Role Of METTL3-dependent N6-methyladenosine MRNA Modification In The Promotion Of Angiogenesis

Objective:To investigate the regulatory effects and mechanisms of METTL3-dependent N6-methyladenosine m RNA modification on hypoxia-induced retinal angiogenesis.Methods:1.A hypoxia cell model of human umbilical vein endothelial cells(HUVECs)was established using 1%O₂ in vitro.The experiment included three groups for hypoxic culture:control group(Ctrl),24 h hypoxic culture group and 48 h hypoxic culture group.A mouse model of oxygen-induced retinopathy(OIR)was established using 75%O₂ in vivo.The experiment included two groups:control group(Ctrl)and hypoxic group.Colorimetric quantification and dot blot assay were used to detect the abundance of m⁶A modification at the cellular and animal levels.q RT-PCR and western blot were used to detect the expression changes of the key regulatory enzymes of m⁶A modification at the cellular and animal levels.HUVECs were transfected with scrambled(Scr)si RNA,HIF-1?si RNA(Si1?),HIF-2?si RNA(Si2?),and HIF-1?+HIF-2?si RNA(double knockdown,DKD),q RT-PCR and western blot were performed to detect METTL3 expression under normoxic and hypoxic conditions.2.At the cellular level,HUVECs were transfected with scrambled(Scr)si RNA,METTL3(M3)si RNA?,pc DNA 3.1 Vector,and pc DNA 3.1-METTL3.MTT assay,Ki67 staining,Transwell assay and tube formation assay were used to detect HUVECs viability,proliferation,migration and tube formation abilities.At the animal level,endothelial-specific METTL3 knockout mice(METTL3^flox/flox;Cdh5-Cre)and control mice(METTL3^+/+;Cdh5-Cre)were used to construct corneal alkali burn model and OIR model.The number of corneal or retinal neovascularization was detected by Isolectin B4(IB4)staining assay.3.METTL3 was overexpressed in HUVECs,m⁶A RNA immunoprecipitation followed by high-throughput sequencing(Me RIP-seq)technology,bioinformatics,m⁶A-q PCR and western blot were used to explore the potential target gene of METTL3.MTT assay,Ki67 staining,Transwell assay and tube formation assay were used to detect HUVECs activity,proliferation,migration and tube formation abilities.4.m⁶A readers(YTHDF1,YTHDF2,YTHDC2 or YTHDC1)were overexpressed respectively in HUVECs,western blot and RIP-q PCR assay were used to detect the expression levels of target genes.The target reader was overexpressed in METTL3-knockdown HUVECs,western blot assay was used to verify the target genes.Ki67staining,Transwell assay and tube formation assay were used to verify the m⁶A target reader at the cellular level.The translation initiation factor was inhibited in m⁶A target reader-overexpression HUVECs,Western blot was used to verify the regulatory effect of m⁶A target reader on the target genes.Results:1.Colorimetric quantification and dot blot assays showed that the level of m⁶A modification was significantly up-regulated in HUVECs and mouse retinas following hypoxic stress,which was caused by increased METTL3 levels.q RT-PCR and western blot assays revealed that METTL3 levels were significantly up-regulated in HUVECs and mouse retinas response to hypoxia both at m RNA and protein levels compared with their respective controls.Moreover,in contrast to the Scr group,hypoxia-induced METTL3 m RNA and protein expressions were abrogated in HIF-si1?,HIF-si2?,or HIF-DKD group.2.METTL3 silencing induced a substantial decrease of m⁶A levels,while METTL3overexpression induced a marked increase of m⁶A levels both under normoxic and hypoxic conditions.MTT(3-[4,5-Dimethythiazol-2-yl]-2,5-diphenyl tetrazolium bromide)assays showed that METTL3 silencing led to a marked reduction of HUVEC viability,Ki67 staining assays showed that METTL3 silencing decreased HUVEC proliferation,Transwell and Matrigel tube formation assays showed that METTL3silencing attenuated the migration and tube formation ability of HUVECs.METTL3overexpression increased the viability,proliferation,migration,and tube formation of HUVECs.Moreover,in the OIR mice model,the pathological neovascularization achieved the maximum amount at P17.The Mettl3-ec KO mice had smaller avascular areas and pathological neovascular tufts compare with the control(Mettl3^+/+Cdh5-Cre)mice.In the alkali burn injury mice model,the area of corneal neovascularization in Mettl3-ec KO mice was smaller than that in the control mice at all detected time points.3.Me RIP followed by sequencing revealed that 574 genes were differentially m⁶A methylated between METTL3 overexpression and control group,including 429 m⁶A hypermethylated genes and 145 m⁶A hypomethylated genes.GO analysis showed that regulation of transcription from RNA polymerase II promoter was the most important molecular function of the up-regulated m⁶A methylated transcripts induced by METTL3 overexpression.Pathway analysis showed that 8 signaling pathways were associated with the up-regulated m⁶A methylated transcripts induced by METTL3overexpression.Notably,Wnt signaling pathway was ranked the Top 1 signaling pathway that was potentially affected by METTL3 overexpression.Me RIP-seq maps of individual transcripts showed the hypermethylated sites in METTL3 overexpression group in comparison with the control group for some members of Wnt signaling pathway such as DVL1,LRP6,and CRAM1.The m⁶A abundance of LRP6 and DVL1was markedly decreased upon METTL3 knockdown as shown by gene specific m⁶A-q PCRs.METTL3 knockdown decreased the levels of LRP6 and DVL1.4.Overexpression of YTHDF1 but not YTHDF2,YTHDC2,or YTHDC1 led to increased levels of LRP6 and DVL1 in HUVECs.RIP-q PCR analysis revealed that LRP6 and DVL1 were the target genes of YTHDF1.Overexpression of YTHDF1recovered the decreased levels of LRP6 and DVL1 in METTL3-knockdown HUVECs.Overexpression of YTHDF1 could partially rescue the reduction of endothelial angiogenic effects induced by METTL3 knockdown.Rapamycin treatment markedly inhibited the increase of LRP6 and DVL1 levels in YTHDF1-overexpressed cells,indicating YTHDF1 mediates the translation of LRP6 and DVL1 in a cap-dependent manner.Conclusion:1.The level of m⁶A modification was significantly up-regulated in endothelial cells and mouse retinas following hypoxic stress,which was caused by increased METTL3levels.2.METTL3 silencing or METTL3 overexpression altered endothelial cell viability,proliferation,migration,and tube formation in vitro.METTL3 knockout in vivo decreased avascular area and pathological neovascular tufts in oxygen-induced retinopathy model and inhibited alkali burn-induced corneal neovascularization.3.Mechanistically,METTL3 exerted its angiogenic role by regulating Wnt signaling through the m⁶A modification of target genes(e.g.,LRP6 and DVL1).4.METTL3 enhanced the translation of LRP6 and DVL1 in a YTHDF1-dependent manner.Collectively,this study suggests that METTL3-mediated m⁶A modification is an important hypoxic stress response mechanism.Targeting m⁶A through its writer enzyme METTL3 is a promising strategy for the treatment of angiogenic diseases.