transwell migration, and tube formation assays, respectively. RNA m6A level was examined through a commercial kit. RNA and protein levels of genes were measured through RT-qPCR and western blot assays, respectively. VEGF secretion level was tested through ELISA assay. JAK2 mRNA stability was detected through actinomycin D assay. The relationship of METTL3, IGF2BP1, and JAK2 was investigated through bioinformatics analysis, MeRIP, RIP, RNA pull-down, and luciferase reporter assays. An AS mouse model was established to examine the effect of METTL3 knockdown on AS development in vivo. The angiogenetic activity was examined through chick chorioallantoic membrane assay in vivo. The results showed that METTL3 was highly expressed in ox-LDL-induced dysregulated HUVECs. METTL3 knockdown inhibited cell proliferation, migration, tube formation, and VEGF expression/secretion in ox-LDL-treated HUVECs, hampered AS process in vivo, and prevented in vivo angiogenesis of developing embryos. METTL3 positively regulated JAK2 expression and JAK2/STAT3 pathway in an m6A dependent manner in HUVECs. IGF2BP1 positively regulated JAK2 expression through directly binding to an m6A site within JAK2 mRNA in HUVECs. METTL3 knockdown weakened the interaction of JAK2 and IGF2BP1. METTL3 exerted its functions through JAK2/STAT3 pathway. In conclusion, METTL3 knockdown prevented AS progression by inhibiting JAK2/STAT3 pathway via IGF2BP1.
Highlights
Atherosclerosis (AS), characterized by the deposition of lipids and other materials in and on the artery walls (AS plaques), is closely associated with the increased risks of multiple life-threatening cardiovascular diseases such as stroke, heart attack, and myocardial infarction (Khatana et al, 2020; Munjal and Khandia, 2020)
The outcomes showed that METTL3, METTL14, and FTO expression levels were noticeably upregulated in HUVECs exposed to Oxidized low-density lipoprotein (ox-LDL) compared to the control group (Figure 1E)
Consistent with our outcomes, a recent study demonstrated that METTL3 functioned as a crucial player in the response to hemodynamic forces and atherogenic stimuli in endothelial cells and METTL3 knockdown prevented the development of AS in the in vivo AS model induced by partial carotid-artery ligation (Chien, et al, 2021)
Summary
Atherosclerosis (AS), characterized by the deposition of lipids and other materials in and on the artery walls (AS plaques), is closely associated with the increased risks of multiple life-threatening cardiovascular diseases such as stroke, heart attack, and myocardial infarction (Khatana et al, 2020; Munjal and Khandia, 2020). Ox-LDL can regulate AS development through multiple mechanisms, including inducing the activation and dysfunction of endothelial cells (Pirillo et al, 2013; Di Pietro et al, 2016; Khatana et al, 2020). A deep insight into the molecular mechanisms underlying ox-LDL-induced endothelial dysfunction might contribute to a better understanding of the pathogenesis of AS. METTL3, an m6A writer, is involved in the regulation of multiple biological processes (e.g., proliferation, migration, and apoptosis) and disease (e.g., cancer, ischemic heart disease, and diabetes) progression (Liu et al, 2020a; Zheng et al, 2019). METTL3 has been reported to be related to the dysfunction of vascular endothelium (Wang et al, 2020a; Yao et al, 2020). The roles and molecular basis of METTL3 in the development of AS are poorly defined
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