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Pleiotropic Action of Renal Cell Carcinoma-Dysregulated miRNAs on Hypoxia-Related Signaling Pathways

  • Zsuzsanna Lichner
    Affiliations
    Department of Laboratory Medicine, Keenan Research Centre in the Li Ka Shing Knowledge Institute St. Michael's Hospital, Toronto, Ontario, Canada
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  • Salvador Mejia-Guerrero
    Affiliations
    Department of Laboratory Medicine, Keenan Research Centre in the Li Ka Shing Knowledge Institute St. Michael's Hospital, Toronto, Ontario, Canada
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  • Monika Ignacak
    Affiliations
    Department of Laboratory Medicine, Keenan Research Centre in the Li Ka Shing Knowledge Institute St. Michael's Hospital, Toronto, Ontario, Canada
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  • Adriana Krizova
    Affiliations
    Department of Laboratory Medicine, Keenan Research Centre in the Li Ka Shing Knowledge Institute St. Michael's Hospital, Toronto, Ontario, Canada
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  • Tian Tian Bao
    Affiliations
    Department of Laboratory Medicine, Keenan Research Centre in the Li Ka Shing Knowledge Institute St. Michael's Hospital, Toronto, Ontario, Canada
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  • Andrew H.F. Girgis
    Affiliations
    Department of Laboratory Medicine, Keenan Research Centre in the Li Ka Shing Knowledge Institute St. Michael's Hospital, Toronto, Ontario, Canada
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  • Yousef M. Youssef
    Affiliations
    Department of Laboratory Medicine, Keenan Research Centre in the Li Ka Shing Knowledge Institute St. Michael's Hospital, Toronto, Ontario, Canada
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  • George M. Yousef
    Correspondence
    Address reprint requests to George M. Yousef, M.D., Ph.D., F.R.C.P.C. (Path), M.Sc., M.B.B.Ch., Department of Laboratory Medicine, St. Michael's Hospital, 30 Bond St., Toronto, ON, M5B 1W8, Canada
    Affiliations
    Department of Laboratory Medicine, Keenan Research Centre in the Li Ka Shing Knowledge Institute St. Michael's Hospital, Toronto, Ontario, Canada

    Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada
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Published:February 10, 2012DOI:https://doi.org/10.1016/j.ajpath.2011.12.030
      The von Hippel-Lindau (VHL) gene is lost in ∼70% of all renal cell carcinomas (RCCs); however, increasing evidence supports the involvement of alternative mechanisms in the regulation of VHL expression, including suppression by microRNAs (miRNAs). miRNAs are small, noncoding RNA molecules that regulate gene expression through binding to target mRNAs. In this study, we found that miRNAs, which are dysregulated in cases of RCC, can target multiple members of RCC-related signaling pathways. Importantly, both VHL and the hypoxia-inducible factor 1-α gene are experimentally validated and are likely direct targets of miR-17-5p and miR-224, as shown by both luciferase assay and Western blot analysis. We found a negative correlation between miR-17-5p and its two predicted targets, VEGF-A and EGLN3, and between miR-224 and its targets SMAD4 and SMAD5 in RCC specimens, suggesting that downstream signaling pathways are also modulated by clear cell RCC-dysregulated miRs. Results from our bioinformatics analysis show that a single miRNA molecule can target multiple components of the same pathway and that multiple miRNAs can target the same molecule. Our results also indicate that miRNAs represent a mechanism for the inactivation of VHL in cases of RCC and can elucidate a new dimension in cancer pathogenesis. As such, miRNAs exemplify new potential therapeutic targets with a significant effect on both tumor growth and metastatic potential.
      Renal cell carcinoma (RCC) is the most common adult kidney neoplasm,
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      Cancer statistics, 2009.
      with the clear cell RCC (ccRCC) subtype accounting for ∼70% of cases. At the molecular level, this subtype has been associated with loss of the von Hippel-Lindau (VHL) tumor suppressor protein. VHL acts as an E3 ubiquitin ligase. The best-understood VHL target, the transcription factor hypoxia-inducible factor 1-α (HIF1α), is destabilized by VHL under normoxic conditions and targeted for ubiquitination and proteasomal degradation. During hypoxia, HIF1α translocates from the cytoplasm into the nucleus, where it initiates transcription of a number of growth and proangiogenic factors, including vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF),
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      Loss of VHL function occurs in all hereditary forms and in ∼77% of sporadic cases of ccRCC. Other mechanisms of VHL down-regulation have been reported, including point mutations of the gene and hypermethylation of the VHL promoter.
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      • Kaelin Jr, W.G.
      The von Hippel-Lindau tumor suppressor protein: new insights into oxygen sensing and cancer.
      MicroRNAs (miRNAs) are a class of small, single-stranded, noncoding RNAs that regulate gene expression at the posttranscriptional level by binding to the 3′ untranslated region (3′-UTR) of the target gene and causing suppression of translation and/or mRNA degradation. miRNAs play important roles in essential processes such as cell differentiation, growth, and cell death.
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      Differential expression profiling of microRNAs and their potential involvement in renal cell carcinoma pathogenesis.
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      Micro-RNA profiling in kidney and bladder cancers.
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      Microarray analysis of microRNA expression in renal clear cell carcinoma.
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      Identification of a microRNA panel for clear-cell kidney cancer.
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      MicroRNA profiling of clear cell renal cell cancer identifies a robust signature to define renal malignancy.
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      MicroRNA 130 family regulates the hypoxia response signal through the P-body protein DDX6.
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      have been recently documented.
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      The VHL-dependent regulation of microRNAs in renal cancer.
      Inactivation of VHL by miRNAs has been predicted by bioinformatics analysis
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      • Romaschin A.D.
      • Honey R.J.
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      Differential expression profiling of microRNAs and their potential involvement in renal cell carcinoma pathogenesis.
      and experimentally confirmed in chronic lymphocytic leukemia.
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      • Lee Y.K.
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      • Kay N.E.
      Aberrant regulation of pVHL levels by microRNA promotes the HIF/VEGF axis in CLL B cells.
      Recent data also suggest the presence of oxygen-independent mechanisms of HIF1α regulation
      • Liu W.
      • Xin H.
      • Eckert D.T.
      • Brown J.A.
      • Gnarra J.R.
      Hypoxia and cell cycle regulation of the von Hippel-Lindau tumor suppressor.
      and that HIF1α and HIF2α can be regulated by miRNAs. miR-199a targets and represses HIF1α in cardiac myocytes,
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      • He M.
      • Sayed D.
      • Vashistha H.
      • Malhotra A.
      • Sadoshima J.
      • Vatner D.E.
      • Vatner S.F.
      • Abdellatif M.
      Downregulation of miR-199a derepresses hypoxia-inducible factor-1alpha and Sirtuin 1 and recapitulates hypoxia preconditioning in cardiac myocytes.
      and HIF1α is also down-regulated by members of the miR-17-92 cluster.
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      Understanding miRNA regulation of tumor initiation and progression is essential to improve diagnosis, prognosis, and treatment options of RCC.
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      In this study, we examine the hypothesis that miRNAs represent a new element, contributing to RCC pathogenesis through direct control of VHL, HIF1α, and their downstream targets. In addition, this might have therapeutic implications, especially for the advanced, therapy resistant, metastatic cases. We followed a combined bioinformatics and experimental approach to identify candidate miRNAs that are dysregulated in kidney cancer and predicted to target VHL and other key molecules in RCC pathogenesis, including HIF1α and VEGF. We experimentally validated the ability of two of these miRNAs (miR-17-5p and miR-224) to target VHL and consequently affect the level of its immediate downstream target, HIF1α. Further analysis showed that miR-224 can affect both tumor cell proliferation and migration.

      Materials and Methods

      Cell Line Models and miRNA Transfection

      The following human cell lines were used for analysis: kidney cancer cell lines 786-0, ACHN, and CAKI-1; transformed embryonic kidney HEK293; and ovary adenocarcinoma SKOV-3. All cell lines were purchased from ATCC (Manassas, VA). Cell lines were grown in Dulbecco's modified essential medium supplemented with 10% fetal bovine serum, penicillin/streptomycin, and sodium pyruvate in a humidified atmosphere at 37°C under 5% CO2. CAKI-1 cells were grown in McCoy's media, supplemented with 10% fetal bovine serum, nonessential amino acids, and penicillin/streptomycin. Transfections were performed in 6-well plates, using siPORT-NeoFX transfection agent (#AM4511; Ambion, Austin, TX) as recommended by the manufacturer. All transfections were performed in three independent biological parallels, and each transfection has three technical parallels.
      Synthetic miRNA mimics and inhibitors were purchased from Applied Biosystems (Foster City, CA). Ectopic overexpression of transfected miRNAs was verified by quantitative RT-PCR (qRT-PCR) reaction. Mean value and SD of three independent transfections are shown in Results. The P value was calculated with a Student's two-tailed t-test.
      A 312-bp segment of the 3′-UTR of the VHL gene containing the predicted interaction sites for miR-17-5p and miR-224 was amplified from genomic DNA by PCR with the use of Phusion High-Fidelity DNA Polymerase (New England Biolabs Inc., Ipswich, MA). The product was purified (QIAquick PCR purification kit; Qiagen, Mississauga, ON) and inserted into pMIR-REPORT expression reporter vector (Applied Biosystems), using the HindIII and SpeI enzymatic restriction sites. Cloning primer sequences were as follows: VHL 5′-CCCACTAGTACATCCGTAGCGGTTGGTGA-3′ (forward) and 5′-CCCAAGCTTACAGCACTTCAACAAGAGTGGACCG-3′ (reverse). Identity of the insert was confirmed by sequencing. Luciferase activity was measured by Dual-Light Luciferase and β-Galactosidase Reporter Gene Assay System (Applied Biosystems). Cells were transfected with 0.5 mg of pMIR-VHL, pMIR-REPORT β-Gal, or pMIR-REPORT control vector, according to the manufacturer's protocol (JetPRIME; PolyPlus, Illkirch, France). Pre-miR-224 or pre-miR-17-5p was transfected at a final concentration of 30 nmol/L. Luciferase and β-galactosidase activities were measured 48 hours after transfection. pLightSwitch-SMAD4 (Sma- and Mad-related protein 4) 3′-UTR luciferase reporter construct coding the 3′-UTR of SMAD4 was purchased from SwitchGear Genomics (Menlo Park, CA).

      Western Blot and ELISA Analyses

      Western blot analysis was performed as described earlier. The following antibodies were used for this study: anti-VHL (Calbiochem, Gibbstown, NJ), anti-phosphatase and tensin homologue (PTEN; #9552; Cell Signaling Technology Inc., Danvers, MA), anti-VEGF-A (#07-1420; Millipore, Billerica, MA,), anti-SMAD4 (Cell Signaling Technology Inc., Pickering, Canada), anti-β-actin (Clone AC-74; Sigma-Aldrich, St Louis, MO), and anti-α-tubulin (Cell Signaling Technology Inc.), followed by the appropriate secondary antibodies coupled to peroxidase (Jackson ImmunoResearch Laboratories Inc., West Grove, PA). Western blot analyses were directly pictured and quantified by the VersaDoc Imaging System (Bio-Rad, Hercules, CA), using QuantityOne band density quantification program. Intensity values were normalized against siPORT transfection control. Band intensity was normalized against the endogenous control (β-actin or α-tubulin). Enzyme-linked immunosorbent assay (ELISA) analysis was performed with 50 μg of total protein per well with the use of the Human/Mouse Total HIF1-α DuoSet IC ELISA assay (R&D Systems, Minneapolis, MN).

      Cell Growth Analysis

      Cells were seeded in 96-well plates at a density of 3000 cells per well and allowed to attach overnight. On the day of the assay cell growth medium was replaced with low-serum (0.5%) medium for 4 hours and were consequently transfected with synthetic miRNA precursor or inhibitor molecules (Applied Biosystems) at the final concentration of 25 nmol/L, following the manufacturer's protocol. Cell growth was monitored for 5 days with the use of MTT assay.

      Cell Migration Analysis

      Migration analysis was performed with the cell wound healing assay. Cells (3 × 105/well) were seeded in 6-well plates and transfected the next day with miR-224 at the final concentration of 25 nmol/L. Twenty-four hours later cells were starved (0.5% fetal bovine serum) and were allowed to reach confluence overnight. Subsequently, a wound was made in each well with the use of a sterile micropipette tip, and cell migration was monitored for 8 hours under a microscope adapted with an incubation chamber at 37°C and with a 0.5% CO2 supply. Photographs were taken at several time points and were used for analysis of cell motility by ImageJ software version 1.41 (National Institutes of Health, Bethesda, MD).

      qRT-PCR Analysis

      Total RNA was isolated by RNeasy RNA isolation kit (Qiagen, Toronto, ON, Canada), mature miRNAs were individually reverse-transcribed by TaqMan miRNA Reverse Transcription Kit (Applied Biosystems), and miRNA relative expression was quantified by specific TaqMan miRNA Assays (Applied Biosystems), using TaqMan Fast Universal PCR Mix (Applied Biosystems). Ectopic and endogenous miR-17-5p and miR-224 levels were normalized against RNU44 expression. mRNAs were randomly reverse-transcribed by High-Capacity RNA-to-cDNA Kit (Applied Biosystems), and relative expression was measured by Power SYBR Green PCR Master Mix (Applied Biosystems). Relative expression values were calculated with the ΔΔCt method against RPLP0 and HPRT1 endogenous controls. The following primers were used: VEGF-A, 5′-CTTGCCTTGCTGCTCTACCT-3′ (forward) and 5′-GTGATGATTCTGCCCTCCTC-3′ (reverse); EGLN3, 5′-AGCTTCCTCCTGTCCCTCAT-3′ (forward) and 5′-CCACCATTGCCTTAGACCTC-3′ (reverse); HIF1AN, 5′-AACCCATTCCTCACCCATCA-3′ (forward) and 5′-TCCACCTCTTTTGGCAAGCA-3′ (reverse); PTEN, 5′-CACAATTCCCAGTCAGAGGCGC-3′ (forward) and 5′-GCTGGCAGACCACAAACTGAGGA-3′ (reverse); HPRT1, 5′-TTGCTGACCTGCTGGATTAC-3′ (forward) and 5′-TCTCCACCAATTACTTTTATGTCC-3′ (reverse); RPLP0, 5′-GGCGACCTGGAAGTCCAACT-3′ (forward) and 5′-CCATCAGCACCACAGCCTTC-3′ (reverse); SMAD4, 5′-TGCATAGTTTGATGTGCCATAG-3′ (forward) and 5′-TCCCATCCAATGTTCTCTGTA-3′ (reverse); and SMAD5, 5′-TTCCCAGCCTATGGATACAA-3′ (forward) and 5′-GCTTCTCCAACACGATTGTTTA-3′ (reverse).

      Tissue Validation

      Fresh frozen ccRCC tissues were tested for the presence of inverse correlation between expression levels of miRNAs and their predicted targets by qRT-PCR analysis. Areas of pure tumor tissues were verified by a pathologist. The use of tissues was approved by the Research Ethics Board of St Michael's Hospital, Toronto, ON. PCR primers and experimental conditions of both targets and miRNAs are as shown above. All target expression values were normalized against the geometrical mean value of HRPT1 and RPLP0 expression. miR-224 and miR-17-5p expressions were normalized against the endogenous control small nuclear RNA RNU44.

      Bioinformatics-Based Target Prediction Analysis

      The most current version of miRecords as of the date of analysis was used to identify miRNA recognition sites in 3′-UTR of VHL (see Supplemental Table S1 at http://ajp.amjpathol.org). MiRecords compiles data obtained by 11 different prediction programs. Only targets predicted by at least three programs were included in the analysis. For all other targets, we used three individual programs, TargetScan 5.1, PicTar, and TargetCombo (Union query). Results are derived from “conserved sites for miRNA families broadly conserved among vertebrates” option of TargetScan 5.1 and “target prediction for all human microRNAs based on conservation in mammals (human, chimp, mouse, rat, dog)” option of PicTar. Non-3′-UTR recognition sites were ignored. Nonconserved sites were not analyzed. Target prediction results were validated against a random gene list (Figure 1; see also Supplemental Figure S1 at http://ajp.amjpathol.org).
      Figure thumbnail gr1
      Figure 1The ccRCC hypoxia-related signaling pathways can be regulated by miRNAs, as predicted by multiple prediction programs. Key molecules along the pathway are shown in bold with their targeting miRNAs in the attached boxes. miR-15 and miR-17 family members and let-7/miR-98 are among the main regulators of ccRCC-related signaling in silico.

      Results

      ccRCC-Dysregulated miRNAs Are Predicted to Target the VHL-Hypoxia Pathways in Silico

      We performed target prediction analysis for key genes that are involved in ccRCC pathogenesis. A total of 149 miRNAs are predicted to be involved in the regulation of ccRCC pathogenesis, based on search for miRNA target sites that are conserved among mammals (Figure 1). Of these, 55 miRNAs are reported to be dysregulated in ccRCC.
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      • Bao T.T.
      • Grigull J.
      • Youssef Y.M.
      • Girgis A.H.
      • Diamandis M.
      • Fatoohi E.
      • Metias M.
      • Honey J.
      • Stewart R.
      • Pace K.T.
      • Bjarnason G.A.
      • Yousef G.M.
      miRNA profiling for clear cell renal cell carcinoma: biomarker discovery and identification of potential controls and consequences of miRNA dysregulation.
      • Yi Z.
      • Fu Y.
      • Zhao S.
      • Zhang X.
      • Ma C.
      Differential expression of miRNA patterns in renal cell carcinoma and nontumorous tissues.
      Cluster analysis of the miRNA/target interactions is shown in Supplemental Figure S1 (available at http://ajp.amjpathol.org). We identified four miRNA/target clusters with potential importance in ccRCC (Figure 2; see also Supplemental Figure S1 at http://ajp.amjpathol.org). Interestingly, multiple genes that regulate the proteasomal degradation of HIF1α are among the predicted targets, including HIF1α, HIF2α, HIFβ, VHL, HIF1α inhibitor (HIF1AN; FIH-1), cullin 2 (CUL2), Egl nine homolog 1 (EGLN1), EGLN2, and ring-box 1 (RBX1). In addition, the immediate targets of HIFα-HIFβ-p300 transcriptional factor complex are predicted to be regulated by ccRCC-dysregulated miRNAs [including VEGF, transforming growth factor A (TGF-A) and epidermal growth factor receptor; platelet derived growth factor β and its receptor (PDGFβ and PDGFRβ); erythropoietin, glucose transporter 1, and IL-6]. Among the downstream signaling pathways, KRAS and the mitogen-activated protein kinase kinase kinase 1 in the MAPK pathway; phosphatidylinositol 3-kinase (PI3K), and AKT in the PI3K/AKT pathway; TGF-β; mammalian target of rapamycine (mTOR), tuberous sclerosis complex 1 eukaryotic translation initiation factor 4E in the mTOR pathway are predicted to be under direct miRNA regulation (Figure 1, Figure 2).
      Figure thumbnail gr2
      Figure 2miRNA network is predicted to regulate hypoxia-related signal cascades in ccRCC. A: Cluster analysis of the miRNA/target interactions in ccRCC. 3′-UTRs of hypoxia-related genes were searched for miRNA target recognition sequences by multiple programs. The four main clusters shown here are extracted from (available at http://ajp.amjpathol.org). Color intensity indicates the number of predicting algorithms for each interaction. B: Genes responsible for oxygen sensing, VHL-HIF1α axis, and the immediate targets of HIF1α and PI3K-AKT, RAS-RAF-MEK-ERK, and mTOR pathways are predicted to be regulated by ccRCC dysregulated miRNAs.
      Importantly, our analysis indicates that a single miRNA can target the same (or closely related) signaling pathway at multiple points through multiple targets (see Supplemental Figure S1 at http://ajp.amjpathol.org). For example, miR-141/200 can target p300, EGLN1, DNA-damage-inducible transcript 4-like, VEGF-A, VEGF-D, VEGF-E, VEGFR2, 3-phosphoinositide dependent protein kinase-1, PI3K, RAS, MAPK members, PTEN, TSC1, Ras homolog enriched in brain, tuberous sclerosis 1, and ribosomal protein S6 kinase 70kDa polypeptide 1. Moreover, the same molecule can be targeted by multiple ccRCC-dysregulated miRNAs. For example, HIF1α can be targeted by miR-17-5p, miR-106b, miR-519, miR-138, miR-224, miR-18, miR-19, miR-9, and miR-93. These observations raise the possibility that altering the expression of miRNAs can have a significant effect on a signaling pathway, even if the translation of the individual targets is not dramatically reduced.

      miR-17-5p Targets the VHL-HIF1α Axis

      VHL and HIF1α are key molecules in the hypoxia response, and most ccRCC cases have documented inactivation of VHL. The VHL transcript features a relatively long 3′-UTR region of 2016 bp. With the use of a minimum of three different algorithms, 181 miRNAs are predicted to target the 3′-UTR of the VHL mRNA. Of these, 61 miRNAs have been reported to be dysregulated in ccRCC (see Supplemental Table S1 at http://ajp.amjpathol.org). Interestingly, VHL can be targeted by the miR-15 family, miR-211/204, miR-224, and miR-17-92 miRNA cluster in silico. The miR-17-92 miRNA cluster is known to be a body of oncogenic miRNAs and has been shown to be overexpressed in a number of different cancers.
      • Kanzaki H.
      • Ito S.
      • Hanafusa H.
      • Jitsumori Y.
      • Tamaru S.
      • Shimizu K.
      • Ouchida M.
      Identification of direct targets for the miR-17-92 cluster by proteomic analysis.
      miR-17-5p is the best-studied member of the cluster. Ectopic expression of a single member of the cluster, miR-17-5p, was sufficient to drive a proliferative signal in HEK293T cells
      • Cloonan N.
      • Brown M.K.
      • Steptoe A.L.
      • Wani S.
      • Chan W.L.
      • Forrest A.R.
      • Kolle G.
      • Gabrielli B.
      • Grimmond S.M.
      The miR-17-5p microRNA is a key regulator of the G1/S phase cell cycle transition.
      by targeting >20 genes involved in the transition between G1 and S cell cycle phases. miR-17-5p expression was also shown to induce proliferation in kidney cancer cell lines
      • Chow T.F.
      • Mankaruos M.
      • Scorilas A.
      • Youssef Y.
      • Girgis A.
      • Mossad S.
      • Metias S.
      • Rofael Y.
      • Honey R.J.
      • Stewart R.
      • Pace K.T.
      • Yousef G.M.
      The miR-17-92 cluster is over expressed in and has an oncogenic effect on renal cell carcinoma.
      and to promote migration and invasion of breast cancer
      • Li H.
      • Bian C.
      • Liao L.
      • Li J.
      • Zhao R.C.
      miR-17-5p promotes human breast cancer cell migration and invasion through suppression of HBP1.
      and hepatocellular carcinoma cell lines.
      • Yang F.
      • Yin Y.
      • Wang F.
      • Wang Y.
      • Zhang L.
      • Tang Y.
      • Sun S.
      miR-17-5p Promotes migration of human hepatocellular carcinoma cells through the p38 mitogen-activated protein kinase-heat shock protein 27 pathway.
      Both HIF1α and VHL are predicted targets of miR-17-5p. To determine whether VHL and HIF1α could be directly regulated by miR-17-5p, we used two independent experimental approaches: a luciferase reporter assay to detect direct interaction between miR-17-5p and VHL or HIF1α 3′-UTRs, and measuring changes of protein levels of these targets upon altering the expression level of the miR-17-5p. SKOV3 (ovarian cancer cell line), HEK293 (human embryonic kidney cell line), ACHN, and 786-0 (RCC cell lines) exhibited low expression of miR17-5p and miR-224; therefore, they were used for miRNA ectopic overexpression studies to confirm miRNA/target interactions. CAKI-1 RCC cell line expresses high levels of miR-17-5p and miR-224; thus, it was used to study the effect of miRNA knockdown by anti-miRs (see Supplemental Figure S2A at http://ajp.amjpathol.org). Efficient miRNA transfection was confirmed by qRT-PCR quantification of the miRNA in transfected and untransfected cells.
      In the first approach, we cloned the miR-17-5p recognition sequences of VHL and HIF1α 3′-UTRs immediately downstream of a luciferase reporter gene, creating pMIR-VHL and pMIR-HIF1α vectors, respectively. The SKOV-3 cell line was selected for the transfection studies because of its low endogenous miR-17-5p expression (see Supplemental Figure S2A at http://ajp.amjpathol.org). When the pMIR-VHL construct was cotransfected with synthetic miR-17-5p mimics (will be referred to as miR-17-5p), it led to 62% reduction in luciferase activity (Figure 3A). Luciferase activity was partially restored when cotransfecting miR-17-5p and its inhibitor (anti-miR-17-5p), whereas no significant change of luciferase expression was observed when pMIR-VHL was cotransfected with a random pre-miRNA pool (RPP). Similarly, luciferase activity dropped to 17% when pMIR-HIF1α was cotransfected with miR-17-5p into SKOV-3 cells. Cotransfection of miR-17-5p and its inhibitor (anti-miR-17) partially rescued luciferase activity (Figure 3B). All transfection experiments included the cotransfection of the pMIR-REPORT β-galactosidase control vector to assess transfection efficiency. This indicates that miR-17-5p can directly and specifically interact with the predicted target recognition sites of the mRNA encoding VHL and that HIF1α is also a likely direct target of miR-17-5p.
      Figure thumbnail gr3
      Figure 3The VHL-HIF1α axis is targeted by miR-17-5p. VHL and HIF1α 3′-UTRs were tested for direct interaction with miR-17-5p. In the luciferase reporter assay, cotransfection of the reporter vectors and miR-17-5p reduced luciferase activity, indicating direct interaction between miR-17-5p and VHL (A) and HIF1α (B). Mean luminescence and SD values are indicated. Luminescence was normalized against pMIR-VHL transfection. C: Transfection of synthetic miR-17-5p decreased VHL protein level as shown by Western blot analysis. Cells were treated with siPORT transfection agent or RPP (random pre-miRNA pool) as negative controls. All transfections were performed in triplicates; mean band density and SD values are indicated. *P < 0.05.
      The interaction of miR-17-5p with VHL was also examined at the protein level by Western blot analysis. SKOV-3 cells were transfected with synthetic miR-17-5p precursor at two different concentrations. VHL protein level showed marked decrease (0.45 times) on the ectopic expression of miR-17-5p (Figure 3C). Control untreated cells and cells transfected with transfection agent only or with a RPP did not affect VHL protein level. Cotransfection with anti-miR-17 was only partially able to rescue luciferase activity. The partial recovery might be because miRNA precursors and inhibitors are chemically different. Taken together, the ccRCC dysregulated miR-17-5p is likely a direct regulator of VHL that acts at the level of posttranscriptional inhibition.

      miR-17-5p Can Target Multiple Key Components of Hypoxia-Related Signaling Pathways

      To elucidate the effect of miR-17-5p on ccRCC downstream of VHL-HIF1α, we searched for additional targets along the hypoxia-related pathways. According to our combined target predictions, six other members of the ccRCC pathogenesis pathway can be potentially targeted by miR-17-5p: HIF1AN, EGLN3, VEGF-A, PI3K, PTEN, and mitogen-activated protein kinase kinase kinase 1. HIF1AN and EGLN3 are regulators of the ubiquitin ligase activity of VHL-Cullin-RING complex, whereas VEGF-A is an immediate target of the HIF1α/HIF1β transcription factor complex. VEGF induces signaling through mitogen-activated protein kinase cascade and PI3K pathway (including PI3K and the inhibitor PTEN). To confirm the bioinformatics analysis, we overexpressed miR-17-5p in cell lines and measured the subsequent changes in the mRNA level of the predicted targets. Although miRNAs act posttranscriptionally, recent evidence supports that they usually also interfere with the stability of their target mRNAs.
      • Cheng C.
      • Li L.M.
      Inferring microRNA activities by combining gene expression with microRNA target prediction.
      • Gennarino V.A.
      • Sardiello M.
      • Avellino R.
      • Meola N.
      • Maselli V.
      • Anand S.
      • Cutillo L.
      • Ballabio A.
      • Banfi S.
      MicroRNA target prediction by expression analysis of host genes.
      • Guimbellot J.S.
      • Erickson S.W.
      • Mehta T.
      • Wen H.
      • Page G.P.
      • Sorscher E.J.
      • Hong J.S.
      Correlation of microRNA levels during hypoxia with predicted target mRNAs through genome-wide microarray analysis.
      PTEN, EGLN3, HIF1AN, and VEGF-A were selected to validate target analysis results in vitro. The 786-0 and CAKI-1 RCC cell lines were transiently transfected with miR-17-5p, anti-miR-17-5p, or a pool of random miRNAs. We observed decreased mRNA levels of all four predicted targets in 786-0 cells on miR-17-5p overexpression (Figure 4A). CAKI-1 cells did not show significant reduction of the mRNA of the targets (data not shown). This could be explained because CAKI-1 cells have high endogenous miR-17-5p expression levels; thus, its biological effect cannot be enhanced by further ectopic expression of synthetic miR-17-5p (see Supplemental Figure S2A at http://ajp.amjpathol.org). However, transfection of CAKI-1 cells with anti-miR-17 (miR-17 inhibitor) led to the elevation of HIF1AN, VEGF-A, and EGLN3 expression, compared with transfection reagent-only control (Figure 4B). These results further validate that multiple hypoxia-related genes are regulated by miR-17-5p.
      Figure thumbnail gr4
      Figure 4miR-17-5p transfection leads to significant drop of the level of the mRNA of four predicted targets: HIF1AN, VEGF-A, EGLN3, and PTEN. A: miR-17-5p overexpression caused down-regulation of all four targets in the 786-0 cells. B: Transfection with anti-miR-17-5p led to elevation of the expression of PTEN, HIF1AN, VEGF-A, and EGLN3 in the CAKI-1 RCC cell line. RPLP0 and HPRT1 were used as endogenous controls for the qRT-PCR reactions. SD values were calculated from triplicate transfections.

      miR-17-5p Regulates PTEN and VEGF-A at the Protein Level

      We next examined whether miR-17-5p can regulate PTEN at the protein level. Changes of PTEN protein expression in response to altered miR-17-5p level were evaluated by Western blot analysis in the HEK293T and 786-0 cell lines. Western blot quantification was normalized against β-actin. The 786-0 cells exhibited 0.56 times decrease in PTEN level when transfected with miR-17-5p (Figure 5A). Transfection of a random pre-miRNA pool did not lower PTEN level. In the HEK293 cells, the level of PTEN decreased by 33% on cell transfection with miR-17-5p and increased by 63% in cells transfected with anti-miR-17-5p (Figure 5B). Control RPP transfection did not alter PTEN protein expression.
      Figure thumbnail gr5
      Figure 5Overexpression of miR-17-5p alters PTEN and VEGF-A protein levels. A: Western blot analysis shows that elevated miR-17-5p level lowered PTEN protein expression in 786-0 and HEK293 cell lines. B: Quantification of Western blot data. C: Western blot analysis shows that elevated miR-17-5p level resulted in reduction of VEGF-A protein expression in the 786-0 ccRCC cell line. VEGF-A protein was undetectable in HEK293 cells. All transfections were performed in triplicates.
      We then examined whether miR-17-5p regulates VEGF-A at the protein level, by measuring the changes in VEGF-A protein level in 786-0 cells on miR-17-5p transfection. Overexpression of miR-17-5p resulted in 26% decrease of VEGF-A protein level, compared with cells treated with transfection agent (Figure 5C). The RPP alone did not alter the VEGF-A protein levels.

      VHL Is a Direct Target of the ccRCC-Dysregulated miR-224

      miR-224 was among the top ccRCC-dysregulated miRNAs in our preliminary study.
      • White N.M.
      • Bao T.T.
      • Grigull J.
      • Youssef Y.M.
      • Girgis A.H.
      • Diamandis M.
      • Fatoohi E.
      • Metias M.
      • Honey J.
      • Stewart R.
      • Pace K.T.
      • Bjarnason G.A.
      • Yousef G.M.
      miRNA profiling for clear cell renal cell carcinoma: biomarker discovery and identification of potential controls and consequences of miRNA dysregulation.
      It showed an overall 21.06-fold overexpression in the cancer specimens, compared with adjacent, healthy kidney cortex in 93.3% of the specimens. miR-224 is a known oncogenic miRNA with reported overexpression in many cancers. It has been reported to regulate TGFβ-mediated proliferation of granulosa cells
      • Yao G.
      • Yin M.
      • Lian J.
      • Tian H.
      • Liu L.
      • Li X.
      • Sun F.
      MicroRNA-224 is involved in transforming growth factor-beta-mediated mouse granulosa cell proliferation and granulosa cell function by targeting Smad4.
      and proliferation of ovarian cancer cells. However, the involvement of miR-224 in hypoxia and RCC has not yet been examined. Our bioinformatics analysis indicates miR-224 recognition sites in the 3′-UTR of VHL, but binding sites for miR-224 in the HIF1α 3′-UTR were not predicted. To experimentally validate if miR-224 can target the VHL-HIF1α axis, we used luciferase assay in the SKOV-3 and HEK293 cell lines. pMIR-VHL was transfected into HEK293 cells either alone or with miR-224 precursor. Cotransfection of miR-224 and pMIR-VHL led to 50% suppression of the reporter signal (Figure 6A). Luciferase expression was not changed when pMIR-REPORTER empty vector or a random miRNA precursor pool was cotransfected with the reporter plasmid. These results show that miR-224 specifically targets the VHL 3′-UTR in vitro.
      Figure thumbnail gr6
      Figure 6miR-224 is a potential regulator of the VHL-HIF1α axis. A: Cotransfection of miR-224 and the reporter vector pMIR-VHL resulted in 50% reduction of luciferase activity. Luciferase activity was normalized against β-galactosidase activity. B: Western blot analysis shows decreased VHL protein level in HEK293 cells on miR-224 transfection. Control cells were transfected with either transfection reagent alone (siPORT) or a random pool of pre-miRNAs (RPP). β-Actin was used as loading control. C: miR-224 transfection resulted in decrease of HIF1α protein (as quantified by ELISA). All transfections were performed in triplicates. ELISA was performed in three technical parallels (n = 9). D: miR-224 alters the expression of two of its targets, SMAD4 and SMAD 5. In the CAKI-1 cells with high miR-224 endogenous expression, anti-miR-224 resulted in elevation of SMAD4 levels (left panel). The 786-0 cells have low endogenous miR-224. Transfecting with miR-224 resulted in reduced SMAD4 (middle panel) and SMAD5 (right panel) expression. E: Western blot analysis shows decreased SMAD4 protein level in the 786-0 cells on miR-224 transfection and elevated SMAD4 protein level on anti-miR-224 transfection of CAKI-1 cells. Transfection reagent alone (siPORT) or a random pool of pre-miRNAs (RPP) did not alter SMAD4 expression. α-Tubulin was used as loading control.
      To further validate the ability of miR-224 to target VHL, we transfected SKOV-3 cells with miR-224 and quantified the protein levels by Western blot analysis. Transfection of miR-224 resulted in down-regulation of VHL protein expression in a dose-dependent manner (Figure 6B). No significant reduction of protein level was observed when cells were treated with transfection reagent or with a random pre-miRNA pool. Interestingly, transfection of miR-224 caused 35% decrease of HIF1α protein level despite the lack of predicted recognition sequence in its 3′-UTR, promoter, or intronic regions (Figure 6C). No alteration of HIF1α expression was observed when the cells were transfected with transfection agent only, random miRNA precursor pool, or specific inhibitors. This observation raises the possibility of indirect regulation of HIF1α by miR-224 or the presence of a less-conserved binding site that is not recognized by predicting algorithms.

      miR-224 Inhibits the TGFβ Pathway Members SMAD4 and SMAD5 in Vitro

      To search for additional targets of miR-224 that could modulate ccRCC pathogenesis, we used TargetScan 5.1 and PicTar programs and analyzed the 500 top targets, sorted by total context score. Targets were grouped to signaling pathways by Database for Annotation, Visualization, and Integrated Discovery (DAVID) functional annotation program. Surprisingly, miR-224 was predicted to regulate several members of the TGFβ pathway, including SMAD4 and SMAD5, with two conserved miR-224 recognition sites on each 3′-UTR. We next examined the effect of altering miR-224 expression on these two targets experimentally. We used two RCC cell lines, CAKI-1 and 786-0. With the use of qRT-PCR analysis, CAKI-1 cells showed 10-fold higher miR-224 expression than 786-0 cells. Therefore, CAKI-1 cells were transiently transfected with anti-miR-224, 786-0 cells were transfected with synthetic miR-224 mimics, and SMAD4 and SMAD5 expression was measured by qRT-PCR 48 hours after transfection. We observed a 1.47-fold increase of SMAD4 mRNA level on anti-miR-224 transfection of CAKI-1 cells, compared with cells treated with transfection agent only (Figure 6D, left panel). However, miR-224 transfection decreased SMAD4 mRNA level to 0.7-fold in the 786-0 cell line (Figure 6D, middle panel). These results support our target prediction. SMAD5 also showed 0.64 times reduction on miR-224 overexpression in 786-0 cells (Figure 6D, right panel). We did not, however, observe significant alteration of SMAD5 mRNA expression when CAKI-1 cells were transfected with anti-miR-224 (data not shown).
      To address the miR-224 regulation of SMAD4 at the protein level, CAKI-1 and 786-0 cells were transfected with anti-miR-224 or miR-224, respectively, and SMAD4 levels were analyzed by Western blot analysis. As expected, SMAD4 protein level was reduced after anti-miR-224 transfection by 0.65 times, whereas a 1.42-fold increase of SMAD4 protein was detected on miR-224 transfection (Figure 6E). Together, these data validate the initial bioinformatics analysis.
      To further prove the direct interaction between SMAD4 and miR-224, 786-0 cells were transfected with pLightSwitch-SMAD4-3′-UTR luciferase reporter construct (pLS-SMAD4) that carried the full 3′-UTR region of SMAD4, including both miR-224 recognition elements. Luciferase activity showed a significant (P > 0.05) decrease when the pLS-SMAD4 construct was cotransfected with miR-224 but not in the control transfections (data not shown).

      miR-17-5p and miR-224 Expression Shows Inverse Correlation to Their Targets in ccRCC Specimens

      Although ccRCC cell lines are acceptable and valuable models of ccRCC, ectopic expression of both the miRNA and its target recognition site in the same cell might not reflect a physiological state and can lead to false validation of the interaction. Therefore, validation of miRNA recognition site in a less artificial system is necessary. To address this issue, we performed a preliminary in vivo analysis by correlating the expression of miR-17-5p and miR-224 with the mRNA levels of four predicted targets: VEGF-A and EGLN3 (miR-17-5p targets) and SMAD4 and SMAD5 (miR-224 predicted targets). Patient samples were tested by qRT-PCR quantification. As shown in Figure 7A, 24 ccRCC samples were ranked according to their miR-17-5p expression. They were classified into two groups: 12 samples with higher expression and 12 with lower expression. Specimens were then additionally ranked according to the expression level of VEGF-A or EGLN3 (Figure 7B), and specimens with higher miR-17-5p expression were indicated by black bars. Both VEGF-A and EGLN3 mRNA expression showed inverse correlation with miR-17-5p levels (specimens with higher miR-17-5p expression showing relatively lower target expression and vice versa), thus supporting the presence of the miRNA-gene interaction in vivo (Figure 7B).
      Figure thumbnail gr7
      Figure 7miR-17-5p and miR-224 expression shows reverse correlation with their targets in ccRCC specimens. A: ccRCC samples were ranked according to their miR-17-5p expression. Specimens with higher miR-17-5p levels are indicated by black bars. White bars represent the specimens with lower miR-17-5p expression. All SD bars were calculated from technical parallels. B: ccRCC specimens were ranked according to the expression of two miR-17-5p predicted targets: VEGF-A and EGLN3. Black bars represent samples with higher miR-17-5p levels. White bars represent samples with lower miR-17-5p expression. C: miR-224 levels of ccRCC specimens were quantified by qRT-PCR and were correlated to the expression of two miR-224 targets: SMAD4 and SMAD5. Black dots indicate the expression of miR-224, and gray dots indicate the expression of SMAD4 or SMAD5 in the different ccRCC specimens. Correlation coefficient is −0.78 for VEGF-A and −0.679 for EGLN3. RPLP0 and HPRT1 were used as endogenous controls.
      However, expression of PTEN and HIF1AN were apparently independent of miR-17-5p (data not shown). A possible explanation is that PTEN and HIF1AN are tumor suppressors with very low expression in the ccRCC samples; thus, the added effect of miRNAs cannot be shown with accuracy. Alternatively, it is possible that the mRNA level does not always faithfully reflect miRNA effect, and miR-17-5p only affects the protein levels of these genes.
      Finally, SMAD4 and SMAD5 mRNA expressions were correlated to miR-224 levels in ccRCC specimens. First, miR-224 expression was quantified by qRT-PCR from fresh frozen tissue. miR-224 exhibited a strong correlation with miR-17-5p expression, as expected from the initial miRNA microarray data whereby both miR-224 and miR-17-5p were overexpressed in ccRCC samples (see Supplemental Figure S2B at http://ajp.amjpathol.org). SMAD4 and SMAD5 mRNA levels were quantified from the ccRCC specimens by qRT-PCR, and the expression values were plotted against miR-224 expression. miR-224 showed inverse correlation to SMAD4 and SMAD5, with a correlation coefficient of −0.78 and −0.689, respectively (Figure 7C).

      Discussion

      In the present work, we provide the first experimental evidence that miRNAs are actively involved in the regulation of ccRCC-related pathways. Uncovering the role of these small noncoding RNA molecules is an important step toward the understanding of ccRCC tumorigenesis. We found that VHL and HIF1α, the critical molecules of ccRCC pathogenesis, are potential targets of miRNAs that are dysregulated in kidney cancer. Furthermore, our results suggest that miR-17-5p has a pleiotropic effect on several signal transduction pathways downstream of the VHL-HIF1α axis. In addition, our in vivo data suggest that at least two members of the TGFβ pathway, SMAD4 and SMAD5, are down-regulated by miR-224. SMAD4 is a likely direct target of miR-224, based on the luciferase reporter assay. SMAD4 is the only co-SMAD in mammals and is a central molecule in the communication of TGFβ receptor and the downstream transcriptional regulation.
      Recent work by our laboratory and others identified a group of miRNAs to be dysregulated in ccRCC. Interestingly, many of the dysregulated miRNAs are predicted to act on hypoxia-related pathways. miR-17-5p is a known oncogene in many cancers, including rhabdomyosarcoma, neuroblastoma, and lung, pancreatic, and breast cancers,
      • Li H.
      • Bian C.
      • Liao L.
      • Li J.
      • Zhao R.C.
      miR-17-5p promotes human breast cancer cell migration and invasion through suppression of HBP1.
      • Mestdagh P.
      • Bostrom A.K.
      • Impens F.
      • Fredlund E.
      • Van Peer G.
      • De Antonellis P.
      • von S tedingk K.
      • Ghesquiere B.
      • Schulte S.
      • Dews M.
      • Thomas-Tikhonenko A.
      • Schulte J.H.
      • Zollo M.
      • Schramm A.
      • Gevaert K.
      • Axelson H.
      • Speleman F.
      • Vandesompele J.
      The miR-17-92 microRNA cluster regulates multiple components of the TGF-beta pathway in neuroblastoma.
      and is a member of the first miRNA cluster to be described as oncogenic. miR-17-5p has already been shown to be up-regulated in kidney cancer,
      • Gottardo F.
      • Liu C.G.
      • Ferracin M.
      • Calin G.A.
      • Fassan M.
      • Bassi P.
      • Sevignani C.
      • Byrne D.
      • Negrini M.
      • Pagano F.
      • Gomella L.G.
      • Croce C.M.
      • Baffa R.
      Micro-RNA profiling in kidney and bladder cancers.
      • Kanzaki H.
      • Ito S.
      • Hanafusa H.
      • Jitsumori Y.
      • Tamaru S.
      • Shimizu K.
      • Ouchida M.
      Identification of direct targets for the miR-17-92 cluster by proteomic analysis.
      and we have recently reported an oncogenic effect of the miR-17-5p in ccRCC.
      • Chow T.F.
      • Mankaruos M.
      • Scorilas A.
      • Youssef Y.
      • Girgis A.
      • Mossad S.
      • Metias S.
      • Rofael Y.
      • Honey R.J.
      • Stewart R.
      • Pace K.T.
      • Yousef G.M.
      The miR-17-92 cluster is over expressed in and has an oncogenic effect on renal cell carcinoma.
      Besides regulating cell cycle, migration, proliferation, and invasion, miR-17-5p has been shown to modulate the differentiation of mesenchymal stem cells through a coherent feed-forward loop
      • Liu Y.
      • Liu W.
      • Hu C.
      • Xue Z.
      • Wang G.
      • Ding B.
      • Luo H.
      • Tang L.
      • Kong X.
      • Chen X.
      • Liu N.
      • Ding Y.
      • Jin Y.
      MiR-17 Modulates osteogenic differentiation through a coherent feed-forward loop in mesenchymal stem cells isolated from periodontal ligaments of patients with periodontitis.
      and to shift the E2F transcriptional balance away from the proapoptotic E2F1 and toward the proliferative E2F3 transcriptional network.
      • Woods K.
      • Thomson J.M.
      • Hammond S.M.
      Direct regulation of an oncogenic micro-RNA cluster by E2F transcription factors.
      In silico target prediction indicated that VHL and HIF1α are both candidate targets of miR-17-5p and miR-224. The potential regulation of HIF1α by miR-17-5p is of particular interest. HIF1α and HIF2α are the best-studied downstream targets of the VHL-Cullin-RING ubiquitin ligase complex. Now, it is clear that HIF1α and HIF2α have different properties and distinct actions in hypoxia-related tumorigenesis. Because miR-17-5p seems to target both the suppressor VHL and the oncogenic HIF1α, we speculate that this regulation also has importance under physiological conditions, probably in the fine-tuning of the target expression.
      Our miR-224 results are not unprecedented. miR-224 was found to be overexpressed in many malignancies, including hepatocellular carcinoma, colorectal cancer, papillary thyroid carcinoma, prostate cancer, ovarian cancer, and acute myeloid leukemia.
      • Li Z.
      • Lu J.
      • Sun M.
      • Mi S.
      • Zhang H.
      • Luo R.T.
      • Chen P.
      • Wang Y.
      • Yan M.
      • Qian Z.
      • Neilly M.B.
      • Jin J.
      • Zhang Y.
      • Bohlander S.K.
      • Zhang D.E.
      • Larson R.A.
      • Le Beau M.M.
      • Thirman M.J.
      • Golub T.R.
      • Rowley J.D.
      • Chen J.
      Distinct microRNA expression profiles in acute myeloid leukemia with common translocations.
      Many of these malignancies are related to hypoxia-induced genes
      • Wu X.Y.
      • Fu Z.X.
      • Wang X.H.
      • Shen W.
      Identification of differential proteins in colon cancer SW480 cells with HIF1-alpha silence by proteome analysis.
      or directly linked to HIF1α-VEGF signaling.
      • Giatromanolaki A.
      • Fiska A.
      • Pitsiava D.
      • Kartalis G.
      • Koukourakis M.I.
      • Sivridis E.
      Erythropoietin receptors in endometrial carcinoma as related to HIF1{alpha} and VEGF expression.
      Note that other genes that are predicted targets of miR-224, such as the HIF1AN or the prolyl-hydroxylase domain protein 2, are involved in HIF1α down-regulation.
      We used both in vitro and in vivo approaches to experimentally validate the bioinformatics predictions. Both luciferase assay and Western blot analysis showed that miR-17-5p is able to target VHL and HIF1α. Luciferase assay is a valid method to show direct interaction between a miRNA and its target. However, this technique has certain limitations. It requires the simultaneous overexpression of the miRNA and the target site in the same cell, and such artificial conditions do not validate that the miRNA/target interaction occurs in vivo. Western blot analysis has the advantage of testing the miRNA effect at the protein level. However, it cannot distinguish clearly between direct and indirect effects of miRNAs. To address the existence of the predicted miRNA/target interactions in vivo, miR-17-5p expression was correlated with the mRNA expression of downstream members of hypoxia pathway in ccRCC patients' samples. Several recent studies have proved the relevance of comparing mRNA and miRNA levels for miRNA target prediction.
      • Cheng C.
      • Li L.M.
      Inferring microRNA activities by combining gene expression with microRNA target prediction.
      • Gennarino V.A.
      • Sardiello M.
      • Avellino R.
      • Meola N.
      • Maselli V.
      • Anand S.
      • Cutillo L.
      • Ballabio A.
      • Banfi S.
      MicroRNA target prediction by expression analysis of host genes.
      • Guimbellot J.S.
      • Erickson S.W.
      • Mehta T.
      • Wen H.
      • Page G.P.
      • Sorscher E.J.
      • Hong J.S.
      Correlation of microRNA levels during hypoxia with predicted target mRNAs through genome-wide microarray analysis.
      • Lionetti M.
      • Biasiolo M.
      • Agnelli L.
      • Todoerti K.
      • Mosca L.
      • Fabris S.
      • Sales G.
      • Deliliers G.L.
      • Bicciato S.
      • Lombardi L.
      • Bortoluzzi S.
      • Neri A.
      Identification of microRNA expression patterns and definition of a microRNA/mRNA regulatory network in distinct molecular groups of multiple myeloma.
      • Sales G.
      • Coppe A.
      • Bicciato S.
      • Bortoluzzi S.
      • Romualdi C.
      Impact of probe annotation on the integration of miRNA-mRNA expression profiles for miRNA target detection.
      The great advantage of the method is that it is based on the analysis of a single transcriptome; thus, it avoids prediction of targets that are not present in the conditions of interest. In our patient specimens, VEGF-A and EGLN3 exhibited strong inverse correlation with miR-17-5p. Although PTEN and HIF1AN did not show the same negative correlation with miR-17-5p in the patient samples, PTEN protein level still reacted to the altered expression of miR-17-5p in RCC and HEK293 cell line models.
      Another important finding of the present work is that a single miRNA, miR-17-5p, is able to regulate multiple targets along the same signaling pathway. miR-17-5p, miR-224, miR-200 family, miR106a/b, miR-21, miR-221, miR-199a, and miR-214 seem to have prominent roles in ccRCC pathogenesis. They are predicted to target the VHL-HIF1α axis along with the targets of HIFα/HIFβ transcription factors: VEGF, transforming growth factor, platelet-derived growth factor, facilitated glucose transporter member 1, the downstream PI3K/AKT and RAS/RAF/MEK/ERK pathways, and the energy and nutrient-sensing mTOR pathway. This implies that even subtle alterations of these miRNAs can result in a significant effect on the outcome of the regulated pathway and on the pathogenesis of ccRCC. This also indicates that miRNAs can control the hypoxia-related VHL-HIF1α axis and the common cancer-related pathways in a tight regulatory network.
      Pleiotropic miRNA regulation of a biological process or phenomenon is not restricted to ccRCC. The control of cell proliferation by miRNAs and their effect on multiple regulatory steps is well established. Oncogenic miRNAs may facilitate cell cycle entry and progression by targeting cyclin-dependent kinase (CDK) inhibitors or transcriptional repressors of the retinoblastoma family. However, tumor suppressor miRNAs induce cell cycle arrest by down-regulating multiple components of the cell cycle machinery.
      • Bueno M.J.
      • Perez de Castro I.
      • Malumbres M.
      Control of cell proliferation pathways by microRNAs.
      • Chen D.
      • Farwell M.A.
      • Zhang B.
      MicroRNA as a new player in the cell cycle.
      miR-885-5p regulates both CDK2 and minichormosome maintenance complex component 5,
      • Afanasyeva E.A.
      • Mestdagh P.
      • Kumps C.
      • Vandesompele J.
      • Ehemann V.
      • Theissen J.
      • Fischer M.
      • Zapatka M.
      • Brors B.
      • Savelyeva L.
      • Sagulenko V.
      • Speleman F.
      • Schwab M.
      • Westermann F.
      MicroRNA miR-885-5p targets CDK2 and MCM5, activates p53 and inhibits proliferation and survival.
      whereas miR-302 targets cyclin-de-2 and CDK2/4.
      • Lin S.L.
      • Chang D.C.
      • Ying S.Y.
      • Leu D.
      • Wu D.T.
      MicroRNA miR-302 inhibits the tumorigenecity of human pluripotent stem cells by coordinate suppression of the CDK2 and CDK4/6 cell cycle pathways.
      miRNAs establish complex feedback circuits that keep cell cycle under tight control
      • Feng M.
      • Yu Q.
      miR-449 regulates CDK-Rb-E2F1 through an auto-regulatory feedback circuit.
      • Sylvestre Y.
      • De Guire V.
      • Querido E.
      • Mukhopadhyay U.K.
      • Bourdeau V.
      • Major F.
      • Ferbeyre G.
      • Chartrand P.
      An E2F/miR-20a autoregulatory feedback loop.
      or are responsible for proliferation control in cancer.
      Although the interaction of a single miRNA with a single target can be investigated and quantified in vitro, it tells little about its biological significance, because every 3′-UTR is probably controlled by a group of miRNAs in vivo. Thus, it is likely that a certain combination of multiple miRNA activities determine the expression of their target genes.
      • Peter M.E.
      Targeting of mRNAs by multiple miRNAs: the next step.
      It will be of great interest to focus the miRNA research on regulatory networks rather than on individual interactions between miRNA and strongly predicted targets. This comprehensive approach might also open the door to new therapeutic applications.
      In conclusion, we provide the first evidence that miRNAs target multiple key molecules along the hypoxia pathway. We found the presence of direct interactions between miR-17-5p and miR-224 with VHL and HIF1α. This hypothesis is supported in vitro at the mRNA and protein levels, as well as in vivo, in ccRCC specimens. We also show that the same miRNA can control multiple targets along the same pathway and that multiple miRNAs can target the same molecule. The importance of our findings is that miRNAs and their respective targets may provide a new line of diagnostic and/or prognostic tumor biomarkers and can represent the basis for new therapeutic strategies in cancer.

      Supplementary Data

      Figure thumbnail grsu1
      Supplemental Figure S1Cluster analysis of the miRNA-target interactions. The 3′-UTRs of RCC pathogenesis-related genes were analyzed for miRNA-target recognition sequences with the use of multiple algorithms. The figure shows the plot of the ccRCC-dysregulated miRNAs against the main hypoxia-related genes. Colors indicate the number of prediction algorithms for a given miRNA/target interaction.
      Figure thumbnail grsu2
      Supplemental Figure S2Expression of miR-17-5p and miR-224 in RCC tissues. A: miR-17-5p endogenous expression levels in all cell lines used in this study. Expression values were normalized against the mammalian small nuclear RNA, RNU44. B: miR-224 expression was quantified from 24 ccRCC specimens. Specimens were ranked according to their miR-224 expression, and samples with higher miR-17-5p expression levels are indicated by black bars. White bars represent specimens with lower miR-17-5p expression levels. Expression values were normalized against RNU44.

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