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

• Main Authors: BAI Qin, CHEN Yanhua, LU Yao, WU Huang, WEN Aiqing
• Format: Article
• Language: zho
• Published: Editorial Office of Journal of Third Military Medical University 2021-05-01
• Series: Di-san junyi daxue xuebao
• Subjects: mettl3; endothelial cells; cell migration; m6a modification
• Online Access: http://aammt.tmmu.edu.cn/Upload/rhtml/202011144.htm

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 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. 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 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. Conclusion Knockdown 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.