Differential Gene Expression and microRNA Profiling of Papillary Type 1 and 2 Renal Cell Carcinomas
Anil V Parwani, Somak Roy, Milon Amin, Rajiv Dhir, Sheldon Bastacky, William A LaFramboise. University of Pittsburgh, Pittsburgh, PA
Background: Papillary renal cell carcinoma (PRCC) is a distinct type of renal cell carcinoma with two distinct subtypes (Type 1 and Type 2). PRCC type 2 is associated with more aggressive clinicopathological features and a worse outcome. Our aim was to perform microRNA and mRNA expression analysis of the two subtypes of PRCC to evaluate differences in microRNA and gene expression profiles.
Design: Total RNA was purified from macrodissected specimens (n=15) using the Qiagen miRNeasy kit with spectrophotometric absorption: 260/280 >1.8 and RIN value >8.0. RNA was labeled with Hy3 for hybridization on miRCURY LNA arrays (Exiqon, Woburn, MA) for 18 hours followed by stringent wash and scan with probe readout classification against the Sanger miRBase (version 11.0). Message RNA underwent cDNA synthesis and in vitro transcription using the Ambion WT Expression assay (Ambion Inc, Austin, TX) followed by fragmentation and hybridization on Human Exon 1.0 ST arrays (Affymetrix Corp.,Santa Clara, CA) for 18hrs. Washing, staining and scanning of arrays was performed (Affymetrix Fluidics Station 450, Scanner 3000) after hybridization and signal intensity calculated by Microarray Suite version 5.0 (MAS 5.0). Statistical comparisons (ANOVA) were performed (Partek Genomics Suite, St. Louis, MO) with false discovery rate: (q value=0.1), -2.0>fold-change>2.0 and removal of comparisons within 5% of background signal intensity.
Results: A total of 86 (52 decreased, 34 up increased) and 210 (79 decreased, 131 increased) transcripts were significantly altered in PRCC type 1 and 2, respectively. Functional annotation revealed 7 tumor suppressor and 2 oncogenes in PRCC type 1 and 10 tumor suppressor and 8 oncogenes that demonstrated significantly altered mRNA expression. miRNA expression profiling showed 118 and 169 significant alterations in PRCC type 1 and 2, respectively. Of the 35 miRNAs with significant microRNA expression profile common to both tumors, 8 were expressed differentially in both tumors (miR-569, miR-22, miR-152, miR-105, miR-140-3p, let-7e, miR-26a, miR-30b, let-7a).
Conclusions: A significant number of transcripts were altered in PRCC types 1 and 2 with differential expression of both tumor suppressor and oncogenes. Similarly, unique microRNA expression profiles are detectable for these two subtypes. Differential gene expression and microRNA analysis can distinguish between PRCC subtypes. Further analysis of the functional pathways combined with targeted sequencing will offer new insights into neoplastic progression of PRCC with implications for targeted therapy.
Category: Genitourinary (including renal tumors)
Tuesday, March 5, 2013 9:30 AM
Poster Session III # 89, Tuesday Morning