A Molecular Epidemiology Study Of Genetic Variants Associated With Alternative Polyadenylation In The Risk Of Colorectal Cancer

Colorectal cancer（CRC）is a malignant tumor associated with environmental and genetic factors and its high incidence and mortality cause great harm to human health.Therefore,early identification of genetic markers in high risk population is of great significance for cancer prevention and treatment.Genome-wide association studies（GWASs）have identified more than 140 CRC susceptibility loci and made important progress in revealing cancer genetic susceptibility.However,the susceptibility loci so far identified could partially explain the heritability of CRC and a large number of predisposition loci remain to be discovered.In addition,the exploration of their biological machanisms has been hampered because many of them locate in non-coding regions.Alternative polyadenylation（APA）refers to a process in which some reasons influence the selection of polyadenylation signal sites（PASs）and result in transcripts with different lengths of 3’untranslated regions（3’UTR）.APA is a crucial post-transcriptional regulatory mechanism that affects a variety of diseases,including cancer.Meanwhile,some genetic variants in PASs or APA-related regulatory elements have been found to alter APA regulation and thus affect cancer progression.Therefore,our study aimed to systematically identify genetic variants affecting APA process in human cancers and define them as cancer APA quantitative trait loci（apaQTL）.Subsequently,by integrating CRC apaQTLs with a large case-control study and a variety of biological assays,our study tried to identify apaQTL variants associated with CRC risk in the Chinese population.This study might provide a novel perspective for elucidating the etiology of CRC and pave the foundation for screening of high-risk population.This dissertation mainly includes the following two parts:PartⅠIdentification and characterization of genetic variants associated with alternative polyadenylation in human cancersObjective:By integrating genomic,transcriptome and epigenetic data,our study aimed to identify genetic variants affecting APA process in 32 human cancers and provide a new insight for further understanding the mechanism of genetic variants in cancers.Methods:Genotype data,APA data and other clinical data（age,sex,tumor stage）in 32 tumor samples were downloaded from The Cancer Genome Atlas（TCGA）database.Through conducting linear regression analysis,we evaluated the associations between single nucleotide polymorphisms（SNPs）and APA events.After correcting a series of confounding factors,SNPs with a false discovery rate（FDR）<0.05 were defined as cancer apaQTLs.Next,the functional characterizations of cancer apaQTLs were systematically performed,including genome distribution of cancer apaQTLs,enrichment analyses of RNA-related features and chromatin regulatory elements,functional annotations of target genes and enrichment analyses of GWAS loci.Results:1.We performed cancer apaQTL analyses by using genotype and RNA-seq data in 32 cancer types derived from 9,082 TCGA individuals.After correcting covariants including age,sex,tumor stage and population structure,we totally identified 703,331 apaQTLs related with 16,571 APA events（FDR<0.05）.2.Functional characterizations of cancer apaQTLs were systematically performed.Firstly,the genome distribution results indicated that cancer apaQTLs were mainly enriched in 3’UTR when compared with non-apaQTLs and many of them were located in the transcription end sites.Mechanistically,cancer apaQTLs might contribute to APA changes through altering poly（A）motifs,binding sites of RNA-binding protein（RBP）and chromatin regulatory elements.In addition,cancer apaQTL-related genes（a Genes）were closely relevant to cancer signaling pathways.Finally,cancer apaQTLs were found to be preferentially enriched in cancer risk loci,suggesting that it could explain a significant proportion of disease heritability.Conclusions:In this project,a pan-cancer atlas of alternative polyadenylation quantitative trait loci was constructed based on multi-omics data from TCGA and functional characterizations were also systematically performed.Collectively,our study not only designs a rich library of cancer apaQTLs,but also provides the foundation for understanding the etiology of cancer.Part Ⅱ Association and mechanism study of genetic variants associated with alternative polyadenylation for colorectal cancer susceptibility in the Chinese populationObjective:Based on the identified CRC apaQTLs,this study aimed to identify genetic variants associated with CRC risk through influencing APA process by using a large population study and a variety of biological assays.Furthermore,the results might help to illustrate the underlying mechanisms of genetic variants contribute to CRC risk.Methods:Based on the results in PartⅠ,functional characterizations of CRC apaQTLs were performed,including genome distribution,enrichment analyses of RBP and chromatin regulatory elements,functional annotations of target genes and enrichment analyses of GWAS loci.Then,a two-stage,multicentre case-control study in the Chinese population was used to identify apaQTLs associated with CRC risk.Finally,multiple biological assays such as 3’-Rapid-amplification of c DNA ends,RNA pulldown,cell proliferation experiments were used to further explore the mechanism of apaQTLs and the risk of CRC.Results:1.Based on the results in PartⅠ,a total of 24,681 apaQTLs affecting 725 APA events（FDR<0.05）were identified.2.Compared with non-apaQTLs,CRC apaQTLs were significantly enriched in 3’UTR and downstream of genes（P<0.0001）.In addition,CRC apaQTLs were also enriched in various RBP and chromatin regulatory elements（histone modification marks and transcription factors,P<0.0001）.Functional characterizations of a Genes revealed that they were involved in CRC-related signaling pathways.Finally,the integration of apaQTLs and GWAS data showed that apaQTLs were significantly enriched in CRC GWAS loci compared with non-apaQTLs（OR=3.08,95%CI=2.83-3.36,P<0.0001）.3.A total of 1,201 apaQTLs conferred a significant genetic predisposition to CRC in the European population.Variant rs1020670 located in 12p11.21 region had the highest potential and showed a significant association with the APA event of DNM1L.Therefore,we selected it as the functional candidate for further interpretation.Next,a two-stage case-control study further validated that rs1020670 contributed to an increased risk of colorectal cancer in the Chinese population.Compared with the individuals with C allele,those with G allele had a high CRC risk(OR=1.22,95%CI=1.13-1.32,P=1.35×10^-6).4.Biological assays indicated that rs1020670[G]shortened the 3’UTR of DNM1L by promoting the binding of CSTF2T,thus increasing its m RNA stability and ultimately leading to the up-regulation of m RNA and protein expression.Additionally,differential expression results indicated that the expression level of DNM1L was increased in CRC tumor tissues,and further experiments proved that the up-regulation of DNM1L might significantly enhanced the proliferation abilities of CRC cellsConclusions:By integrating CRC apaQTLs and population studies,an apaQTL variant rs1020670（C>G）was proved to be significantly associated with CRC risk in the Chinese population.Finally,biological experiments indicated that variant could contribute to an increased risk of CRC by promoting aberrant alternative polyadenylation of DNM1L.In general,our results provided a novel perspective for elucidating the etiology of CRC.