Molecular classification of renal cell carcinoma subtypes using microRNA signatures

Background: Renal cell carcinoma (RCC) is composed of various morphologically and cytogenetically distinct subtypes, the most prevalent of which are clear cell RCC (ccRCC, 75-80%), papillary RCC (papRCC, 10-15%) and chromophobe RCC (chRCC, 5%). Upper urinary tract urothelial cell carcinomas (UUT-UCCs) are uncommon and account for only 5-10% of urothelial carcinomas. Distinguishing between the subtypes is usually made by morphologic assessment, which is not always accurate.

Objective: Our aim was to identify microRNA (miRNA) signatures that can distinguish the different RCC subtypes accurately.

Methodology: A total of 27 different subtype cases were analyzed. MiRNA microarray analysis was performed on FFPE tissues of three common RCC subtypes (18 ccRCCs, 3 chRCC and 5 papRCC), on 1 UUT-UCC and were compared to 20 normal tissue samples. Results were validated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis with miRNA specific primers. Microarray data were analyzed by standard approaches. Relative expression for qRT-PCR was determined using the ΔΔCt method. Experiments were done in triplicate and an average was calculated. Fold change was expressed as a log2 value.

Results: We identified 434 miRNAs that were significantly deregulated (DE) in all kidney tumours compared to the normal tissues. A total of 126 miRNAs (29%) had increased expression while 303 (69.8%) had decreased expression in RCC. Out of these, 94 were co-up-regulated and 218 were co-down-regulated among chRCC, papRCC and ccRCC. Of these, 89 and 203 were co-up- and co-down-regulated between RCCs and UUT-UCCs, respectively. We detected 11, 44 and 24 up-regulated miRNAs, which were specific for ccRCC, chRCC and papRCC, respectively. Furthermore, 19, 18 and 8 miRNAs were uniquelly down-regulated in ccRCC, chRCC and papRCC, respectively. We also detected 89 and 203 co-up- and co-down-regulated miRNAs between kidney cancer and UUT-UCCs. Five miRNAs were up-regulated specifically in renal tumours and 49 in UUT-UCCs, whereas 15 miRNAs were down-regulated specifically in renal tumours and 89 in UUT-UCCs, respectively.

Discussion: We present novel deregulated miRNAs in RCC and we have built an accurate molecular classification method among its most prevalent RCC subtypes using microRNA signatures.