Abstract:AIM: To investigate the role and mechanism of methyltransferase-like 3(METTL3)-mediated N6-methyladenosine(m6A)methylation modification in regulating biological activity of vascular endothelial cells in the pathogenesis of choroidal neovascularization.
METHODS: Human umbilical vein endothelial cells(HUVEC)cultured in vitro were divided into the following groups: control group(normal culture), low density lipoprotein(LDL)group, fluorescence-labelled LDL(Dil-LDL)group, 12.5μg/mL and 25μg/mL oxidized LDL(ox-LDL)groups, 12.5μg/mL and 25μg/mL fluorescence-labelled ox-LDL(Dil-ox-LDL)groups, DMSO group, STM2457(METTL3 inhibitor)group, DAPT group; and monkey retina-choroidal endothelial cells(RF/6A)cultured in vitro were divided into control group, DMSO group, 12.5 μg/mL ox-LDL group, and DAPT group. Endocytosed lipoprotein level was examined through fluorescence microscopy. RNA m6A methylation level was detected through a dot blot assay. Protein and RNA levels of METTL3 or angiogenesis-related markers were measured through Western blot assays and real-time quantitative polymerase chain reaction(RT-qPCR), respectively. METTL3 expression and localization were investigated through immunofluorescence. Cell migratory and tube formation capacities were assessed through transwell migration and tube formation assays, respectively.
RESULTS: Endocytosed lipoprotein levels in HUVECs exposed to Dil-LDL, 12.5μg/mL and 25μg/mL Dil-ox-LDL groups were significantly higher than those in the control group. 12.5μg/mL and 25μg/mL ox-LDL groups significantly increased m6A methylation(all P<0.05), METTL3 protein expression(all P<0.01), and cell migration and angiogenesis capacities(all P<0.01). METTL3 mRNA level was significantly unregulated in the 12.5μg/mL ox-LDL group(P<0.05). In comparison to the DMSO group, the addition of STM2457 caused significant decrease in m6A methylation level(P<0.05), expression of VEGF and other angiogenesis-related markers(all P<0.05), cell migration and angiogenesis capacities(all P<0.01)and the expression of NICD(P<0.05). However, there were no significant differences in METTL3 protein and mRNA levels(all P>0.05). The expression of VEGF and NICD(all P<0.05), as well as the ability of cell migration and angiogenesis of RF/6A, was all significantly decreased in the DAPT group compared to the DMSO group(all P<0.01).
CONCLUSION: METTL3-mediated m6A methylation modification promotes angiogenesis in vascular endothelial cells via the Notch signaling pathway in the pathogenesis of choroidal neovascularization.
