Analysis Of N6-adenosine Methylation Profile And Differential Expression Of CircRNA In Colorectal Cancer

Research background and purposeColorectal cancer(CRC)is one of the most common malignant tumors and a major cause of cancer-related death in the world.Due to the occult onset of CRC,atypical clinical symptoms,and the lack of effective and non-invasive tumor screening markers,about 20% of CRC patients are in the middle and advanced stage,and over half of the patients will have progression and distant metastasis during the course of the disease.The cure rate can reach up to 90.0% in patients at the early stage of the disease,but advanced colorectal cancer patients with distant metastasis have a 5-year survival rate of only 14%.The diagnosis and treatment of CRC brings great economic burden to individuals,their families and society.Multiple genes and signal pathways are involved in the development of CRC.Abnormal expression of circular RNA and abnormal level of N6-adenylate methylation(M6A)modification are associated with tumorigenesis and cancer progression.In this study,we jointly analyzed and screened the M6 A methylation and expression profile of circular RNA in colorectal cancer and predicted the functional mechanism,in order to further explore the potential pathogenesis of colorectal cancer and provide effective tumor screening markers and new therapeutic targets.Methods1.The cancer tissues and adjacent normal tissues of colorectal cancer patients who underwent surgery in the first affiliated Hospital of Chengdu Medical College from June2020 to June 2021 were collected.2.Methylated RNA immunoprecipitation sequencing and RNA sequencing were used to analyze the methylation profile and expression profile of circRNA in colorectal cancer and adjacent normal tissues.Diff Reps software and edge R software were used to screen the circular RNA with differential methylation and differential expression.3.CircRNAs with significant differential expression was selected,and qRT-PCR technique was used to verify the accuracy of high-throughput sequencing results.4.Bioinformatics analysis: GO functional analysis and KEGG signal pathway were used to predict the potential biological functions of the host genes of differentially methylated and differentially expressed circRNA.5.To further explore the clinical correlation between the host gene of the circRNAs with differential methylation and differential expression and colorectal patients by searching database(Gene Expression Profiling Interactive Analysis,GEPIA).6.CircRNA-miRNA interaction and miRNA-m RNA interaction are predicted by Target Scan and miRanda data software.According to the theory of competitive endogenous RNA(ceRNA)regulation mechanism,cytoscape(version 3.8.0)software is used to draw and visualize circRNA-miRNA-m RNA regulation and control network.7.The protein coding potential of circular RNA was predicted by searching the website http: // bigd.big.ac.cn/lgc.Result1.Through high-throughput sequencing technology,we identified 7990 circRNAs,of which 2894 circRNAs(36.22%)underwent m6 A methylation modification.In colorectal cancer tissues,a total of 4340 m6 A peaks were detected,corresponding to2651 circRNAs.A total of 3216 m6 A peaks were detected in adjacent tissues,corresponding to 2129 circRNAs.The unique m6 A peaks in adjacent normal tissues were 578,corresponding to 243 circRNAs,whereas the unique m6 A peaks in cancer tissues were 1702,corresponding to 765 circRNAs.Approximately 50% of the circRNA contains only one m6 A peak.About 80% of the circRNA consists of exon sequences.Compared with non-m6 A circRNA,m6 A occurs more often on circRNA composed of single exons,and all exons in m6 A circRNA are longer in length than those in non-m6 A circRNA.2.A total of 336 differential methylation peaks were found in colorectal cancer,with 171 upregulated and 165 downregulated methylation peaks.There were 247 differentially methylated circRNAs,with 130 hyper-and 117 hypo-methylated.Differentially methylated circRNA were widely distributed on autosomes,but the differentially hyper-methylated circRNA is distributed on chromosome 6,7 and 10,and the hypo-methylated circRNA is distributed on chromosomes 1,2 and 12.3.The GO analysis of the host genes of the differential methylated circRNA was mainly correlated with cellular macromolecular metabolic processes,cell cycle,protein binding,and RNA binding.KEGG analysis was mainly enriched in DNA replication,RNA transport,protein processing,cancer pathways,c GMP-PKG,and tight junction signaling pathways.4.Cancer tissues had 877 differentially expressed circRNAs,including 522 downregulated and 355 upregulated circRNAs.The qRT-PCR validation results were consistent with the expression trend of the sequencing results.The GO analysis showed that the host genes of the differentially expressed circRNA were associated with the macromolecular binding capacity,macromolecular metabolic processes,and cellular localization.The KEGG analysis was mainly enriched in base excision repair,protein processing,adhesion junctions,and c GMP-PKG signaling pathways.5.There was a potential relationship between m6 A methylation modification and circRNA expression,and the correlation analysis found no correlation between m6 A methylation and circRNA expression.6.Joint analysis of Me RIP-seq and RNA-seq data found that a total of 30 circRNAs were differentially methylated and differentially expressed,and searching public databases found that 33%(10/30)of the host genes of the circRNA had abnormal expression in colorectal cancer tissues,the decrease of some genes expression was closely related with the reduced overall survival of patients.7.In the ceRNA regulatory network,the expression of m RNA was positively correlated with that of circRNA.By searching the tumor database,32 target m RNA genes showed abnormal expression in various malignancies,of which 28 m RNA were abnormally expressed in CRC tissues.8.According to the prediction analysis of the protein coding potential of circular RNA,850(10.64%)circRNAs out of 7990 total circRNA had protein coding potential.Of the 2,894 m6 A circRNAs,575(19.87%)of circRNAs had protein-coding potential.Of the 224 differentially methylated circRNAs,101(45.09%)circRNAs were predicted to have protein-coding potential.Conclusion1.There were significant differences in the m6 A methylation modification profiles of circRNA between colorectal cancer tissues and adjacent cancer tissues,and GO functional analysis and KEGG signaling pathway analysis predicted that host genes of differentially methylated circRNA are closely associated with tumors.2.There were significant differences in the expression profiles of circRNA between colorectal cancer tissues and adjacent cancer tissues,and GO functional analysis and KEGG signaling pathway analysis predicted that host genes of differentially expressed circRNA are closely associated with tumors.3.Host genes of the differentially methylated and differentially expressed circular RNA are strongly associated with colorectal cancer.4.Through the ceRNA regulatory network,differentially methylated and differentially expressed circular RNA positively regulates the expression of multiple m RNA of related target genes to colorectal cancer.5.The M6 A methylation modification increases the protein-coding potential of the circular RNA.