Systematic Identification And Functional Clarification Of Regulatory Genetic Variants Associated With The Prognosis Of Pancreatic Cancer

Objective:Pancreatic cancer(PC)is a malignant tumor with extremely poor prognosis.It is of great importance to identify prognostic molecular markers for carrying out targeted follow-up and precise treatment and thus improving the prognosis of pancreatic cancer.With the continuous development and widespread application of high-throughput sequencing technology and microarray technology,researchers have conducted many association studies between gene expression and the prognosis of pancreatic cancer.However,most of the previous studies were carried out with small sample size,and there may be great heterogeneity between these studies.Therefore,it was necessary to conduct a meta-analysis on the published results of the associations between gene expression and the prognosis of pancreatic cancer to identify genes with prognostic significance in pancreatic cancer.Furthermore,bioinformatic analysis and population association studies were needed to identify functional genetic variants regulating these prognostic genes.Finally,biological function experiments should be carried out to confirm the underlying mechanisms behind these associations.This study aimed to systematically identify prognostic genetic variants with potential clinical application prospects in pancreatic cancer and explore the underlying biological mechanism.Methods:Firstly,a meta-analysis on 7 published independent datasets was performed to discover genes associated with the prognosis of pancreatic cancer.Subsequently,Expression Quantitative Trait Loci(e QTL)analysis was used to identify the genetic variants regulating the m RNA expression of these prognostic genes.A two-stage population association study in a total of 893 patients with pancreatic cancer was then carried out to analyze the association between the candidate genetic variants and the prognosis of pancreatic cancer.A series of bioinformatic tools such as Haploview,Regulome DB,Haploreg and Cistrome were then utilized to perform functional annotation on candidate genetic variant rs4887783 and the SNPs in strong linkage disequilibrium(LD)(r2?0.8)with it to identify the potential functional SNP.Dual-luciferase reporter gene assay was conducted to explore the effect of rs4887783 alleles on the promoter activity of RFWD3 gene.Electrophoretic mobility shift assay and super-shift assay were performed to explore how rs4887783 SNP affected the binding affinity of transcription factors to the promoter region of RFWD3.Finally,Cell Counting Kit-8(CCK-8)and Transwell assays were carried out to analys the impacts of RFWD3 gene expression changes on proliferation and migration abilities of pancreatic cancer cells.Result:A total of 128 genes significantly associated with the prognosis of pancreatic cancer were preliminarily found at the threshold of q-value<0.001 in our meta anylysis.Interestingly,higher expression of RFWD3 was significantly associated with poor prognosis of pancreatic cancer with a hazard ratio of 1.50(95%Confidence Interval,CI:1.25-1.79,q-value=6.90×10^-4).Moreover,data from The Cancer Genome Atlas(TCGA)database showed that the SNP rs4887783 located in the 5'-Untranslated Region(UTR)of RFWD3 was a significant cis-e QTL of RFWD3 in pancreatic cancer.A two-stage population association study showed that rs4887783 genotypes were associated with the prognosis of pancreatic cancer,the G allele increased the risk of death,with a hazard ratio of 1.27(95%CI:1.12-1.43,P_adjust=0.0001).Bioinformatic functional annotation indicated that the rs4887783 SNP may be a potential functional SNP.Dual-luciferase reporter gene assay showed that rs4887783 SNP exhibited allele-specific regulatory activity.Electrophoretic mobility shift assay and super-shift assay showed that rs4887783-G allele had stronger specific binding ability to the REST transcription factor.Transient overexpression or silencing of RFWD3 did not affect the proliferation of pancreatic cancer cells,but affected the migration ability of pancreatic cancer cells.Conclusions:Through the comprehensive combination of meta-analysis,bioinformatic analyses,population association studies and biological function experiments,we found that rs4887783 SNP affected the prognosis of pancreatic cancer through regulating the binding affinity of REST transcription factor which affected RFWD3 gene transcription and the migration ability of pancreatic cancer cells.This genetic variant has potential clinical application prospects,it provides insights and clues for precise medicine and individualized treatment of pancreatic cancer.