M6A Modification Of EphA2 And VEGFA In Colon Cancer On The Role And Mechanism In The Development

N6-methyladenosine(m6A)is the most common chemical modification on RNA,and it regulates various biological processes of RNA.In the nucleus,the methylation enzyme with METTL3-METTL14 as the core catalyzes the formation of m6 A,and it can also be removed by demethylase FTO or ALKBH5.This dynamic and reversible balance maintains the m6 A modification on RNA.However,a class of protein(YTHDFs,IGF2 BPs,etc.)in cells can specifically recognize m6 A modification and then regulate RNA function.In recent years,it has been found that m6A-related regulatories are abnormally expressed in a variety of tumors and play a key role in the occurrence and development of tumors.However,its regulatory mechanism involved in the deterioration of colon cancer is not clear.The purpose of this study was to explore the biological function and regulatory mechanism of m6 A modification in colon cancer.According to bioinformatics prediction,four methyltransferases(METTL3,METTL16,RBM15 and VIRMA)and six recognition proteins(YTHDF1,YTHDF2,HNRNPA2B1,HNRNPC and IGF2BP2/3)were up-regulated in colon cancer,and METTL3 was significantly up-regulated in colon cancer.However,there was no significant difference among other m6 A binding proteins.In addition,Kaplan-Meier analysis of survival showed that patients with high METTL3 expression had poor overall survival.After confirming that methyltransferase METTL3 was the key enzyme for m6 A modification in colon cancer,qRT-PCR and Western blot were used to confirm that METTL3 mRNA and protein level were highly expressed in colon cancer tissues.In addition,Dot Blot assay demonstrated a marked increase in overall m6 A levels in colon cancer tissues.Secondly,the expression of METTL3 in normal intestinal epithelial cells and six colon cancer cell lines were detected by qRT-PCR and Western blot,and HCT-116 with high METTL3 expression were selected for the experiment.The METTL3-deleted colon cancer heterozygous cell line was constructed by Crispr/Cas9 technology,the qRT-PCR,Western blot and Dot Blot proved that the expression level of METTL3 mRNA and protein was successfully down-regulated,and the expression level of m6 A decreased significantly.RNA-seq high-throughput sequencing and m6 A modification websites SRAMP,RMBase2.0and BERMP predict the target genes regulated by m6 A modification,Me RIP-PCR and luciferase reporter system confirmed that the m6 A modification of EphA2 and VEGFA by METTL3.The results of Dot Blot,qRT-PCR and Western blot showed that m6 A modification was significantly down-regulated,and the expressions of EphA2 and VEGFA mRNA and protein were significantly down-regulated,which further confirmed that EphA2 and VEGFA were regulated by m6 A modification.The correlation analysis of all m6 A recognition proteins with EphA2 and VEGFA by bioinformatics showed that IGF2 BPs level was positively correlated with EphA2 and VEGFA,indicating a potential positive regulatory mechanism.In addition,the analysis of GEPIA online database showed that IGF2BP2 and IGF2BP3 had high expression level in colon cancer.The qRT-PCR and Western blot confirmed the interference efficiency of two specific si RNAs targeting IGF2BP2/3,and found that interfering IGF2BP2 reduced the expression of EphA2 mRNA and protein,while interfering IGF2BP3 inhibited the expression of VEGFA mRNA and protein.The mRNA stability experiment showed that METTL3,IGF2BP2 and IGF2BP3 affected the half-life of EphA2 and VEGFA mRNA,IGF2BP2 combined with m6A-methylated EphA2,and IGF2BP3 recognized m6A-methylated VEGFA to enhance the stability of both mRNA and prevent its degradation.According to RNA-Seq data analysis,EphA2 and VEGFA are enriched in cell proliferation and vascular system development.The results of MTT assay and clone formation showed that METTL3 deletion inhibited cell proliferation,and compared with the cells overexpressing EphA2 or VEGFA alone,overexpressed EphA2 and VEGFA at the same time had stronger proliferation ability.Transwell migration and invasion test confirmed the role of METTL3 in the migration and invasion of colon cancer cells.The results showed that METTL3 deletion significantly inhibited the migration and invasion of HCT-116 cells,and whether the overexpression of EphA2 and VEGFA alone or in combination,could save the migration and invasion of HCT-116 cells.The Vasculogenic mimicry(VM)experiment showed that HCT-116 cells produced some various sizes tubular structures in the matrix gel.However,the tubular structures were not formed after METTL3 deletion,the tubular structures could be formed again when the target genes EphA2 and VEGFA was expressed individually or jointly.In vivo,the tumorigenesis experiment in nude mice showed that the deletion of METTL3 resulted in the decrease of EphA2 and VEGFA expression,and affected the tumor growth and angiogenesis.Then,we detected the signal pathways related to cell proliferation and vascular mimicry in KEGG pathway analysis by Western blot,and clarified that METTL3 targeted EphA2 and VEGFA promoted HCT-116 cells proliferation and VM formation through PI3K/AKT/m TOR and ERK1/2 signal pathways in vivo and in vitro.To sum up,m6 A plays an important role in the occurrence and development of colon cancer.In vivo and in vitro,it was clarified that METTL3 acted on the target genes EphA2 and VEGFA by m6 A modification,and two different recognition proteins IGF2BP2 and IGF2BP3 recognized methylated EphA2 and VEGFA and participated in the two signal pathways(PI3K/AKT/m TOR and ERK1/2)to jointly promote the proliferation of colon cancer cells and the formation of VM.The results of this study enrich the functionality of m6 A modification,provide a new perspective for understanding the molecular mechanism of colon cancer,and provide a new idea for the prevention and treatment of colon cancer,which has potential application value.