Using state-of-the-art sequencing technology, we have quantified 463 genome-widely expressed miRNAs in ccRCC, normal kidney and ccRCC cell lines. Overall, the miRNome of ccRCC tumors resembled that of the ccRCC cell lines. Quantitative differential expression analysis allowed the identification of a 100-miRNA signature distinguishing ccRCC from normal kidney. We identified 21 miRNAs discriminating tumors of favorable prognosis patients (non-recurrent) from those with a less favorable prognosis of which patients with metastatic disease at time of diagnosis had the worst prognosis and all died within 1.5 years from RCC; four other miRNAs seemed associated with disease-specific death. Quantitative deep-sequencing technology allowed the discovery of novel miRNAs which have not been reported before in any species and were shown to be present in normal kidney tissue and tumors.
Of the most abundantly expressed miRNAs, miR-21, miR-451, miR-125a and miR-204 together contributed to 27% and 18% of total miRNAs in ccRCC and normal kidney tissue. MiR-21, the most abundantly expressed miRNA accounting for 14% of the total miRNA in ccRCCs and 5% in normal kidney, has been found to be overexpressed in many human cancer and to act as an oncogene by targeting tumor suppressor PTEN in various cancers . Of interest, the highly overexpressed MiR-451 and miRNA-27a have been reported to regulate multi-drug resistance (MDR) in carcinoma cell lines , and since RCC often express the MDR phenotype these two miRNAs might indeed be important in contributing to the RCC phenotype.
One hundred of 463 (21%) genome-widely expressed miRNAs showed significant expression changes in RCC. Recently, dysregulation of miRNAs in ccRCC has been reported [9–17]. Of these studies, 7 studies made use of array-based platforms, one of a miRNA PCR-primer array platform  whereas only Weng et al  applied deep sequencing technology to assess whether expression profiles in 3 ccRCC differed between stored frozen versus fixed samples. The latter investigators found that formalin-fixed paraffin-embedded tissue could be used to profile small RNAs. Mostly, small numbers of ccRCC tumors were used for expression profiling. Some studies used normal kidney as control tissue, other studies compared miRNA expression profiles between ccRCC and different kidney tumor histologies.
Our data confirm the ccRCC miRNAs findings reported in the literature [9–17] () but there are also discrepancies with regard to reported dysregulated miRNAs in other studies and the dysregulated miRNAs found by us utilizing deep sequencing technology. For instance, Nakada et al  have reported 43 miRNAs differentially expressed in ccRCC and 15 miRNAs were also found by us. Chow et al  reported 80 differentially expressed miRNAs of which only 15 overlapped with our 100 miRNAs. Despite the large extent of overlapping results, the discrepancies between (combined) results from earlier array studies and our current deep sequencing results may arise from differences in sample preparation, different technologies and the quantitative accuracy of the applied deep sequencing technology. However, the direction of the differential expression of miRNAs found by us and others did not differ except for the fact that one study did not report the direction for all of the dysregulated miRNAs. Moreover, in our study a highly stringent selection criterion of ≥95% tumor cell content was used for miRNA library construction which may further enhanced the sensitivity of miRNA detection.
A multitude of studies have investigated the mRNA targets of one single miRNAs and the comprehensive, publicly available miRecord database enabled us to identify the various currently validated targets of the list of differentially expressed miRNAs as identified by us. Using DAVID database enabled us in silico prediction of the combined sets of upregulated or downregulated miRNAs and their potential target networks of genes and signaling pathways. Target and pathway prediction suggested involvement of the deregulated miRNAs in a variety of biological processes involved in malignant behavior of cells and in the transformation of normal cells to malignant cells. To understand functional implications of the target genes, enriched biological pathways predicted according to DAVID bio-informatics database were identified. Interestingly, the top enriched pathways are apoptosis and transcriptions. The deregulated miRNAs and their top enriched pathways were in particular involved in apoptosis and transcription suggesting that these deregulated miRNAs may indeed play roles in a variety of biological processes involved in malignant behavior of cells and in the transformation of normal cells to malignant cells. Based on this integrative approach, our data provide an important platform for future investigations aiming at characterizing the role of specific miRNAs in ccRCC pathogenesis.
We tested whether upregulated miRNAs could be efficiently knocked down by using antagomirs and this was affirmed. The second question was, whether knocking down of upregulated miRNAs would affect proliferation and/or apoptosis in one RCC cell line used. Both miR-210 and miR-27a were linked to predicted target genes which are of interest with regard to cell proliferation and apoptosis (see ).
Our exploratory experiments showed that proliferation and apoptosis of SKRC7 cells were not affected by knocking down miR-210 or miR-27a following transfection with specific antagomirs. Data in other tumor types suggested that miR-210 is indeed involved in proliferation. Interestingly, upregulation of miR-210 was also reported in other malignancies, including breast and head and neck cancer, and found to correlate with prognosis [22,23], although in some studies downregulation of miR-210 was found in cancer, e.g. breast cancer in comparison with benign breast epithelium and esophageal cancer . Expression of miR-210 might merely reflect the hypoxia status known to be typically present in ccRCC and serve as a surrogate marker for tumor hypoxia because miR-210 is the most robustly induced miRNA under hypoxia [18,22,23]. This might explain the lack of effect on proliferation or apoptosis in our cell line miR-210 knock down experiments, but differences in miR-210 target expression of FGFRL1 might be another explanation.
Two other studies suggested that miR-27a may be involved in the development of tumor drug resistance  and we were interested in miR-27a because of its involvement in MDR/P-glycoprotein expression in cancer cells, a typical characteristic of many RCCs.
The lack of effect on proliferation and/or apoptosis after knocking down miR-210 and miR-27a in one ccRCC cell line may indicate that the effect of these miRNAs on the endpoints chosen do not apply to ccRCC and/or to this particular cell line only, or that the conditions to affect proliferation and/or apoptosis require a more intricate interplay of more factors. Discrepancies between individual cancer cell lines and tumors may be determined by cell-specific differences in expression levels of miRNA target genes or other cellular c.q. exogenous factors.
Of the dysregulated miRNAs miR-122 showed the largest fold-change in expression level in RCCs. Although expression level of miR-122 is much lower than that of miR-21, the clear cut expression change in RCC suggests that it may serve as a novel biomarker to distinguish tumor from normal kidney tissue. Notably, 5 downregulated miRNAs miR-532 and miR-362, miR-500, miR-501, miR-502 are clustered and together encoded in one intron of a renal specific gene voltage-gated chloride ion channel CLCN5, and have been reported to be downregulated in ccRCC .
Differentially expressed miRNAs as we found in our limited subgroup of metastatic RCC not only serve as potential prognostic markers, but also even as possible therapeutic targets. Of the 12 miRNAs which discriminated between non-recurrent and metastatic prognostic sub-groups, miR-221/222 has been reported in many other types of human cancers including prostate carcinoma . MiR-130b has been suggested to regulate expression of the tumor suppressor gene RUNX3  whereasmiR-146a has been shown to play an important role in oncogenic transformation of immune cells in mice model .
The downregulated miR-204, which has been found to be also dysregulated head and neck cancer , displayed very high expression in NRCs with expression level of on average 5.8% (range 1.1 to 8.2%), of the total miRNAs, but gradually decreased following the sub-groups order of no recurrence, recurrence and metastatic sub-groups. Of interest, miR-204, located on chromosome 9 in intron 6 of the potential Ca2+ channel TRPM  may proof to be a robust classifier if validated in an independent set of ccRCC/NRC samples.
A key advantage of deep sequencing is that it is a powerful tool allowing the discovery of novel miRNAs that cannot be detected using array-based technology. The use of our deep sequencing data and two additional pipelines allowed discovery of 21 novel miRNAs which generated perfect secondary structures indicating that deep sequencing is indeed a powerful tool to identify novel miRNAs of as yet unknown function. Future studies are warranted to proof that they are bonafide miRNAs.
Characterization of the miRNome of clear cell renal cell carcinomas by deep sequencing enabled to precisely quantify expression levels of miRNA and identify dysregulated miRNAs in RCC that may serve as novel diagnostic marker. Several of the differentially expressed miRNAs potentially target networks of genes and signaling pathways that may be involved in the malignant transformation of normal kidney cells and pathophysiology of ccRCC. Our data provide an important platform for future investigations aimed at characterizing the role of specific miRNAs in ccRCC pathogenesis.