The Role Of A-to-I RNA Editing In Colorectal Cancer And Its Correlation With The Gut Microbiota

Objective: Colorectal cancer(CRC)is one of the most common malignant tumors worldwide.The changes in both epigenetics and the gut microbiota have been shown to play important roles in the occurrence and development of CRC.RNA editing is a type of epigenetics that refers to the insertion,deletion,or substitution of nucleotides in RNA.By far studies have indicated the important role of RNA editing in CRC,but the clinical significance of RNA editing in CRC diagnosis remains to be further explored.Emerging evidence suggested the effects of the gut microbiota on epigenetic changes in CRC,but there remains unclear about the correlation between RNA editing and the gut microbiota.Therefore,we explored the role of RNA editing in CRC diagnosis through transcriptome sequencing and verified its functional impact on CRC cell lines.In addition,we preliminarily explored the relationship between RNA editing and the gut microbiota by integrating 16 S r RNA gene sequencing data.Methods:(1)Sample recruitment: a total of 136 CRC patients who underwent tumor resection in the Affiliated Hospital of Jiangnan University from 2016 to 2019 were recruited to collect paired samples of fresh tumor and adjacent normal tissues.(2)Transcriptome sequencing and analysis: 26 tumor tissues and 16 adjacent normal tissue samples(including 7pairs of paired tissue samples)were processed and sequenced by transcriptome sequencing.Compared with publicly available transcriptome sequencing data of two cohorts(Wuhan cohort,PRJNA658001,and American cohort,PRJNA608970),analysis was performed to screen differential RNA editing sites.Selected differential editing sites were verified using targeted sequencing.(3)Wild-type and edited forms of selected genes overexpressed in HCT116 cells to verify the effects of RNA editing on CRC function cells.(4)The 16 S r RNA sequencing data were analyzed to screen differential microbiota.Then correlation analysis between RNA editing and the gut microbiota was conducted.The microbes significantly related to the RNA editing sites were selected to verify the effect on cell functions and RNA editing enzyme expression by co-culture with cells.Finally,we explored the effects of knockdown and overexpression of ADAR on the editing level of RNA editing sites.Results:(1)In multiple CRC transcriptome sequencing data,the expression of RNA editing enzyme ADAR in tumors was found significantly higher than that in normal tissues(p<0.05).Through data analysis of three CRC populations,we found 25 common RNA differential edits(p<0.05),of which 20 were up-regulated and 5 were down-regulated in the tumor group.The missense editing sites were IGFBP7(c.232A>G,p.R78G)and IGFBP7(c.284A>G,p.K95R).Besides,by screening the missense editing sites of two Chinese cohorts we found that BLCAP(c.5A>G,p.Y2C),BLCAP(c.14A>G,p.Q5R),and BLCAP(c.44A>G,p.K15R)also showed significant differences(p<0.05)in addition to IGFBP7.The targeted sequencing confirmed RNA differential editing of the above sites except for the intron region.the RNA editing level of editing sites of IGFBP7 and BLCAP were combined to generate a classifier.The AUC is 0.9061(p<0.05)for the prediction of the occurrence of CRC.Meanwhile,we found that RNA editing had a better classification effect compared with the classifier generated by the expression level.(2)At the cellular level,the IGFBP7 edited type significantly reduced the proliferation,cell colony formation,cell migration,cell invasion,and cell apoptosis of CRC cells compared with the wild type(p<0.05).BLCAP edited types BLCAP(p.Y2C)and BLCAP(p.Q5R)could promote the proliferation of CRC cells(p<0.05)and all edited types could promote cell colony formation(p<0.05).Besides,BLCAP(p.Q5R)promoted cell invasion(p<0.05).In addition,the apoptosis of all edited types had been significantly reduced compared with the wild type(p<0.05).(3)The results of correlation analysis between microbiota relative abundance and RNA editing levels showed that BLCAP(p.K15R),BLCAP(p.Q5R)and BLCAP(p.Y2C)were significantly negatively correlated with Parabacteroides(BLCAP(p.Y2C): r =-0.3431,p<0.05;BLCAP(p.Q5R): r =-0.4231,p<0.05;BLCAP(p.K15R): r =-0.3546,p<0.05).The results of the co-culture of Parabacteroides distasonis(P.ditasonis)with CRC cell line HCT116 and non-cancer cell line HCo Epi C showed that it had a significant inhibitory effect on the proliferation of HCT116cells(p<0.05)and had no significant effect on HCo Epi C.Furthermore,the co-culture data showed that P.ditasonis significantly inhibited the expression of ADAR m RNA in HCT116cells(p<0.05).The editing level of BLCAP(p.Q5R)showed an upward trend after overexpressing ADAR.Similarly,the editing level of BLCAP(p.Q5R)and BLCAP(p.Y2C)had a significant downward trend after successfully knocking down ADAR(p<0.05).Conclusion: This study found and verified RNA editing sites with differences in multiple CRC cohorts.The combination of RNA editing levels of IGFBP7 and BLCAP showed satisfying predictive performance on the diagnosis of CRC.Further exploration found that IGFBP7 and BLCAP edited types had significant effects on the function of CRC cells.In addition,the correlation between microbiota relative abundance and RNA editing level found that BLCAP(p.Y2C),BLCAP(p.Q5R),and BLCAP(p.K15R)were significantly negatively correlated with Parabacteroides.Co-culture with P.distasonis could specifically inhibit the proliferation of CRC cells and significantly inhibit the ADAR expression of CRC cells,indicating that the gut microbiota might influence the occurrence and development of CRC by regulating RNA editing.