The Transcriptional Regulation Mechanism Of CCAT2 In Prostate Cancer Cell Lines

Objectives In the prostate cancer cell line DU145,the transcriptional regulation of long non-coding gene CCAT2 was explored by detecting the promoter activity of 153 bp segments encompassing the G/T SNP site and in which the especial transcription factors could bind.Methods Construction of plasmids were based on the pGL3-Basic-LUC vector and the pGL3-Promoter-LUC vector.The 153 bp CCAT2 gene promoter fragment was inserted into the reporter gene vector.All of those were named as pGL3-Basic-153Gs-LUC,pGL3-Basic-153Ts-LUC,pGL3-Promoter-153Ts-LUC,pGL3-Promoter-153Gs-LUC,pGL3-Promoter-TRANS-153Ts-LUC and pGL3-Promoter-TRANS-153Gs-LUC,respectively.The reporter gene activities driven by G/T-type CCAT2 promoter functional elements were measured 24 hours post-transfection via the Dual-Luciferase? Reporter Assay System.A candidate transcription factor DDX5 expression was screened by western-Blot.We hypothesized that DDX5 could bind CCAT2 promoter regions.To test this hypothesis,we performed chromatin immunoprecipitation(ChIP)-quantitative PCR(qPCR)assays to measure the binding activity.Results After 24 hours of transfection,the reporter activities were measured driven by the series of 153 bp fragment in the prostate cancer cell line DU145 cells.Comparing with the relative reporter activity of the pGL3-Basic-153Gs-LUC transfected group,G/T 153 bp CCAT2 fragments were both effective at driving reporter gene(P <0.05,respectively),and T allele fragment was shown significantly higher relative activity than T form(P <0.05),whereas there was no statistical difference in the relative activity of T allele CCAT2 promoter and SV40 promoter.Based on the pGL3-Promoter-LUC reporter gene plasmid vector,the relative reporter activity after the insertion of the G/T 153 bp region following SV40 promoter were increased(P<0.05,respectively),and the increasing level driven by Tallele fragment was higher than that of the G-type as well(P<0.05).However,there was no statistical difference in the relative activity of the reporter gene driven by both the reversed insertion of the Gs/Ts 153 bp promoter functional segment and SV40 promoter comparing with SV40 promoter only.Western-Blot experiments showed that DDX5 protein was expressed in DU145 and LNCaP cell lines.The ChIP qPCR assay results showed that the DDX5 protein binds to the promoter region in CCAT2 and that the T-type promoter has a higher binding activity in the DU145 cell line(P<0.05).Conclusions In the prostate cancer DU145 cell line,a 153 bp fragment of the CCAT2 promoter encompassing rs6983267 SNP site has promoter activity.The T allele promoter activity is higher than that of the G-form.This difference might be related to the differential binding activity with DDX5.