Real-time quantitative PCR analysis of Wnt signalling in human prostate cancer

ABSTRACT: A comprehensive expression analysis of Wnt signaling genes was performed in a panel of prostate cancer cell lines and tissue specimens using TaqMan low density arrays. The effect of DNA methylation on gene expression was investigated using DNMT inhibitor 5-Aza-CdR. Tissue specimens from a range of disease states were used to represent the stepwise progression of prostate cancer, including benign prostatic hyperplasia (BPH), histologically benign epithelium adjacent to tumor (HB), pre-invasive high-grade prostatic intraepithelial neoplasia (HGPIN) and primary localized tumors categorized into low- and high-grade disease. Fifteen Wnt signaling related genes were idenified with significantly altered expression in prostate cancer; the majority of which were upregulated in tumors. Notably, histologically benign tissue from men with prostate cancer appeared more similar to tumour (r=0.76) than to BPH (r=0.57) (P<0.001). Overall, the expression profile was highly similar between tumors of high (M-bM-^IM-%7) and low (M-bM-^IM-$6) Gleason scores. Pharmacological demethylation of PC-3 cells reactivated 38 genes (M-bM-^IM-%2-fold); 40% of which inhibit Wnt signaling. qPCR gene expression profiling using TLDA Human Wnt Gene set v1.0 microfluidic card (Applied Biosystems, Foster City, CA).The TLDA consisted of 4 identical 96-gene sets preconfigured in a 384-well format. Three different malignant cell lines were profiled LNCaP, 22Rv1 and PC3 (+/- 5-Aza-2M-bM-^@M-^YDeoxycytidine). One benign cell line was included for comparative puposes: PWR1E. Patient samples were obtained from FFPE tissue sections from men who underwent radical prostatectomy or transrectal resection of the prostate. To overcome the limited amount of RNA obtained from FFPE tissues, RNA samples were pooled. Five different pools were generated: high grade prostate cancer (Gleason score M-bM-^IM-%7), low grade prostate cancer (Gleason score M-bM-^IM-$6), HGPIN, HB and BPH. Each pool consisted of DNase-treated total RNA (100ng), isolated from microdissected tissue from 4 individual cases, selected on the basis of similar histological and clinical features and previous epigenetic characterization in our laboratory. Two Biological replicates for each pool were prepared. The high capacity cDNA Archive Kit (Applied Biosystems) was used to reverse transcribe pooled RNA (400ng). TaqManM-BM-. reactions were performed in duplicate on a 7900HT Sequence Detection System. RQ data from TLDAs were analyzed using Real Time StatMinerM-BM-. v3.1 (Integromics, Granada, Spain).

ORGANISM(S): Homo sapiens

SUBMITTER: Antoinette Perry 

PROVIDER: E-GEOD-33557 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress