| Background:Colorectal cancer(CRC)is a common malignant tumor of the digestive system,the incidence and mortality of which are on the rise in recent years in China.Apolipoprotein B mRNA editing catalytic polypeptide(APOBEC)family is a kind of gene editing enzyme that can specifically converse cytosine in genome into uracil.They are key participants in the innate immune response and antiviral response of human body under physiological conditions.However,on condition of chronic inflammation,abnormal overexpression of the APOBEC family occurs.Human genome can be accidentally injured,which results in the APOBEC characterised mutation,namely APOBEC mutation signatures.The current researches on the mutation signatures of APOBEC mainly focuses on APOBEC3 B,whose mutation tag is TCW(the underlined base is the mutant one,others are the flanking ones,and W stands for A or T).Previous studies have shown that APOBEC mutation signatues are significantly enriched in bladder cancer,head and neck squamous cell carcinoma,breast cancer,cervical cancer,lung cancer,but the enrichment of APOBEC mutation signatures in CRC remains unknown.In recent years,mutation signature sequences of other family members of APOBEC becoming increasingly clear,and the basic and epidemiological studies on the correlation between APOBEC and CRC are emerging endlessly.Hence,it is urgent to carry out comprehensive and systematic epidemiological studies to explore the role of APOBEC in the occurrence and development of CRC.Objectives:Starting from the mechanism of mutation of APOBEC,we aimed to determine the enrichment of mutation signature of each APOBEC family member in CRC patients.Then,the relationship between APOBEC and the development of CRC was comprehensively discussed from the four following aspects: the relationship between the differential expression of circRNAs and APOBEC,the differences of the expression of APOBEC among different disease stages,the relationship between the differential expression of APOBEC and prognosis of CRC patients,and the innate genetic susceptibility of APOBEC.Methods:Full exon capture sequencing was performed on tissue samples from 16 CRC patients to obtain somatic mutation information of tumor tissues and paracancer adenomatous polyps.The characteristic mutation signature of each APOBEC family member were determined by referring to the latest literature.The enrichment of mutation signature of each APOBEC familigy member was determined by one side Fisher exact test.The APOBEC family member with the most significant mutagenesis was identified by comparing the proportion s of samples significantly enriched with APOBEC mutation signature.The results above were then verified with the data from The Cancer Genome Atlas(TCGA)public database.In addition,full transcriptional sequencing data from tissue samples of 19 patients were used for circular RNA(circRNA)prediction by CIRI and find_circ software.differentially expressed circRNAs between cancer and adjacent tissues were screened by edgeR package of R software.Gene set enrichment analysis(GSEA)was conducted to predict the function of the differentially expressed circRNAs.A case-control study was conducted in 846 subjects from Changhai Hospital.56 patients of hemorrhoid were taken as the control group,51 patients of adenomatous polyp and 739 patients of CRC were taken as the case groups.The expression levels of activation induced cytidine deaminase(AID),APOBEC3 A,APOBEC3B,APOBEC3 G and uracil DNA glycosylase(UNG)between the case groups and control group were evaluated by immunohistochemical stain scoring.Kruskal-Wallis test was conducted to compare the differences in the expression levels of the above molecules among all stages of CRC development,so as to identify the potential biological markers of adenoma polyp or CRC occurrence.Cohort study was conducted in the above 739 CRC patients.The expression levels of AIDAPOBEC3 A,APOBEC3B,APOBEC3 G,and UNG in the tumor tissue were taken as exposure factors.All patients were followed to collect their survival and recurrence information.Kaplan-Meier analysis was conducted to study the effects of exposure factors on overall survival and tumor free survival of CRC patients.Multi-factor Cox regression model was used to screen independent influencing factors on overall survival and tumor free survival of CRC patients.A case-control study was conducted in 2044 subjects from Changhai Hospital and Yangpu Central Hospital.1240 people who came for physical examination were taken as the control group,164 patients with colorectal adenomatous polyp and 640 CRC patients were taken as case groups.Genomic DNA was extracted from peripheral blood of research participants,Taqman probe realtime PCR was conducted to measure the genotype of SNPs including rs12157810,rs2267401,rs12628403,and rs3890995.Logistic regression was conducted to compare the distribution of SNP allele frequency between the control group and the case group,to make clear the role of studied SNPs in CRC.Results:1.In the CRC tissue samples that were sequenced by us,the proportion of AID or APOBEC3 D mutation signatures(WRC and WWC)of the total single nucleotide mutation(SNV)was significantly higher than that of other members of the APOBEC family.This result is faithfully verified in the data of 489 CRC exon capture sequencing data from TCGA database.Similar results were obtained from the same analysis of the paracancer adenomatous polyps samples.Enrichment analysis indicated that samples with significant enrichment of AID and APOBEC3 D mutation signatures was the most,accounting for 25%and 18.8%,respectively.In the exon sequencing data from TCGA database,samples with significant enrichment of AID and APOBEC3 D mutation signatures was the most,accounting for 13.1% and 5.3%,respectively,while samples with significant enrichment of APOBEC3 D mutation signature was relatively small,accouting for only 1.6%.2.As predicted by CIRI and find_circ software,7701 circRNAs were detected in the total transcriptome sequencing data.Among them,25 circRNAs were differentially expressed between cancer and adjacent tissues in CRC patients.5 circRNAs were significantly enriched with more gene sets in GSEA,including X:131749306|131794466,9:16435555|16437524,6:160397979| 160410846,14:96833467|96860735,and12:55700899|55701154.Among them,the expression of X:131749306|131794466,6:160397979|160410846,and 14:96833467|96860735 was positively correlated with the expression of most genes in the C2 gene set,while the expression of 9:16435555|16437524and 12:55700899|55701154 was negatively correlated with the expression of most genes in the C2 gene set.Wherein,The gene set enriched in 6:160397979|160410846 with the lowest false discovery rate(FDR),namely CHANG CORE SERUM RESPONSE UP was very similar to the gene set REACTOME VIF MEDIATED DEGRADATION OF APOBEC3 G in gene composition.3.The histochemical scores(H-score)of AID and APOBEC3 G in the lesions of CRC patients were significantly higher than those of colorectal adenomatous polyp patients and healthy control(P <0.001),while the difference of H-score of these two genes between the lesions of adenomatous polyp patients and the control group was not statisticallysignificant.(P = 0.349 and 0.440,respectively).The H-score of APOBEC3 B in the lesions of CRC patients and adenomatous polyps was significantly higher than that of the control group(P <0.001),while there was no significant statistical difference in the H-score of APOBEC3 B in the lesions of CRC patients and adenomatous polyps(P=0.421).By contrast,there was no statistically significant difference in the H-score between APOBEC3 A and UNG among the three groups(P>0.005).4.High expression of AID and low expression of UNG can significantly increase the recurrence rate of CRC patients(P=0.017 and 0.044,respectively).High expression of APOBEC3 B or APOBEC3 G can significantly improve the overall survival rate of CRC patients(APOBEC3B P=0.020,APOBEC3 G P=0.009),and reduce postoperative recurrence rate(APOBEC3B P=0.002,APOBEC3 G P=0.002).Stratified analysis by tumor location showed that high expression of APOBEC3 B and APOBEC3 G significantly improved overall survival rate of colon cancer patients(P<0.001)and reduced postoperative recurrence rate(APOBEC3B P=0.0314,APOBEC3 G P<0.001).High expression of APOBEC3 A and APOBEC3 B significantly reduced postoperative recurrence rate in patients with rectal cancer(P = 0.024 and 0.039,respectively).Multivariate Cox regression analysis showed that high expression of APOBEC3 G was an independent protective factor for the overall survival of colon cancer patients(HR=0.36,95%CI=0.20-0.63),after excluding the effects of age,gender,postoperative chemotherapy or not,TNM stage,tumor differentiation degree,preoperative serum carcinoembryonic antigen,and preoperative serum glycosin antigen 19-9.5.In patients with APOBEC3 B deletion mutation,the CC genotype of rs12157810 SNP in APOBEC3 A promoter region was a risk factor for CRC occurrence in healthy people(adjusted odds ratio,AOR=10.03,95% CI=3.11-32.32).After all the subjects of the study were further stratified by gender,the distribution of rs12157810 genotypes in female patients with APOBEC3 B deletion mutations was not significantly different among the control group,adenomatous polyp patients,and CRC patients.However,CC genotype of rs12157810 was a risk factor for CRC occurrence in healthy male people with APOBEC3 B deletion mutation(AOR = 3.49,95% CI = 3.49-218).Conclusions:1.AID is the APOBEC family member with the most significant mutagenesis function in the occurrence of CRC.The mutagenesis of AID is an early molecular event in the occurrence of CRC.2.Five circular RNAs were found to be differentially expressed significantly between the cancer tissue and the tissue adjacent to cancer in CRC patients.Functional prediction analysis showed that circular RNAs that might play a key role in the occurrence of CRC were X:131749306|131794466,9:16435555|16437524,6:160397979|1604108466,14:96833467| 96860735,and 12:55700899|55701154.Among them,6:160397979|160410846 may be related to the degradation of APOBEC3 G.3.AID,APOBEC3 B and APOBEC3 G may be potential biological markers for CRC,and APOBEC3 B may be potential biological markers for adenomatous polyps.4.High expression of AID and low expression of UNG can significantly increase the recurrence rate of CRC patients.High expression of APOBEC3 B and APOBEC3 G are markers for good prognosis of colon cancer patients,among which high expression of APOBEC3 G is an independent protective factor for overall survival of colon cancer patients.5.In population with APOBEC3 B deletion mutation,CC genotype of APOBEC3 A promoter region SNP rs12157810 in is a risk factor for CRC.This carcinogenic effect has gender differences,more significant in male. |
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