Transcriptional Regulation Between CCAT2 And AR In Prostate Cancer Cells

Objective To study the transcriptional regulation between long chain non-coding RNA CCAT2 and androgen receptor(AR)in prostate cancer cell line and explore the regulation mechanism between them.Methods Prostate cancer cell lines DU145,LNCa P and PC3 were cultured.The c DNA primers of AR,CCAT2 and DDX5 gene were designed and amplificated by high fidelity PCR.The PCR fragments were then subcloned into pc DNA3.1 expression vector and were transfected into cells by Lipofectamine 3000.The total RNA was extracted,and the abundance of CCAT2,AR and other related genes were detected by q PCR.AR promoter primers were designed.Using DU145 cell genome as template,the DNA fragment(-2334～ +719)of AR gene promoter was amplified by high fidelity enzyme.The luciferase reporter gene vectors driven by AR promoter fragment was constructed by Hind III and Xho I sites.The reporter gene driven by full-length and seriers of the AR promoter truncation were cotransfected into cells with expression plasmids CCAT2 and DDX5.The relative promoter activity of AR was detected by reporter gene analysis.DDX5 gene was mutated by site directed mutagenesis,and the effect of DDX5 on AR transcription was analyzed by q PCR and reporter gene analysis.The binding activity of DDX5 in AR promoter was analyzed by chromatin immunoprecipitation technique.Results The plasmids of reporter gene driven by DNA fragment of AR promotor and CCAT2 expression plasmids were successfully constructed.Real time PCR results showed that G-CCAT2 inhibited AR m RNA levels to 0.43 folds in PC3 cells(P<0.05).In DU145 cells and LNCa P cells,G-CCAT2 activated AR m RNA by 2.4 and 1.5 folds respectively(P< 0.05).The results of luciferase reporter gene showed that G-CCAT2 inhibited the relative promoter activity of AR to 0.52 times in PC3 cells(P<0.05).In DU145 and LNCa P cells,GCCAT2 activated the relative promoter activity of AR by 3.7 folds and 12.5 folds respectively(P<0.05).The effect of CCAT2 on AR promoter activity reversed by deletting AR promoter sequence from-1779 to-1448.The results of chromatin immunoprecipitation showed that DDX5 had binding activity in AR promoter.The results of luciferase reporter gene showed that DDX5 could inhibite the relative activity of AR promoter by 3.4 times(P<0.05)and DN-DDX5 activate the relative activity of AR promoter by 5.6 times(P<0.05)Conclusion CCAT2 could regulate AR transcription activity in prostate cancer cells with the response region of-1779 to-1448.DDX5 could function and its helicase activity may play important role in the regulation of CCAT2 on AR transcription in PC3 Cells.Figure18;Table 9;Reference 123...