Alternative Polyadenylation Of BID In Esophageal Squamous Cell Carcinoma

Background&Aims:The majority of human genes have multiple polyadenylation sites(PASs),which are differentially utilized through the process of alternative polyadenylation(APA).Dysregulation of APA contributes to numerous diseases,including cancer,but specific genes subject to APA that impact oncogenesis have not been well-characterized and many cancer APA landscapes remain underexplored.This study was to describe the APA landscape in esophageal squamous-cell carcinoma(ESCC)and investigate the functions of APA-regulated genes and their potential roles as prognostic indicators for ESCC.Methods:We used Dynamic Analyses of APA from RNA-seq(DaPars)to define both the 3'UTR APA profile in ESCC and to identify 3'UTR shortening events that may drive tumor progression.PAS was confirmed by 3'-rapid-amplification of cDNA ends(3'-RACE).Dual-luciferase reporter assays were utilized to study the relationship between 3'UTR length and expression.RNA level expression was detected by quantitative real-time PCR(qRT-PCR)and protein level was quantified by western blot or immunohistochemistry(IHC)assays.We used CRISPR/Cas9 genome-editing to delete the extended region of 3'UTR of candidate gene.Effect of 3'UTR shortening on cell proliferation was detected by CCK-8 reagent and colony formation assays in vitro and by xenograft tumor formation assays in vivo.Kaplan-Meier analysis and log-rank test were used to compare overall survival and hazard ratios(HRs)were calculated using Cox models.Results:We observed that the BID 3'UTR is recurrently shortened and that BID mRNA levels are significantly higher in four distinct squamous cell carcinoma datasets.Moreover,system correlation analysis reveals that CstF64 is a candidate upstream regulator of BID 3'UTR length.Mechanistically,we provide evidence that a shortened BID 3'UTR promotes proliferation of ESCC cells by disrupting ceRNA crosstalk resulting in downregulation of the tumor suppressor gene ZFP36L2.Finally,our in vitro and in vivo data is supported by human patient data whereby short 3'UTR of BID(HR,2.07;95%CI,1.10-3.88;P=0.024)and low expression of ZFP36L2(HR,1.91;95%CI,1.14-3.19;P=0.014)are prognostic of survival in ESCC.Conclusions:These findings identify BID as a recurrent 3'UTR shortening event in squamous cell carcinoma.CstF64 is upregulated in ESCC and is an upstream regulator of BID APA.BID 3'UTR shortening is beneficial to ESCC because of disrupted ceRNA crosstalk with the tumor suppressor ZFP36L2.In addition,BID 3'UTR shortening and ZFP36L2 downregulation are two prognostic indicators of ESCC patients.