A Study On The Gene Variation Detection Methods And The Role Of Gene Variation In Changing Gene Regulation By HOXB13 In Prostate Cancer

Cancer has become the main cause of human death worldwide.As a complex disease,excavating,screening and annotating cancer-related genetic variation sites are of great significance for the prediction,diagnosis and treatment of cancer.A large number of genetic variation sites that are significantly associated with cancer risk have been discovered through genome-wide association analysis(GWAS).However,the risk variation loci discovered by GWAS are usually common mutations with a variation frequency greater than 1%,excluding rare mutation loci that may have greater genetic effects.Furthermore,whether these risk variant sites discovered by GWAS are causal sites that lead to disease risk still requires a lot of functional analysis and screening work.A large number of studies have shown that most risk variant sites mainly affect downstream gene expression by changing the binding of specific transcription factors,thereby leading to disease risk.Therefore,this project starts from the transcription factors that are closely related to the risk of occurrence and development of cancer.By analyzing the ChIP-seq data of tumor clinical samples,we discover the mutation sites that affect the binding of transcription factors and systematically explore regulatory mutation sites that affect key transcription factor binding.HOXB13 transcription factor plays an important regulatory function in prostate cancer.Previous reports have shown that the prostate cancer risk SNP site rs339331 mediates HOXB13 to regulate the expression of the downstream target gene RFX6,thereby affecting cancer risk.This study focused on the regulatory role of HOXB13 transcription factor and comprehensively screened SNP sites that mediate the regulatory role of HOXB13.We obtained HOXB13 ChIP-seq data from 42 clinical samples of prostate patients,and analyzed all SNP sites present in the HOXB13 chromatin binding peak.Then we evaluated the differences between alleles for binding ability to HOXB13 to screen mutation sites that can affect HOXB13 binding.Furthermore,combined with the RNA-seq data of HOXB13 knockdown cells,4814 potential HOXB13-mediated gene regulatory SNPs were obtained based on changes in adjacent genes or interacting genes.Subsequently,the regulatory activities of 9628 alleles of these 4814 SNPs were evaluated through massive parallel reporter analysis(MPRA)experiments,and 190 SNP sites with regulatory activity and allelic differences were obtained.These sites may play an important role in the occurrence of the disease by affecting the regulatory reprogramming of the HOXB13 gene in prostate cancer.In addition,42.6%of the SNVs among these 190 mutation sites have a mutation frequency of less than 1%in the population.Therefore,these variants with gene regulatory functions discovered in our research work can complement the risk sites discovered by GWAS analysis,laying a good foundation for the comprehensive discovery and elucidation of the molecular mechanisms of cancer genetic susceptibility risks.Genetic variation plays an important role in human disease risk.It is of great significance to interpret the function of genetic variation and elucidate its pathogenic mechanism.Gene editing is commonly used to construct genetic variation in research.At present,CRISPR/Cas9 technology has been widely used for targeted gene editing in various organisms.However,how to accurately detect genetic variations introduced by gene editing is still a problem that needs to be solved.This project developed a rapid and accurate method for evaluating gene editing based on qPCR technology,called getPCR(genome editing test PCR).This method takes advantage of the sensitivity of Taq DNA polymerase to nucleotide mismatches at the 3’end of the primer during the initiation of DNA replication.It uses a primer that spans the Cas9 cleavage site with 3-5 3’ end bases to sense and discriminate the indel sequences.By this,it can selectively amplify and quantify the proportion of wild-type sequences,thereby achieving a quantitative assessment of gene editing.This method can not only accurately detect random indel mutations introduced by NHEJ,but also can accurately quantify the base variations introduced by HDR or base editor methods using primers matching the anticipated target sequences.They have good application potential in gene editing variation evaluation and genotyping of cell clones.In order to improve the ability of indel-sensitive primers to distinguish edited sequences in getPCR,we improved the specificity of the Taq polymerase through semi-rational molecular evolution and obtained a highly specific Taq with three amino acid substitutions of S577A,W645R and 1707V.When this highly specific Taq DNA polymerase is used for getPCR analysis,the specificity for detecting indel or single-base variations is greatly improved,completely eliminating amplification signals from mismatched templates,and greatly improving the accuracy of quantification.Furthermore,we combined the advantages of digital PCR(dPCR)in absolute quantification of gene detection to detect gene editing and genetic variation through the getPCR method on the dPCR platform.We found that compared with the existing GEF-dPCR method that uses fluorescent probes to distinguish between gene-edited sequences and wild-type sequences,our get-dPCR method based on highly specific Taq DNA polymerase has a stronger ability to identify genetic variants.In the detection of indel and single base variations,this method can completely distinguish between wild sequences and mutated sequences.This not only allows for accurate detection,but also solves the problem of non-specific signals of fluorescent probes from mismatched templates,also known as "raindrops" problem in the GEF-dPCR method.This demonstrates the superior accuracy of our get-dPCR method in assessing both gene editing and single-base DNA variation.