The Differential Expression Profile Of M6A-Modified CircRNAs And The Analysis Of Function And Prognosis Of Their Parental Genes For Colorectal Cancer

Background: Colorectal cancer(CRC)is one of the most common malignant tumors in human digestive system.According to the statistics,more than 1.9 million CRC new cases and 0.9 million CRC deaths were added in the world in 2020,accounting for one tenth of all malignant tumors in terms of incidence rate and mortality.Due to the lack of obvious early symptoms in CRC,most patients are diagnosed in the middle to late stage,resulting in poor survival prognosis.Tumor marker detection has the advantages of convenient operation,easy sample acquisition and non-invasive,which can be applied to the early screening and prognosis assessment of cancer.It is worth noting that many studies at home and abroad have found that abnormal level of N6-methyladenosine(m6A)modification occurs in various cancers,which serve as the potential markers of cancers.It is interesting that m6 A modification is commonly present in circular RNA(circ RNA),and this chemical modification plays a crucial role in the biological function of circ RNA,including the initiation of circ RNA translation mediated by m6 A.Aims: In this study,circ RNA was used as an entry point to detect differentially expressed m6A-circ RNA between CRC and adjacent tissue samples by immunoprecipitation and microarray techniques in order to search for potential tumor markers;At the same time,the prognosis and function of their parental genes were analyzed by bioinformatic method,and the expression of their parental genes was further verified in clinical tissue samples through q PCR and Western blot,providing a theoretical basis for further exploring the molecular mechanism of CRC.Methods:(1)The "Sup" RNA(not modified by m6A)and "IP" RNA(modified by m6A)were obtained by immunoprecipitation technology,and the circ RNAs were enriched by Rnase R,and Cy3 was labeled on the "Sup" circ RNA and Cy5 was labeled on the "IP" circ RNA.Then the circ RNAs were fragmented and hybridized on the chip.(2)Based on the TCGA-COAD and GSE106582 cohorts,explore differentially expressed genes at the m RNA level in CRC;Obtaining parental genes with downregulated m6A-circ RNA and m RNA expression in CRC through Veen analysis for gene function enrichment and protein interaction network analysis;Prognostic analysis of parental genes was performed using the Kaplan Meier method,and gene mutations of prognostic related genes in CRC were analyzed using the c Bio Portal tool.(3)Exploring the m RNA and protein levels of TPM1 and GAB1 expression in CRC tissue samples using q PCR and Western blotting techniques,and exploring the correlation between TPM1 expression and clinical factors(binary classification)using Wilcoxon test.Results:(1)A total of 2378 differentially expressed m6A-circ RNAs were detected in CRC and adjacent tissue samples,and 1694 m6A-circ RNAs were downregulated in CRC tissue samples,and most of the differentially expressed m6A-circ RNAs were derived from exon sequences,accounting for 81.75%.(2)Based on the TCGA database,there are 3748 genes downregulated at m RNA level in CRC;Moreover,based on the GSE106582 cohort,5048 genes were found to be downregulated at m RNA level in CRC.(3)Low expression of nine parental genes was associated with poor prognosis in CRC(P<0.05),including ABCD3,ABHD6,GAB1,MIER1,MYOCD,PDE8 A,RPS6KA5,TPM1,and WDR78.Their gene mutation rates were 4%,6%,5%,6%,10%,6%,6%,5%,and 8%,respectively.Among them,TPM1 and GAB1 were downregulated in m RNA and protein levels in CRC tissue samples,and the expression of TPM1 was related to age,gender,clinical stage,and lymph node metastasis.(4)KEGG function enrichment analysis found that m6A-circ RNA and m RNA downregulated parental genes were mainly enriched in cancer related signaling pathways,especially proteoglycans in cancer and calcium signaling pathway;Based on the MCC algorithm,analyzing these parental genes through protein interaction networks,it was found that TPM1 is the core gene with the highest score.Conclusion: The m6 A modification level of circ RNA in CRC tissue samples has changed,and differentially expressed m6A-circ RNA can serve as potential tumor markers;Furthermore,it was found that TPM1 can serve as a potential tumor suppressor gene for CRC,and the downregulation of m6 A modification level of its transcription product hsa＿circ RNA＿035619 may be an important factor in the abnormal expression of TPM1 in CRC.