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PDF file - 107K, Significantly expressed genes in regions that are cytogenetically associated with copy-number aberrations in aberrant versus diploid tumors as based by GSEA and integration of protein data

PDF file - 28K, Broad regions of gains and losses in at least 5% of cases (n=154).

PDF file - 50K, Significantly expressed genes located on the 3p and 5q chromosomal arms among aberrant versus normal kidney and integration of protein and miRNA data in ccRCC

PDF file - 30K, Select genes significantly methylated in tumor versus normal kidney in regions associated with copy-number aberrations

PDF file - 34K, Significantly expressed proteins encoded by genes in regions that are cytogenetically associated with copy-number aberrations in aberrant versus diploid tumors

PDF file - 34K, microRNAs expressed concordantly with copy-number aberrations

PDF file - 74K, Copy-number results in relation to results of previous studies

Supplementary Methods from Multilevel Whole-Genome Analysis Reveals Candidate Biomarkers in Clear Cell Renal Cell Carcinoma

PurposeThe clinical implications of biallelic inactivation ofCDKN2A/Bin clear cell renal cell carcinoma (ccRCC) and relevant dysregulated biological pathways and gene signatures were investigated.Materials and MethodsData were obtained from the TCGA data set and validated using Project GENIE and previously published dataset.CDKN2A/Ballelic status was classified into 3 groups, including biallelicCDKN2A/Binactivation (homozygous deletion or combined heterozygous deletion and mutation), monoallelicCDKN2A/Bloss (heterozygous deletion or mutation) and absentCDKN2A/Ballelic loss. Univariate and multivariate cancer-specific survival and disease-free survival analyses were performed. Integrated analyses of copy number, gene expression (mRNA and miRNA), protein expression and methylation changes were conducted.ResultsOf 440 patients with ccRCC 17 (3.9%) had biallelicCDKN2A/Binactivation and 116 (26.4%) had monoallelicCDKN2A/Bloss.CDKN2A/Ballelic inactivation was associated with late tumor stage, high histological grade, presence of metastasis and greater tumor size. Patients with biallelic deletion ofCDKN2A/Bshowed significantly worse cancer-specific survival and disease-free survival (pMTAPhomozygous deletion was observed inCDKN2A/Bbiallic inactivated tumors (46.7%; pCDKN2A/Bwas observed in biallelic inactivated tumors at the mRNA and protein levels. miR-21 was the most highly expressed miRNA in biallelic inactivated tumors. Biallelic inactivated tumors were significantly enriched for genes related to activation ofATRin response to replication stress and miR-21 target genes.ConclusionsCDKN2A/Bbiallelic inactivation may be a prognostic marker for ccRCC and is associated with distinct dysregulation of gene expression signatures.

Renal cell carcinoma (RCC) is the most common neoplasm of the kidney. We conducted an integrated analysis of copy number, gene expression (mRNA and miRNA), protein expression, and methylation changes in clear cell renal cell carcinoma (ccRCC). We used a stepwise approach to identify the most significant copy number aberrations (CNA) and identified regions of peak and broad copy number gain and loss, including peak gains (3q21, 5q32, 5q34-q35, 7p11, 7q21, 8q24, 11q13, and 12q14) and deletions (1p36, 2q34-q37, 3p25, 4q33-q35, 6q23-q27, and 9p21). These regions harbor novel tumor-related genes and miRNAs not previously reported in renal carcinoma. Integration of genome-wide expression data and gene set enrichment analysis revealed 75 gene sets significantly altered in tumors with CNAs compared with tumors without aberration. We also identified genes located in peak CNAs with concordant methylation changes (hypomethylated in copy number gains such as STC2 and CCND1 and hypermethylated in deletions such as CLCNKB, VHL, and CDKN2A/2B). For other genes, such as CA9, expression represents the net outcome of opposing forces (deletion and hypomethylation) that also significantly influences patient survival. We also validated the prognostic value of miRNA let-7i in RCCs. miR-138, located in chromosome 3p deletion, was also found to have suppressive effects on tumor proliferation and migration abilities. Our findings provide a significant advance in the delineation of the ccRCC genome by better defining the impact of CNAs in conjunction with methylation changes on the expression of cancer-related genes, miRNAs, and proteins and their influence on patient survival. Cancer Res; 72(20); 5273–84. ©2012 AACR.

Tumor microvascular density (MVD) has been shown to correlate with the aggressiveness of several cancers. With the introduction of targeted anti-angiogenic therapy, assessment of MVD has the potential not only as a prognostic but also as a therapeutic marker. The significance of tumor vascularity in clear cell renal cell carcinoma (ccRCC) has been debated, with studies showing contradictory results. Previous studies were limited by manual quantification of MVD within a small area of tumor. Since then, the validity of this method has been questioned. To avoid the inaccuracies of manual quantification, we employed a computerized image analysis, which allowed assessment of large areas of tumor and adjacent normal tissue. The latter was used as an internal reference for normalization. MVD and vascular endothelial growth factor (VEGF) were assessed in 57 cases of ccRCC. Sections were immunostained for CD34 and VEGF. Areas of ccRCC and normal kidney medulla were analyzed within scanned images using software that counted CD34-positive vessels and measured the intensity of VEGF staining. We obtained unadjusted values from tumoral areas and calculated adjusted values as tumor/normal ratios. Unadjusted MVD had no association with clinical outcome. However, similarly to tumor stage, higher adjusted MVD was associated with shorter disease-free survival (log-rank P=0.037, Cox P=0.02). This was significant in univariate and multivariate analyses. MVD did not correlate with tumor stage, pointing to its independent prognostic value. As expected due to the known molecular abnormalities in ccRCC, most tumors showed higher VEGF expression than normal tissue. Higher adjusted VEGF was associated with high tumor grade (P=0.049). The finding of increased MVD as an independent marker of tumor aggressiveness may prove useful in the development of new tests for prognostic and therapeutic guidance. Digital techniques can provide more accurate assessment of immunomarkers and may reveal less obvious associations.

Renal cell carcinoma is the most common neoplasm of the adult kidney. Currently to our knowledge there are no biomarkers for diagnostic, prognostic or predictive applications for renal cell carcinoma. miRNAs are nonprotein coding RNAs that negatively regulate gene expression and are potential biomarkers for cancer.We analyzed 70 matched pairs of clear cell renal cell carcinoma and normal kidney tissues from the same patients by microarray analysis and validated our results by quantitative real-time polymerase chain reaction. We also performed extensive bioinformatic analysis to explore the role and regulation of miRNAs in clear cell renal cell carcinoma.We identified 166 miRNAs that were significantly dysregulated in clear cell renal cell carcinoma, including miR-122, miR-155 and miR-210, which had the highest over expression, and miR-200c, miR-335 and miR-218, which were most down-regulated. Analysis of previously reported miRNAs dysregulated in RCC showed overall agreement in the direction of dysregulation. Extensive target prediction analysis revealed that many miRNAs were predicted to target genes involved in renal cell carcinoma pathogenesis. In renal cell carcinoma miRNA dysregulation can be attributed in part to chromosomal aberrations, co-regulation of miRNA clusters and co-expression with host genes. We also performed a preliminary analysis showing that miR-155 expression correlated with clear cell renal cell carcinoma size. This finding must be validated in a larger independent cohort.Analysis showed that miRNAs are dysregulated in clear cell renal cell carcinoma and may contribute to kidney cancer pathogenesis by targeting more than 1 key molecule. We identified mechanisms that may contribute to miRNA dysregulation in clear cell renal cell carcinoma. Dysregulated miRNAs represent potential biomarkers for kidney cancer.

Background: Kallikrein-related peptidases (KLKs) are a family of serine proteases that have been shown to be dysregulated in several malignancies including ovarian cancer. The control of kallikrein genes and their physiological function in cancer is not well understood. We hypothesized that microRNAs (miRNAs) represent a novel mechanism for post-transcriptional control of KLK expression in cancer. Methods: We first analysed miRNA expression in ovarian cancer in silico. A total of 98 miRNAs were reported to have altered expression in ovarian cancer. Three of these miRNAs were predicted to target KLK10. We experimentally verified the predicted miR–KLK10 interaction using two independent techniques, a luciferase assay with a construct containing the KLK10 3′ untranslated region (UTR), pMIR–KLK10, and measuring KLK10 protein levels after transfection with miRNA. Results: When we co-transfected cells with pMIR–KLK10 and either let-7f, miR-224, or mR-516a, we saw decreased luciferase signal, suggesting that these miRNAs can target KLK10. We then examined the effect of these three miRNAs on KLK10 protein expression and cell growth. Transfection of all miRNAs, let-7f, miR-224, and miR-516a led to a decrease in protein expression and cellular growth. This effect was shown to be dose dependent. The KLK10 protein levels were partially restored by co-transfecting let-7f and its inhibitor. In addition, there was a slight decrease in KLK10 mRNA expression after transfection with let-7f. Conclusion: Our results confirm that KLKs can be targeted by more than one miRNA. Increased expression of certain miRNAs in ovarian cancer can lead to decreased KLK protein expression and subsequently have a negative effect on cell proliferation. This dose-dependent effect suggests that a ‘tweaking' or ‘fine-tuning' mechanism exists in which the expression of one KLK can be controlled by multiple miRNAs. These data together suggest that miRNA may be used as potential therapeutic options and further studies are required.

Accurate assessment of prognosis of clear cell renal cell carcinoma (ccRCC) is key in optimizing management plans to fit individual patient needs. miRNAs are short noncoding single-stranded RNAs that control the expression of target genes and may act as cancer biomarkers. We analyzed the expression of miR-210 in 276 cases of primary ccRCC and compared its expression in 40 pairs of adjacent normal and cancerous tissues. We assessed its expression in primary and metastatic tumors, in the common RCC subtypes, and the benign oncocytoma. The results were validated with an independent data set from The Cancer Genome Atlas. miR-210 was significantly overexpressed in ccRCC compared with normal kidney. miR-210(+) patients had a statistically higher chance of disease recurrence [hazard ratio (HR), 1.82; P = 0.018] and shorter overall survival (HR, 2.46; P = 0.014). In multivariate analysis, miR-210 lost its statistically significant association with shorter disease-free survival and overall survival after adjusting for tumor size and tumor, node, metastasis stage. Papillary RCC showed comparable miR-210 overexpression, whereas decreased up-regulation was seen in chromophobe RCC and oncocytoma. A number of predicted targets that might be involved in carcinogenesis and aggressive tumor behavior were identified. miR-210 is a potential therapeutic target and independent marker of poor prognosis of ccRCC.

MicroRNAs (miRNAs) play a crucial role in tumor progression and metastasis. We, and others, recently identified a number of miRNAs that are dysregulated in metastatic renal cell carcinoma compared with primary renal cell carcinoma. Here, we investigated three miRNAs that are significantly downregulated in metastatic tumors: miR-192, miR-194 and miR-215. Gain-of-function analyses showed that restoration of their expression decreases cell migration and invasion in renal cell carcinoma cell line models, whereas knockdown of these miRNAs resulted in enhancing cellular migration and invasion abilities. We identified three targets of these miRNAs with potential role in tumor aggressiveness: murine double minute 2, thymidylate synthase, and Smad Interacting protein 1/zinc finger E-box binding homeobox 2. We observed a convergent effect (the same molecule can be targeted by all three miRNAs) and a divergent effect (the same miRNA can control multiple targets) for these miRNAs. We experimentally validated these miRNA-target interactions using three independent approaches. First, we observed that miRNA overexpression significantly reduces the mRNA and protein levels of their targets. In the second, we observed significant reduction of the luciferase signal of a vector containing the 3'UTR of the target upon miRNA overexpression. Finally, we show the presence of inverse correlation between miRNA changes and the expression levels of their targets in patient specimens. We also examined the prognostic significance of miR-215 in renal cell carcinoma. Lower expression of miR-215 is associated with significantly reduced disease-free survival time. These findings were validated on an independent data set from The Cancer Genome Atlas. These results can pave the way to the clinical use of miRNAs as prognostic markers and therapeutic targets.

Hereditary Breast and Ovarian Cancer Sydrome (HBOC) accounts for approximately 5-10% of breast and ovarian cancer cases and germline mutations of the BRCA1 and BRCA2 genes confer substantially increased risk. However, the risk of developing cancer, the type of cancer and the associated age at diagnosis vary depending on the type of mutation carried and the individual's ethnic background and gender. Screening of BRCA1 and BRCA2 mutations was conducted on 5, 512 high-risk individuals with a prior probability of carrying a pathogenic mutation (>10% chance) from the Advanced Molecular Diagnostics Laboratory (AMDL) (Mount Sinai Hospital, Toronto). Information on age, type of cancer diagnosed and age at diagnosis, ethnicity and family history were collected. Cumulative incidence competing risk and Fine-Gray proportional hazard regression analyses were generated to determine factors associated with earlier diagnosis of cancer. Among 5,029 women and 480 men who underwent testing, a total of 845 unique variants were identified of which, 289 (34.2%) are pathogenic. There were 603 female mutation carriers, of these, 303 were affected with breast or ovarian cancer (50%), 16 with another cancer (2.25%) and 284 were unaffected (47.1%). We also identified 20 different ethnic groups presenting with at least 3 unique variants. Six ethnic groups carried at least one variant associated with an earlier diagnosis of cancer and differential breast or ovarian cancer incidence. For instance, among the Ashkenazi Jewish cohort, the c.68_69del variant was associated with significantly earlier age at diagnosis for ovarian cancer incidence (Gray's test = 40.6; Fine-Gray HR = 1.12, 95% CI = 1.05 – 1.31). By screening a diverse cohort of high-risk individuals for BRCA1 and BRCA2 mutations, we identified pathogenic variants associated with an earlier age of diagnosis and differential incidence of breast or ovarian cancer among unique ethnic groups. Citation Format: Andrew H. Girgis, Marina Wang, Alexa Fine Kathleen-Rose ZakoorSam Khalouei, George Charames, Jordan Lerner-Ellis. BRCA1 and BRCA2 mutation spectrum across 5, 509 high-risk individuals identifies pathogenic variants associated with ethnicity, age of diagnosis, and type of cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A34.

Renal cell carcinoma (RCC) is the most common neoplasm of the adult kidney. Unlike other cancers, its incidence has risen in the past 20 years. The most common subtype of RCC is clear cell RCC (ccRCC) which accounts for approximately 70–80% of cases. A number of genetic aberrations have been reported to be associated with RCC. These include mutations of the von-Hippel Lindau tumor suppressor (VHL) gene which can be associated with a hereditary form of RCC. Inactivation of VHL leads to the stabilization of hypoxia-inducible factors (HIFs) which activates a number of downstream target proteins and contributes to cell proliferation and migration. Currently, there are no established tumor markers for RCC in clinical practice. Recently, a number of molecular markers have been examined as potential diagnostic and prognostic markers for RCC but none have gained clinical application. The new era of molecular profiling has broadened the potential discovery of biomarkers for RCC. This approach allows simultaneous comparison of thousands of molecules in one experiment which will lead to a better understanding of the pathways that are involved in RCC pathogenesis. Molecular profiling can benefit RCC patients at multiple levels including the improvement of early diagnosis, accurate tumor subclassification, prognosis, and prediction of treatment response. In this chapter, we provide a comprehensive review of the genomics of renal cell carcinoma and describe known genetic alterations that are associated with each RCC subtype. We present the current status of tumor markers in RCC and discuss the use of molecular profiling in RCC through different approaches. We also describe the clinical applications of molecular profiling in RCC and how this approach may improve personalized medicine for RCC patients. Finally, we discuss the concept of“integrated genomics” and how this can be applied to further the understanding of the pathogenesis of RCC.

Clear cell renal cell carcinoma (ccRCC) is the most common tumor of the adult kidney, with an increasing rate of incidence. Recently, exome sequencing studies have revealed that the SWI/SNF (switch/sucrose nonfermentable) members PBRM1 and ARID1A are mutated in ccRCC, and it has also been suggested that aberrant chromatin regulation is a key step in kidney cancer pathogenesis. Herein, we show that down-regulation of another SW/SNF component, ARID1A, occurs frequently in ccRCC. We detected copy number loss of ARID1A in 16% of patients with ccRCC. Immunohistochemistry indicated that 67% of ccRCC (53 of 79) had significantly lower expression of BAF250a, the protein product of ARID1A, than did the matched normal kidney cortex. In parallel, we conducted in silico mRNA expression analysis on 404 ccRCC tumors and 167 normal kidney cortex samples using publicly available databases and confirmed significant down-regulation of ARID1A in 68.8% of patients. We also show that decreased BAF250a protein and ARID1A mRNA expression correlate with tumor stage and grade. Our results indicate that both the protein and mRNA levels of ARID1A are statistically significant prognostic markers for ccRCC. Even after controlling for other confounders in the multivariate analysis, BAF250 retained its prognostic significance. BAF250a IHC is easy to perform and represents a potential biomarker that could be incorporated in laboratory practice to enhance the accuracy of the existing prognostic models.

The kallikrein-related peptidases (KLKs) have been implicated in many types of cancer, including prostate and ovarian.We performed a comprehensive in silico study to characterize the KLK locus using transcriptomic (gene expression) and genomic (mutations and DNA copy number) data in prostate cancer (n=194), serous ovarian cancer (n=506), glioblastoma multiforme (n=206), and sarcoma (n=207) from The Cancer Genome Atlas and independent publicly available datasets to assess KLKs as cancer biomarkers.Overall, there was mRNA overexpression in prostate and serous ovarian cancer and decreased expression in glioblastoma multiforme. There was higher frequency of genomic loss in serous ovarian cancer, and rare KLK gene mutations observed in serous ovarian cancer and GBM. Dysregulation of KLKs correlates with survival: for prostate cancer, a combination of dysregulation of KLK1, 5 and 13 was associated with worse disease-free survival.We conclude that specific dysregulation of KLKs at the genetic and transcriptomic levels have useful prognostic value.

The behavior of clear cell renal cell carcinoma can be difficult to predict. Angiogenesis has proven to be a useful prognostic indicator in different malignancies. Endoglin (CD105) is a new marker of angiogenesis found to have prognostic utility in various tumors. Here, we provide the first automated digital assessment of intratumoral microvascular density in clear cell renal cell carcinoma using endoglin and CD31 and assess their utility as predictors of clinical outcome. Both endoglin and CD31 expression showed association with advanced tumor stage (P = .025 and P = .011, respectively). There was a significant correlation between CD31 and tumor grade (P = .034). Kaplan-Meier survival curves showed that patients with higher endoglin expression had significantly shorter progression-free survival (P = .010). Patients with higher CD31 expression tended to have a worse prognosis, although this was not statistically significant (P = .082). In univariate analysis using endoglin as a continuous variable, increased endoglin was strongly associated with reduced survival (hazard ratio, 1.74; 95% CI, 1.39-2.18; P =

miRNAs are small, nonprotein coding RNAs that are differentially expressed in many malignancies. We previously identified 80 miRNAs that are dysregulated in clear cell renal cell carcinoma. In this study we validated over expression of the miR-17-92 cluster in clear cell renal cell carcinoma and tested the effect of 2 members of this cluster (miR-17-5p and miR-20a) on tumor proliferation. We also elucidated the role of miRNA in clear cell renal cell carcinoma pathogenesis with bioinformatics.miRNA expression was validated by quantitative reverse transcriptase-polymerase chain reaction. The cell proliferation effect of miR-17-5p and miR-20a was tested in a renal adenocarcinoma cell line model. Multiple in silico analyses were done of dysregulated miRNAs.We validated miR-71-92 cluster over expression in clear cell renal cell carcinoma by quantitative reverse transcriptase-polymerase chain reaction. Transfection of miR-20a inhibitor significantly decreased cell proliferation in a dose dependent manner. Transfection of miR-17-5p, which is not endogenously expressed in the ACHN cell line, led to increased cell proliferation compared to control values. This effect was suppressed by miR-17-5p inhibitor. Bioinformatics analysis identified 10 clusters of miRNAs dysregulated in clear cell renal cell carcinoma that followed the same expression patterns. We also identified matching patterns between reported chromosomal aberration in clear cell renal cell carcinoma and miRNA dysregulation for 37.5% of the miRNAs. Target prediction analysis was done using multiple algorithms. Many key molecules in clear cell renal cell carcinoma pathogenesis, including HIFs, mTOR, VEGF and VHL, were potential targets for dysregulated miRNAs.A significant number of dysregulated proteins in clear cell renal cell carcinoma are potential miRNA targets. Also, many clear cell renal cell carcinoma dysregulated miRNAs are phylogenetically conserved.

Background There is a critical need for collaborative measures between Canadian institutions to better facilitate variant analysis and data sharing. Objectives The Canadian Open Genetics Repository (COGR) is a collaborative effort for the collection, sharing and analysis of variants reported by medical diagnostics laboratories across Canada. The project focuses on reaching consensus agreements on variant classification among clinical laboratories through data sharing and analysis and disseminating such information to a large, public data repository. Design/method COGR provides laboratories a custom Variant Assessment Tool, and facilitates sharing through GeneInsight ® , a database capturing variant interpretations, reference sequence data, and gene-disease associations. Agreements and discrepancies for individual variant interpretations were identified, and a voting system was put in place to attempt to reach consensus on discrepant classifications. Results The COGR network currently contains over 3,000 variants across 23 genes associated with 10 diseases. There are 46 variants seen by at least three laboratories with fully consistent classifications. A total of 96 variants had discrepant classifications across at least two laboratories. When targeting the 5 most discrepant variants for review via a presentation and anonymous voting, only 1 variant reached consensus. Conclusions By sharing data among laboratories, consensus classifications could quickly be reached for a subset of variants. However, despite presenting evidence for variant classification and discussions amongst experts in the field, there is still considerable difficulty reaching consensus for ambiguous variants. This highlights the need for structured and rule-based variant review. The COGR is facilitating collaboration between Canadian laboratories and international efforts through data sharing and consensus building.

385 Background: miRNAs play a crucial rule in tumor progression and metastasis. We previously identified miR-192, miR-194 and miR-215 to be down-regulated in metastatic compared to primary clear cell renal cell carcinoma (ccRCC). In this work, we examine the role of miR-192, miR-194, and miR-215 in RCC progression and aggressiveness. Methods: We examined the role of these three miRNAs on tumor cell migration and invasion abilities using RCC cell line models. We performed target prediction analysis and experimentally validated the targets using independent approaches. In addition, we examined the clinical utility of miR-215 as a potential prognostic marker in RCC by measuring miR-215 expression using qRT-PCR in 61 formalin-fixed paraffin-embedded tissues from primary ccRCC and correlated the expression levels with clinical outcome. Results: Restoration of miR-192, miR-194, and miR-215 expression decreased cell migration and invasion in RCC cell lines. Target prediction analysis identified three potential targets of these miRNAs; MDM2, TYMS, and SIP1/ZEB2. We validated the miRNA-target interaction experimentally using three approaches. First by measuring the effect of miRNA overexpression on mRNA and protein levels of the predicted target, then by measuring the effect of miRNA overexpression on a luciferase signal of a vector containing the 3’UTR of the predicted target, and finally, by validating these interactions in vivoby examining the presence of an inverse correlation between miRNA changes and the expression levels of their targets on clinical specimens. In 61 patients with resected ccRCC tumors, we found that low miR-215 expression in the primary was associated with a significantly reduced recurrence-free survival. (26.4 vs. 49.2 months, respectively, p = 0.0320). Conclusions: Our analysis showed that miR-192, miR-194, and miR-215 are involved in RCC metastasis and that miR-215 predicts for recurrence in patients with resected RCC. Our findings pave the way to the clinical use of miRNAs as prognostic markers and potential therapeutic targets.

428 Background: The von Hippel-Lindau (VHL) gene is lost in 70% of clear cell Renal Cell Carcinomas (ccRCC); however, additional mechanisms are proposed to regulate VHL expression, including suppression by microRNAs (miRNAs). miRNAs are a class of naturally occurring, small non-coding RNA molecules that downregulate gene expression of target mRNAs. We demonstrate that ccRCC-dysregulated miRNAs can target multiple members of the ccRCC-related signaling pathways. Methods: miR-17 and miR-224 mimics and inhibitors were transfected into ccRCC cell lines using siPORT (Ambion). PicTar and TargetScan were used for target prediction. Target expression and miRNA expression was analyzed by qRT-PCR (Ambion). Western blot antibodies were purchased from Millipore or Cell Signaling. Cell lines were purchased from ATCC. All methods followed the manufacturer’s protocol. Results: According to our preliminary results, the miRNAs that are dysregulated in ccRCC specimens are predicted to target multiple members of the hypoxia-related pathways. To confirm the in silico analysis, miR-17 and miR-224 were selected for experimental target validation, as they were among the most up-regulated miRNAs in ccRCC. We experimentally validated VHL and HIF1α as likely direct targets of miR-17 and miR-224. Luciferase reporter assay confirmed that miR-17 directly downregulates VHL. Moreover, VHL protein level decreased upon miR-17 and miR-224 transfection. We also established a negative correlation between the expression of miR-17 and two predicted targets VEGF-A, EGLN3 in RCC specimens, and miR-224 and its predicted targets SMAD4 and SMAD5. This suggests that downstream signaling pathways are also modulated by miR-17 and miR-224. These results confirm the most important findings of the bioinformatics analysis: miR-17 targets different molecules along the same signaling pathway and that multiple ccRCC-dysregulated miRNAs can synergistically suppress a single target, which functions in the pathogenesis. Conclusions: Our results indicate that miRNAs possibly regulate hypoxia-related pathways at multiple points. This is of special interest as miRNAs may serve as potential therapeutic targets.