Down-regulation of METTL3-LYN m6A-IGF2BP2 pathway inhibits endothelial cell migration

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Down-regulation of METTL3-LYN m6A-IGF2BP2 pathway inhibits endothelial cell migration

更新时间：2021-08-02

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- Down-regulation of METTL3-LYN m6A-IGF2BP2 pathway inhibits endothelial cell migration
- Journal of Army Medical University Vol. 43, Issue 9, Pages: 845-851(2021)
- 作者机构：
  
  陆军军医大学（第三军医大学）大坪医院输血科
- 作者简介：
- 基金信息：
- DOI：10.16016/j.1000-5404.202011144
  CLC： R322.12;R329.28;R394.3
- Published：2021，
  
  Published Online：15 May 2021，
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[1]白秦,陈燕华,卢尧,等.下调METTL3-LYN m6A -IGF2BP2通路抑制内皮细胞迁移[J].陆军军医大学学报(原第三军医大学学报),2021,43(09):845-851.BAI Qin,CHEN Yanhua,LU Yao,et al.Down-regulation of METTL3-LYN m6A-IGF2BP2 pathway inhibits endothelial cell migration[J].J Amry Med Univ (J Third Mil Med Univ),2021,43(09):845-851.

BAI Qin, CHEN Yanhua, LU Yao, et al. Down-regulation of METTL3-LYN m6A-IGF2BP2 pathway inhibits endothelial cell migration. [J]. Journal of Army Medical University 43(9):845-851(2021)
[1]白秦,陈燕华,卢尧,等.下调METTL3-LYN m6A -IGF2BP2通路抑制内皮细胞迁移[J].陆军军医大学学报(原第三军医大学学报),2021,43(09):845-851.BAI Qin,CHEN Yanhua,LU Yao,et al.Down-regulation of METTL3-LYN m6A-IGF2BP2 pathway inhibits endothelial cell migration[J].J Amry Med Univ (J Third Mil Med Univ),2021,43(09):845-851. DOI： 10.16016/j.1000-5404.202011144.

BAI Qin, CHEN Yanhua, LU Yao, et al. Down-regulation of METTL3-LYN m6A-IGF2BP2 pathway inhibits endothelial cell migration. [J]. Journal of Army Medical University 43(9):845-851(2021) DOI： 10.16016/j.1000-5404.202011144.

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；m6A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m6A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m6A识别蛋白IGF2BP1和IGF2BP2，siRNA敲低m6A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m6A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m6A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m6A修饰位点，调控其mRNA稳定性来发挥作用。

Abstract

ObjectiveTo investigate the effect of methyltransferase like 3 (METTL3) on the migration of human umbilical vein endothelial cells (HUVECs)

and explore the underlying mechanism preliminarily. MethodsThe 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 m6A 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. ResultsThe HUVECs with METTL3 stable knockdown were successfully generated by CRISPR-Cas9. METTL3 knockdown resulted in obviously inhibited cell migration (P<0.05)

remarkably decreased m6A methylation level (P<0.01)

and significantly down-regulation in mRNA level of LYN (P<0.05). But knockdown of the m6A 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. ConclusionKnockdown of METTL3 inhibits the migration of endothelial cells. Mechanistically

IGF2BP2 recognizes and binds to the m6A 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m6A甲基化修饰

Keywords

METTL3endothelial cellscell migrationm6A modification

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Related Institution

Laboratory of Stem Cell Biology and Therapy, National Clinical Research Center for Child Health and Disorders, Key Laboratory of Child Development and Disorders of Ministry of Education, Chongqing Key Laboratory of Pediatrics, Children's Hospital of Chongqing Medical University

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Department of Anesthesiology, Wuhan Central Hospital, Tongji Medical College, Huazhong University of Science and Technology

Department of Urology, the First Affiliated Hospital of Chongqing Medical University

Third Military Medical University

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