Transcriptional Regulation Mechanism Of Dead-box Polypeptide 5 On CCAT2

Objectives In the prostate cancer cell line DU145,the transcriptional regulation mechanism of DDX5(Dead-box polypeptide 5)on long non-coding RNA CCAT2 was explored by detecting the effect of DDX5 on the activity of G-allelic and T-allelic promoters of CCAT2.Methods RNA was extracted from cultured DU145 cells and reverse-transcribed into cDNA.Primers were designed for DDX5 coding sequence.The target fragment was then inserted into pGM-T vector.Agarose gel electrophoresis was used to verify whether it was successful or not.The construction was sequenced and aligned with NCBI databases.The target fragment digested from pGMT vector was subcloned into pcDNA6-flag vector.The pcDNA6-DDX5 expression plasmid was transfected into DU145 cells.The mRNA and protein level of DDX5 was detected by qPCR and Western blot respectively.DU145 cells were co-transfected with reporter gene driving by CCAT2 promoter fragment and pcDNA6-DDX5 plasmids.The Dual-Luciferase?Reporter Assay System was used to explore the relative promoter activity.CCAT2 RNA expression was detected by qPCR.The siRNA designed for DDX5 was verified by qPCR and Western blot.The effect of siDDX5 on CCAT2 promoters was analyzed by using Dual-Luciferase?Reporter Assay System.The pGL3-153Ts-Rluc plasmid was constructed.The effect of DDX5 on CCAT2 G/T promoter activity ratio was measured by Dual-Luciferase?Reporter Assay System.Results The eukaryotic expression plasmid of pcDNA6-DDX5 was successfully constructed.The pcDNA6-DDX5 plasmid was transfected into DU145 cells.The results of qPCR and Western blot showed that,the mRNA and protein expression level of DDX5 both increased.The results of Dual-Luciferase?Reporter Assay System showed that,the relative activity of G-allelic promoter of CCAT2 increased and T-allelic promoter of CCAT2 decreased under overexpression of DDX5(P<0.05).The results of qPCR showed that,the relative expression of total CCAT2 RNA and G-type CCAT2 RNA increased under overexpression of DDX5(P<0.05).The relative expression of U-type CCAT2 RNA decreased(P<0.05).The results of Dual-Luciferase?Reporter Assay System showed that,both of the relative activity of CCAT2 G-allelic promoter and T-allelic promoter decreased under the knockdown of DDX5(P < 0.05).The results of DualLuciferase?Reporter Assay System showed that,the CCAT2 G/T promoter activity ratio increased under overexpression of DDX5(P < 0.05),wheras the CCAT2 G/T promoter activity ratio decreased under the knockdown of DDX5(P < 0.05).Conclusions DDX5 could activate CCAT2 G-allelic promoter activity in DU145 cell line.Figure12;Table8;Reference137...