The Expression And Biological Function Of Methyltransferase-like3 In Colorectal Cancer

Background:Colorectal cancer(CRC)is one of the most common gastrointestinal malignancies in the world.According to statistics,in2018,CRC ranks the third in both incidence and mortality among all kinds of cancers in the world.In China,due to the aging population,changes in dietary habits and other factors,the incidence and mortality of colorectal cancer are also increasing year by year.It is reported that in2020,the incidence rate of CRC in China is the third and the mortality rate is the fifth.Early diagnosis and treatment of colorectal cancer patients will be delayed due to the absence of typical clinical symptoms,so that the patients have entered the middle and late stage when metastasis occurs,resulting in a high recurrence rate and mortality rate of tumors,which seriously threatens human health.Therefore,to find out the molecular mechanism of colorectal cancer occurrence and metastasis is particularly important for the prevention and treatment of colorectal cancer.N6-methyladenosine(m~6A),methylated at the N6 position of adenosine,has been considered as the most pervasive,abundant and conserved internal transcriptional modification within eukaryotic messenger RNA,which can regulate the transcription and translation of mRNA.M~6A is closely related to the development of tumor.M~6A methylation is a dynamic and reversible process coordinated by a series of methyltransferases,demethylases and readers.METTL3 as the core component,interacting with METTL14 and WTAP,constitutes an m~6A methyltransferase complex,which mediates the m~6A modification of mRNA.In recent years,various studies have shown that METTL3 is closely related to acute myeloid leukemia,liver cancer,lung cancer and breast cancer.Objective:To detect the expression and clinical significance of METTL3 in colorectal cancer tissues and adjacent tissues,so as to preliminarily investigate the role and potential mechanism of METTL3 in colorectal cancer cell proliferation.Methods:1.46 cases of CRC and matched normal or benign tissue samples were collected from the First People’s Hospital of ChenZhou from 2014to 2017(normal tissues from tumor tissue adjacent to carcinoma edge are more than 2cm).2.The expression of METTL3 mRNA in colorectal cancer tissues and adjacent tissues of 46 patients were detected by real-time fluorescent quantitative PCR,and the correlation was analyzed between METTL3expression levels and age,tumor size,differentiation degree and lymph node metastasis of colorectal cancer patients.3.We knocked out METTL3 in CRC cells by using plasmid transfection with dual guide-RNA(dual gRNA)(KO group)in contrast to control cells(gRNA Ctrl group).4.M~6A sequencing analysis step is:first,m~6A-modified RNA fragments were present by specific antibody markers,then enriched and purified RNA fragments.High-throughput sequencing was carried out together with the input sample,and the m~6A-modified peak detection and motif predictive analysis were realized by combining bioinformatics.Results:1.The expression level of METTL3 mRNA in CRC tissues was significantly higher than that in adjacent tissues(p<0.05).2.The expression level of METTL3 mRNA was closely related to tumor location(p=0.039)and tumor size(p=0.038),but was not related to patient age,gender,tumor differentiation,invasion depth,lymph node metastasis and TNM grading(p>0.05).3.The expression level of METTL3 protein in KO group was significantly lower than that in gRNA Ctrl group,indicating a good knockout effectence.The results of CCK8 and cell cloning experiments showed that the cell proliferation ability of KO group was significantly decreased compared with that of control group.4.Through m~6A sequencing,it was found that the higher genes in gRNA Ctrl group(such as COL3A1,NBPF14,NBPF25P)were positively regulated genes of METTL3,while the higher genes in KO group(such as ROCK2,TAOK1,SEPT7)were negatively regulated genes of METTL3.In peak specific to KO group,GO functions were mainly concentrated in protein binding,nucleotides and nuleus.The KEGG pathway was mainly enriched in P13K-Akt-signaling-pathway,Small-cell-lung-cancer,Pathways-in-cancer.Conclusions:1.METTL3 mRNA is highly expressed in CRC cells.2.The expression level of METTL3 mRNA in CRC tissues is closely related to tumor location and tumor size(p<0.05).3.METTL3 can promote CRC cell proliferation.