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

Department of Clinical Oncology, Leiden University Medical Center, Leiden, The Netherlands.
PLoS ONE (Impact Factor: 3.23). 06/2012; 7(6):e38298. DOI: 10.1371/journal.pone.0038298
Source: PubMed


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.

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Available from: Jelle J Goeman
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    • "Several microarray based studies have demonstrated 21 to 34 differentially expressed miRNAs between ccRCC and normal kidney tissue [11]. SRNA-Seq studies reported more than 100 differentially regulated miRNAs, some of which might serve as diagnostic and prognostic markers [12] [13]. Nevertheless, these studies lack detailed information about miRNA targets and bioinformatical analysis is often only focused on miRNAs currently known to miRbase. "
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    ABSTRACT: Altered microRNA (miRNA) expression is a hallmark of many cancer types. The combined analysis of miRNA and messenger RNA (mRNA) expression profiles is crucial to identifying links between deregulated miRNAs and oncogenic pathways. Therefore, we investigated the small non-coding (snc) transcriptomes of nine clear cell renal cell carcinomas (ccRCCs) and adjacent normal tissues for alterations in miRNA expression using a publicly available small RNA-Sequencing (sRNA-Seq) raw-dataset. We constructed a network of deregulated miRNAs and a set of differentially expressed genes publicly available from an independent study to in silico determine miRNAs that contribute to clear cell renal cell carcinogenesis. From a total of 1,672 sncRNAs, 61 were differentially expressed across all ccRCC tissue samples. Several with known implications in ccRCC development, like the upregulated miR-21-5p, miR-142-5p, as well as the downregulated miR-106a-5p, miR-135a-5p, or miR-206. Additionally, novel promising candidates like miR-3065, which i.a. targets NRP2 and FLT1, were detected in this study. Interaction network analysis revealed pivotal roles for miR-106a-5p, whose loss might contribute to the upregulation of 49 target mRNAs, miR-135a-5p (32 targets), miR-206 (28 targets), miR-363-3p (22 targets), and miR-216b (13 targets). Among these targets are the angiogenesis, metastasis, and motility promoting oncogenes c-MET, VEGFA, NRP2, and FLT1, the latter two coding for VEGFA receptors.
    Full-text · Article · May 2014 · BioMed Research International
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    • "As indicated by the previous published datasets, miR-182-5p was in a down-regulated expression pattern in RCC compared with normal renal tissue [29,30]. To further validate the expression pattern of miR-182-5p in RCC, we quantified the expression levels of miR-182-5p in 25 pairs of human RCC tissues and adjacent non-tumor tissues by qRT-PCR (Figure 1A). "
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    ABSTRACT: Background Emerging evidence has suggested that dysregulation of miR-182-5p may contribute to tumor development and progression in several types of human cancers. However, its role in renal cell carcinoma (RCC) is still unknown. Methods Quantitative RT-PCR was used to quantify miR-182-5p expression in RCC clinical tissues. Bisulfite sequencing PCR was used for DNA methylation analysis. The CCK-8, colony formation, flow cytometry, and a xenograft model were performed. Immunohistochemistry was conducted using the peroxidase and DAB methods. A miR-182-5p target was determined by luciferase reporter assays, quantitative RT-PCR, and Western blotting. Results miR-182-5p is frequently down-regulated in human RCC tissues. Epigenetic modulation may be involved in the regulation of miR-182-5p expression. Enforced expression of miR-182-5p in RCC cells significantly inhibited the proliferation and tumorigenicity in vitro and in vivo. Additionally, overexpression of miR-182-5p induced G1-phase arrest via inhibition of AKT/FOXO3a signaling. Moreover, FLOT1 was confirmed as a target of miR-182-5p. Silencing FLOT1 by small interfering RNAs phenocopied the effects of miR-182-5p overexpression, whereas restoration of FLOT1 in miR-182-5p -overexpressed RCC cells partly reversed the suppressive effects of miR-182-5p. Conclusions These findings highlight an important role for miR-182-5p in the pathogenesis of RCC, and restoration of miR-182-5p could be considered as a potential therapeutic strategy for RCC therapy.
    Full-text · Article · May 2014 · Molecular Cancer
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    • "They are aberrantly expressed or mutated in cancers, suggesting that they may play a role as a novel class of oncogenes or tumour suppressor genes [11]. Deregulated miRNAs have been found in many tumours, including kidney cancer and carcinomas of the urinary tract [5], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21]; however, not much is known regarding their expression profile in upper tract urothelial carcinomas. Furthermore, in RCC the results are not always consistent [12], [13], [17], [18], [22], [23], [24], [25]. "
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    ABSTRACT: Upper tract urothelial carcinomas (UT-UC) can invade the pelvicalyceal system making differential diagnosis of the various histologically distinct renal cell carcinoma (RCC) subtypes and UT-UC, difficult. Correct diagnosis is critical for determining appropriate surgery and post-surgical treatments. We aimed to identify microRNA (miRNA) signatures that can accurately distinguish the most prevalent RCC subtypes and UT-UC form the normal kidney. miRNA profiling was performed on FFPE tissue sections from RCC and UT-UC and normal kidney and 434 miRNAs were significantly deregulated in cancerous vs. the normal tissue. Hierarchical clustering distinguished UT-UCs from RCCs and classified the various RCC subtypes among them. qRT-PCR validated the deregulated expression profile for the majority of the miRNAs and ROC analysis revealed their capability to discriminate between tumour and normal kidney. An independent cohort of freshly frozen RCC and UT-UC samples was used to validate the deregulated miRNAs with the best discriminatory ability (AUC>0.8, p<0.001). Many of them were located within cytogenetic regions that were previously reported to be significantly aberrated. miRNA targets were predicted using the miRWalk algorithm and ingenuity pathway analysis identified the canonical pathways and curated networks of the deregulated miRNAs. Using the miRWalk algorithm, we further identified the top anti-correlated mRNA/miRNA pairs, between the deregulated miRNAs from our study and the top co-deregulated mRNAs among 5 independent ccRCC GEO datasets. The AB8/13 undifferentiated podocyte cells were used for functional assays using luciferase reporter constructs and the developmental transcription factor TFCP2L1 was proved to be a true target of miR-489, which was the second most upregulated miRNA in ccRCC. We identified novel miRNAs specific for each RCC subtype and UT-UC, we investigated their putative targets, the networks and pathways in which they participate and we functionally verified the true targets of the top deregulated miRNAs.
    Full-text · Article · Mar 2014 · PLoS ONE
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