下调METTL3-LYN m^(6)A-IGF2BP2通路抑制内皮细胞迁移  

Down-regulation of METTL3-LYN m^(6)A-IGF2BP2 pathway inhibits endothelial cell migration

作　　者：白秦 陈燕华 卢尧[1] 吴煌 文爱清[1] BAI Qin;CHEN Yanhua;LU Yao;WU Huang;WEN Aiqing(Department of Blood Transfusion,Daping Hospital,Army Medical University(Third Military Medical University),Chongqing,400042,China)

机构地区：[1]陆军军医大学(第三军医大学)大坪医院输血科,重庆400042

出　　处：《第三军医大学学报》2021年第9期845-851,共7页Journal of Third Military Medical University

摘　　要：目的研究甲基转移酶样蛋白3(methyltransferase like 3,METTL3)对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)迁移能力的影响,并初步探究其作用机制。方法通过在线设计靶向METTL3第1个外显子的sgRNA,构建到lentiCRISPR v2载体;Western blot和免疫荧光染色实验检测METTL3蛋白表达水平;细胞划痕实验和Transwell实验检测细胞迁移能力;m^(6)A甲基化RNA免疫沉淀实验检测细胞迁移相关基因酪氨酸激酶LYN mRNA上m^(6)A甲基化修饰情况;荧光定量PCR(qPCR)检测LYN的表达情况;利用shRNA敲低m^(6)A识别蛋白IGF2BP1和IGF2BP2,siRNA敲低m^(6)A识别蛋白YTHDF2,qPCR检测识别蛋白敲低水平及LYN的mRNA表达情况;RNA免疫沉淀实验进一步检测IGF2BP2与LYN mRNA的结合情况;放线菌素D抑制转录后,qPCR检测LYN mRNA半衰期。结果利用CRISPR-Cas9技术成功构建METLL3基因敲低的HUVECs细胞系。METTL3基因敲低后,内皮细胞的迁移能力下降(P<0.05),细胞迁移相关基因LYN mRNA上m^(6)A甲基化修饰水平显著降低(P<0.01),且LYN mRNA的表达明显下调(P<0.05)。敲低m^(6)A识别蛋白YTHDF2和IGF2BP1,对LYN mRNA的表达量无显著影响(P>0.05),而敲低IGF2BP2,LYN的mRNA表达量显著下调(P<0.01),RNA免疫沉淀实验结果显示IGF2BP2与LYN mRNA结合。mRNA半衰期实验结果表明敲低IGF2BP2降低LYN mRNA稳定性。结论敲低METTL3抑制内皮细胞迁移,其机制可能通过IGF2BP2识别并结合LYN mRNA上m^(6)A修饰位点,调控其mRNA稳定性来发挥作用。Objective To investigate the effect of methyltransferase like 3(METTL3)on the migration of human umbilical vein endothelial cells(HUVECs),and explore the underlying mechanism preliminarily.Methods The sgRNA targeting the first exon of METTL3 was designed with aid of online platform and synthesized,and then inserted into the lentiCRISPR v2 vector.Western blotting and immunofluorescence staining were used to determine the protein expression of METTL3.Cell migration were detected by cell scratch test and transwell chamber assay.Methylated RNA immunoprecipitation was employed to detect m^(6)A modification of cell migration related gene,tyrosine kinase,LYN mRNA.And fluorescent quantitative PCR(qPCR)was adopted to detect the mRNA level of LYN.After recognition proteins IGF2BP1 and IGF2BP2 were knocked down with shRNA technique and YTHDF2 with siRNA,the expression levels of the 3 knockdown molecules and LYN were detected by qPCR.The interaction between LYN mRNA with IGF2BP2 was verified by RNA immunoprecipitation(RIP)assay.qPCR was used to detect the half-life of LYN mRNA after actinomycin D was employed to inhibit the process of transcription.Results The HUVECs with METTL3 stable knockdown were successfully generated by CRISPR-Cas9.METTL3 knockdown resulted in obviously inhibited cell migration(P<0.05),remarkably decreased m^(6)A methylation level(P<0.01),and significantly down-regulation in mRNA level of LYN(P<0.05).But knockdown of the m^(6)A recognition proteins YTHDF2 and IGF2BP1 had no significant effect on the mRNA expression of LYN(P>0.05),while knockdown of IGF2BP2 decreased the LYN mRNA level(P<0.01).RIP assay results indicated that IGF2BP2 interacted with LYN mRNA(P<0.05).LYN mRNA decay in IGF2BP2 knockdown cells was faster than the control cells.Conclusion Knockdown of METTL3 inhibits the migration of endothelial cells.Mechanistically,IGF2BP2 recognizes and binds to the m^(6)A site on LYN mRNA,and then regulates LYN mRNA stability.

关 键 词：甲基转移酶样蛋白3 内皮细胞 细胞迁移 m^(6)A甲基化修饰 

分 类 号：R322.12[医药卫生—人体解剖和组织胚胎学] R329.28[医药卫生—基础医学]