Comprehensive Analysis Of LncRnas N6-Methyladenosine Modification In Colorectal Cancer

Research background and purposeColorectal cancer(CRC)is the third leading malignancy in cancer-related deaths.Epidemiological analysis showed that more than 50%of CRC patients had liver metastasis.With advances in medical technology,the treatment of CRC is being optimized,but there is still no effective treatment for patients with advanced metastatic CRC.Long non-coding RNAs(lncRNAs)can regulate gene expression in a variety of ways and participate in the occurrence and progression of tumors.LncRNAs can be modified by N6-adenosine methylation(m6A)and regulate a variety of physiological and pathological processes.However,the specific mechanisms of lncRNAs and m6A modifications in CRC have not been fully elucidated.Therefore,the establishment of lncRNAs and m6A modified transcription maps in CRC is of great significance for the in-depth study of the role of lncRNAs in the growth,metastasis and invasion of CRC cancer cells.Materials and Methods1.In our hospital,,5 pairs of CRC tissue and paracancer tissue(NC)samples that were not surgically removed before chemotherapy and radiotherapy were collected,and RNA was extracted for RNA-seq and MeRIP-seq.2.After the methylation sites in each sample were identified by MACS software,differential methylation sites were identified by diffReps.Cuffdiff software(part of V2.2.1)was used to obtain the FPKM values of lncRNAs at the transcription level to form the expression profile of lncRNAs,and identify differentially expressed lncRNAs based on multiple changes and P-values between samples.RT-qPCR was used to detect differentially distributed lncRNAs to verify the accuracy of sequencing results.The adj acent lncRNAs genes with different expression and m6A modification were analyzed by GO and KEGG pathways.3.RNA-seq and MeRIP-seq sequencing data were analyzed jointly to determine the correlation between m6A level and lncRNAs expression level in CRC and NC tissues,and to identify differentially methylated and differentially expressed lncRNAs.4.LncRNAs with methylation fold change>7 and expression fold change>2.5 were screened from CRC-related lncRNAs.Based on miRNA-target gene prediction software of Miranda and TargetScan,the combination miRNAs and mRNAs of screened lncRNAs were predicted.Endogenous competing RNA(CeRNA)networks were mapped using Cytoscape(V3.7.1).Differentially expressed lncRNAs associated with CRC were screened and Pearson correlation coefficients between lncRNAs and mRNA expression levels were calculated.Coded and non-coded co-expression(CNC)networks were plotted using Cytoscape(V3.7.1).Result1.A total of 8332 m6A peaks were detected in 6690 lncRNAs in CRC tissues,and 7064 m6A peaks were detected in 5513 lncRNAs in NC tissues.About 91%of lncRNAs had unique m6A modification peaks.According to the location relationship between m6A methylated lncRNAs and mRNA,most lncRNAs contained m6A sites with overlapping exons.The m6A peak showed a higher multiple of change in the bidirectional group of CRC tissues.However,in normal tissues adj acent to cancer,the m6A peak in the natural antisense group had a higher multiple of change.2.There were 383 differentially methylated lncRNAs in CRC,of which 65.5%were hypommethylated,34.5%were hypermethylated and 48.24%were 1-1000 bp in length,mainly distributed on chromosome 1,2,7,11,16 and 19.GO analysis showed that the adj acent genes of lncRNAs with differences in m6A modification were mainly involved in biological processes such as cell cycle arrest,DNA replication,regulation of catabolic process and RNA binding.KEGG analysis showed that these adj acent genes were mainly enriched in glutamate synapse,selenium compound metabolism,calcium signaling pathway and Rap1 signaling pathway.3.RNA sequencing identified 163 differentially expressed lncRNAs in CRC,of which 44 were up-regulated and 119 down-regulated.GO analysis showed that differentially expressed adj acent genes of lncRNAs were related to cell proliferation and development,extracellular matrix decomposition,extracellular domain,and potassium channel regulatory activity.KEGG analysis showed that these adjacent genes were associated with dopaminergic synapses,insulin signal transduction and Wnt signal transduction pathways.4.M6A level of lncRNAs in CRC was positively correlated with m6A level of lncRNAs in NC,and methylation level in CRC group was higher than that in NC group.Methylation was positively correlated with lncRNAs expression in CRC and NC groups.The proportion of non-m6A modified lncRNAs was larger than that of m6A modified lncRNAs.5.We constructed a CeRNA and CNC regulatory network,in which the CeRNA regulatory network predicted the regulatory relationships among 16 lncRNAs,80 miRNAs and 400 mRNAs.The CNC regulatory network predicted regulatory relationships between 12 lncRNAs and 158 mRNAs.Conclusions1.M6A methylation and lncRNAs expression were significantly different between CRC and NC tissues.2.Differentially methylated or expressed adjacent genes of lncRNAs were significantly enriched in biological and signaling pathways related to the occurrence and development of CRC.3.Methylation was positively correlated with lncRNAs expression levels in CRC and NC,and methylation down-regulated lncRNAs expression.4.CeRNA and CNC regulatory network revealed the regulatory relationship among lncRNAs,miRNAs and mRNAs.