Genome-Wide MicroRNA Expression Analysis of Clear Cell Renal Cell Carcinoma by Next Generation Deep Sequencing

Abstract

MicroRNAs (miRNAs), non-coding RNAs regulating gene expression, are frequently aberrantly expressed in human cancers. Next-generation deep sequencing technology enables genome-wide expression profiling of known miRNAs and discovery of novel miRNAs at unprecedented quantitative and qualitative accuracy. Deep sequencing was performed on 11 fresh frozen clear cell renal cell carcinoma (ccRCC) and adjacent non-tumoral renal cortex (NRC) pairs, 11 additional frozen ccRCC tissues, and 2 ccRCC cell lines (n = 35). The 22 ccRCCs patients belonged to 3 prognostic sub-groups, i.e. those without disease recurrence, with recurrence and with metastatic disease at diagnosis. Thirty-two consecutive samples (16 ccRCC/NRC pairs) were used for stem-loop PCR validation. Novel miRNAs were predicted using 2 distinct bioinformatic pipelines. In total, 463 known miRNAs (expression frequency 1–150,000/million) were identified. We found that 100 miRNA were significantly differentially expressed between ccRCC and NRC. Differential expression of 5 miRNAs was confirmed by stem-loop PCR in the 32 ccRCC/NRC samples. With respect to RCC subgroups, 5 miRNAs discriminated between non-recurrent versus recurrent and metastatic disease, whereas 12 uniquely distinguished non-recurrent versus metastatic disease. Blocking overexpressed miR-210 or miR-27a in cell line SKCR-7 by transfecting specific antagomirs did not result in significant changes in proliferation or apoptosis. Twenty-three previously unknown miRNAs were predicted in silico. Quantitative genome-wide miRNA profiling accurately separated ccRCC from (benign) NRC. Individual differentially expressed miRNAs may potentially serve as diagnostic or prognostic markers or future therapeutic targets in ccRCC. The biological relevance of candidate novel miRNAs is unknown at present.

The epigenetic landscape of renal cancer

“…Of note, more mi RNAs were downregulated or silenced in renal tumours than in normal tissue, and this downregulation or silencing [Au: OK?] was often associated with promoter methylation or copy number changes 179,[182][183][184][185][186][187][188] . Many of these mi RNAs target components of networks that are dysregulated by mutations or promoter methylation.…”

MicroRNA-122 Promotes Proliferation, Invasion and Migration of Renal Cell Carcinoma Cells Through the PI3K/Akt Signaling Pathway

“…Recently numerous studies have shown aberrant expression of microRNA-122-5p (miR-122) in human cancer tissues, including RCC (Zhou et al, 2010;Heinzelmann et al, 2011;Osanto et al, 2012), suggesting that it is a candidate tumor activator in RCC. miR-122 is one of the most frequent miRNA isolated in the liver and plays important roles in many aspects of liver physiology, such as stress response (Bhattacharyya et al, 2006) and lipid metabolism (Esau et al, 2006).…”

Identification of miR-125a-5p as a tumor suppressor of renal cell carcinoma, regulating cellular proliferation, migration and apoptosis

“…However, the function and clinical significance of miR-125a-5p in RCC has yet to be explored (15). To determine whether miR-125a-5p is important for the tumorigenesis of RCC, human renal cancer and paired normal tissues were collected and compared for miR-125a-5p expression levels, using quantitative polymerase chain reaction (qPCR).…”