Tumor Biology

, Volume 33, Issue 1, pp 131–140 | Cite as

Exploring the role of miRNAs in renal cell carcinoma progression and metastasis through bioinformatic and experimental analyses

  • Heba W. Z. Khella
  • Nicole M. A. White
  • Hala Faragalla
  • Manal Gabril
  • Mina Boazak
  • David Dorian
  • Bishoy Khalil
  • Hany Antonios
  • Tian Tian Bao
  • Maria D. Pasic
  • R. John Honey
  • Robert Stewart
  • Kenneth T. Pace
  • Georg A. Bjarnason
  • Michael A. S. Jewett
  • George M. Yousef
Research Article


Metastasis results in most of the cancer deaths in clear cell renal cell carcinoma (ccRCC). MicroRNAs (miRNAs) regulate many important cell functions and play important roles in tumor development, metastasis and progression. In our previous study, we identified a miRNA signature for metastatic RCC. In this study, we validated the top differentially expressed miRNAs on matched primary and metastatic ccRCC pairs by quantitative polymerase chain reaction. We performed bioinformatics analyses including target prediction and combinatorial analysis of previously reported miRNAs involved in tumour progression and metastasis. We also examined the co-expression of the miRNAs clusters and compared expression of intronic miRNAs and their host genes. We observed significant dysregulation between primary and metastatic tumours from the same patient. This indicates that, at least in part, the metastatic signature develops gradually during tumour progression. We identified metastasis-dysregulated miRNAs that can target a number of genes previously found to be involved in metastasis of kidney cancer as well as other malignancies. In addition, we found a negative correlation of expression of miR-126 and its target vascular endothelial growth factor (VEGF)-A. Cluster analysis showed that members of the same miRNA cluster follow the same expression pattern, suggesting the presence of a locus control regulation. We also observed a positive correlation of expression between intronic miRNAs and their host genes, thus revealing another potential control mechanism for miRNAs. Many of the significantly dysregulated miRNAs in metastatic ccRCC are highly conserved among species. Our analysis suggests that miRNAs are involved in ccRCC metastasis and may represent potential biomarkers.


Renal cell carcinoma VEGF-A MicroRNA Tumour markers Metastasis Prognosis 



Clear cell renal cell carcinoma


Epithelial to mesenchymal




Quantitative reverse transcription polymerase chain reaction


Renal cell carcinoma


Vascular endothelial growth factor


Acidic ribosomal phosphoprotein


Epidermal growth factor like 7


Focal adhesion kinase


Targeting insulin-like growth factor 1


Matrix metalopeptidase 2


Hypoxia inducible factor 1 alpha subunit


Platelet-derived growth factor B


Platelet-derived growth factor C


Murine double minute 2


Thymidylate synthase


Von Hippel Lindau

Supplementary material

13277_2011_255_MOESM1_ESM.doc (58 kb)
Supplementary Table 1The 40 top dysregulated miRNAs in metastatic ccRCC compared to primary tumours (DOC 58 kb)
13277_2011_255_MOESM2_ESM.doc (176 kb)
Supplementary Table 2Genes involved in Metastasis of different types of cancers are predicted targets of metastatic ccRCC miRNAs (DOC 175 kb)
13277_2011_255_MOESM3_ESM.doc (174 kb)
Supplementary Table 3Clusters of miRNA dysregulated in Metastasis (DOC 174 kb)
13277_2011_255_MOESM4_ESM.doc (116 kb)
Supplementary Table 4Intronic miRNAs and their host genes differentially expressed in ccRCC metastasis (DOC 116 kb)


  1. 1.
    Chow WH, Dong LM, Devesa SS. Epidemiology and risk factors for kidney cancer. Nat Rev Urol. 2010;7:245–57.PubMedCrossRefGoogle Scholar
  2. 2.
    Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin. 2010;60:277–300.PubMedCrossRefGoogle Scholar
  3. 3.
    Remzi M, Javadli E, Ozsoy M. Management of small renal masses: a review. World J Urol. 2010;28:275–81.PubMedCrossRefGoogle Scholar
  4. 4.
    Weiss RH, Lin PY. Kidney cancer: identification of novel targets for therapy. Kidney Int. 2006;69:224–32.PubMedCrossRefGoogle Scholar
  5. 5.
    Chaffer CL, Weinberg RA. A perspective on cancer cell metastasis. Science. 2011;331:1559–64.PubMedCrossRefGoogle Scholar
  6. 6.
    Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100:57–70.PubMedCrossRefGoogle Scholar
  7. 7.
    Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646–74.PubMedCrossRefGoogle Scholar
  8. 8.
    Coghlin C, Murray GI. Current and emerging concepts in tumour metastasis. J Pathol. 2010;222:1–15.PubMedCrossRefGoogle Scholar
  9. 9.
    Klein CA. Parallel progression of primary tumours and metastases. Nat Rev Cancer. 2009;9:302–12.PubMedCrossRefGoogle Scholar
  10. 10.
    Hunter KW, Crawford NP, Alsarraj J. Mechanisms of metastasis. Breast Cancer Res. 2008;10 Suppl 1:S2.PubMedCrossRefGoogle Scholar
  11. 11.
    Kim MY, Oskarsson T, Acharyya S, Nguyen DX, Zhang XH, Norton L, et al. Tumor self-seeding by circulating cancer cells. Cell. 2009;139:1315–26.PubMedCrossRefGoogle Scholar
  12. 12.
    White NM, Fatoohi E, Metias M, Jung K, Stephan C, Yousef GM. Metastamirs: a stepping stone towards improved cancer management. Nat Rev Clin Oncol. 2011;8:75–84.PubMedCrossRefGoogle Scholar
  13. 13.
    Santarpia L, Nicoloso M, Calin GA. MicroRNAs: a complex regulatory network drives the acquisition of malignant cell phenotype. Endocr Relat Cancer. 2010;17:F51–75.PubMedCrossRefGoogle Scholar
  14. 14.
    Sotiropoulou G, Pampalakis G, Lianidou E, Mourelatos Z. Emerging roles of microRNAs as molecular switches in the integrated circuit of the cancer cell. RNA. 2009;15:1443–61.PubMedCrossRefGoogle Scholar
  15. 15.
    Chow TF, Youssef YM, Lianidou E, Romaschin AD, Honey RJ, Stewart R, et al. Differential expression profiling of microRNAs and their potential involvement in renal cell carcinoma pathogenesis. Clin Biochem. 2010;43:150–8.PubMedCrossRefGoogle Scholar
  16. 16.
    Jung M, Mollenkopf HJ, Grimm C, Wagner I, Albrecht M, Waller T, et al. MicroRNA profiling of clear cell renal cell cancer identifies a robust signature to define renal malignancy. J Cell Mol Med. 2009;13:3918–28.PubMedCrossRefGoogle Scholar
  17. 17.
    Weng L, Wu X, Gao H, Mu B, Li X, Wang JH, et al. MicroRNA profiling of clear cell renal cell carcinoma by whole-genome small RNA deep sequencing of paired frozen and formalin-fixed, paraffin-embedded tissue specimens. J Pathol. 2010;222:41–51.PubMedGoogle Scholar
  18. 18.
    White NM, Bao TT, Grigull J, Youssef YM, Girgis A, Diamandis M, et al. miRNA profiling for clear cell renal cell carcinoma: biomarker discovery and identification of potential controls and consequences of miRNA dysregulation. J Urol. 2011;186(3):1077–83.PubMedCrossRefGoogle Scholar
  19. 19.
    Youssef YM, White NM, Grigull J, Krizova A, Samy C, Mejia-Guerrero S, et al. Accurate molecular classification of kidney cancer subtypes using microRNA signature. Eur Urol. 2011;59:721–30.PubMedCrossRefGoogle Scholar
  20. 20.
    Chow TF, Mankaruos M, Scorilas A, Youssef Y, Girgis A, Mossad S, et al. The miR-17-92 cluster is over expressed in and has an oncogenic effect on renal cell carcinoma. J Urol. 2010;183:743–51.PubMedCrossRefGoogle Scholar
  21. 21.
    Fendler A, Stephan C, Yousef GM, Jung K. MicroRNAs as regulators of signal transduction in urological tumors. Clin Chem. 2011;57:954–68.PubMedCrossRefGoogle Scholar
  22. 22.
    Neal CS, Michael MZ, Rawlings LH, Van der Hoek MB, Gleadle JM. The VHL-dependent regulation of microRNAs in renal cancer. BMC Med. 2010;8:64.PubMedCrossRefGoogle Scholar
  23. 23.
    White NM, Bui A, Mejia-Guerrero S, Chao J, Soosaipillai A, Youssef Y, et al. Dysregulation of kallikrein-related peptidases in renal cell carcinoma: potential targets of miRNAs. Biol Chem. 2010;391:411–23.PubMedCrossRefGoogle Scholar
  24. 24.
    Redova M, Svoboda M, Slaby O. MicroRNAs and their target gene networks in renal cell carcinoma. Biochem Biophys Res Commun. 2011;405:153–6.PubMedCrossRefGoogle Scholar
  25. 25.
    White NM, Yousef GM. MicroRNAs: exploring a new dimension in the pathogenesis of kidney cancer. BMC Med. 2010;8:65.PubMedCrossRefGoogle Scholar
  26. 26.
    Heinzelmann J, Henning B, Sanjmyatav J, Posorski N, Steiner T, Wunderlich H, et al. Specific miRNA signatures are associated with metastasis and poor prognosis in clear cell renal cell carcinoma. World J Urol. 2011;29:367–73.PubMedCrossRefGoogle Scholar
  27. 27.
    White NMA, Khella HWZ, Grigull J, Adzovic S, Youssef YM, Honey RJ, Stewart R, Pace KT, Bjarnason GA, Jewett MAS, Evans AJ, Gabril M, Yousef GM. miRNA profiling in metastatic renal cell carcinoma reveals a tumour suppressor effect for miR-215. Br J Cancer. 2011, in press.Google Scholar
  28. 28.
    Sethupathy P, Megraw M, Hatzigeorgiou AG. A guide through present computational approaches for the identification of mammalian microRNA targets. Nat Methods. 2006;3:881–6.PubMedCrossRefGoogle Scholar
  29. 29.
    Baskerville S, Bartel DP. Microarray profiling of microRNAs reveals frequent coexpression with neighboring miRNAs and host genes. RNA. 2005;11:241–7.PubMedCrossRefGoogle Scholar
  30. 30.
    Blenkiron C, Goldstein LD, Thorne NP, Spiteri I, Chin SF, Dunning MJ, et al. MicroRNA expression profiling of human breast cancer identifies new markers of tumor subtype. Genome Biol. 2007;8:R214.PubMedCrossRefGoogle Scholar
  31. 31.
    Harbour JW, Onken MD, Roberson ED, Duan S, Cao L, Worley LA, et al. Frequent mutation of BAP1 in metastasizing uveal melanomas. Science. 2010;330:1410–3.PubMedCrossRefGoogle Scholar
  32. 32.
    Paris PL, Sridharan S, Hittelman AB, Kobayashi Y, Perner S, Huang G, et al. An oncogenic role for the multiple endocrine neoplasia type 1 gene in prostate cancer. Prostate Cancer Prostatic Dis. 2009;12:184–91.PubMedCrossRefGoogle Scholar
  33. 33.
    Bueno MJ, Malumbres M. MicroRNAs and the cell cycle. Biochim Biophys Acta. 2011;1812:592–601.PubMedGoogle Scholar
  34. 34.
    Liu B, Peng XC, Zheng XL, Wang J, Qin YW. MiR-126 restoration down-regulate VEGF and inhibit the growth of lung cancer cell lines in vitro and in vivo. Lung Cancer. 2009;66:169–75.PubMedCrossRefGoogle Scholar
  35. 35.
    Zhu N, Zhang D, Xie H, Zhou Z, Chen H, Hu T, et al. Endothelial-specific intron-derived miR-126 is down-regulated in human breast cancer and targets both VEGFA and PIK3R2. Mol Cell Biochem. 2011;351:157–64.PubMedCrossRefGoogle Scholar
  36. 36.
    Chuang MJ, Sun KH, Tang SJ, Deng MW, Wu YH, Sung JS, et al. Tumor-derived tumor necrosis factor-alpha promotes progression and epithelial-mesenchymal transition in renal cell carcinoma cells. Cancer Sci. 2008;99:905–13.PubMedCrossRefGoogle Scholar
  37. 37.
    Polanski R, Warburton HE, Ray-Sinha A, Devling T, Pakula H, Rubbi CP, et al. MDM2 promotes cell motility and invasiveness through a RING-finger independent mechanism. FEBS Lett. 2010;584:4695–702.PubMedCrossRefGoogle Scholar
  38. 38.
    Mizutani Y, Wada H, Yoshida O, Fukushima M, Nakao M, Miki T. Significance of thymidine kinase activity in renal cell carcinoma. J Urol. 2003;169:706–9.PubMedCrossRefGoogle Scholar
  39. 39.
    Chhabra R, Dubey R, Saini N. Cooperative and individualistic functions of the microRNAs in the miR-23a 27a 24–2 cluster and its implication in human diseases. Mol Cancer. 2010;9:232.PubMedCrossRefGoogle Scholar
  40. 40.
    Pichiorri F, Suh SS, Rocci A, De LL, Taccioli C, Santhanam R, et al. Downregulation of p53-inducible microRNAs 192, 194, and 215 impairs the p53/MDM2 autoregulatory loop in multiple myeloma development. Cancer Cell. 2010;18:367–81.PubMedCrossRefGoogle Scholar
  41. 41.
    Griffiths-Jones S. Annotating noncoding RNA genes. Annu Rev Genom Hum Genet. 2007;8:279–98.CrossRefGoogle Scholar
  42. 42.
    Girijadevi R, Sreedevi VC, Sreedharan JV, Pillai MR. IntmiR: a complete catalogue of intronic miRNAs of human and mouse. Bioinformation. 2011;5:458–9.PubMedGoogle Scholar
  43. 43.
    Wuttig D, Baier B, Fuessel S, Meinhardt M, Herr A, Hoefling C, et al. Gene signatures of pulmonary metastases of renal cell carcinoma reflect the disease-free interval and the number of metastases per patient. Int J Cancer. 2009;125:474–82.PubMedCrossRefGoogle Scholar
  44. 44.
    Finley DS, Pantuck AJ, Belldegrun AS. Tumor biology and prognostic factors in renal cell carcinoma. Oncologist. 2011;16 Suppl 2:4–13.PubMedCrossRefGoogle Scholar
  45. 45.
    Noon AP, Vlatkovic N, Polanski R, Maguire M, Shawki H, Parsons K, et al. p53 and MDM2 in renal cell carcinoma: biomarkers for disease progression and future therapeutic targets? Cancer. 2010;116:780–90.PubMedCrossRefGoogle Scholar
  46. 46.
    Shi YK, Yu YP, Tseng GC, Luo JH. Inhibition of prostate cancer growth and metastasis using small interference RNA specific for minichromosome complex maintenance component 7. Cancer Gene Ther. 2010;17:694–9.PubMedCrossRefGoogle Scholar
  47. 47.
    Honeycutt KA, Chen Z, Koster MI, Miers M, Nuchtern J, Hicks J, et al. Deregulated minichromosomal maintenance protein MCM7 contributes to oncogene driven tumorigenesis. Oncogene. 2006;25:4027–32.PubMedCrossRefGoogle Scholar
  48. 48.
    Ren B, Yu G, Tseng GC, Cieply K, Gavel T, Nelson J, et al. MCM7 amplification and overexpression are associated with prostate cancer progression. Oncogene. 2006;25:1090–8.PubMedCrossRefGoogle Scholar
  49. 49.
    Yoshida K, Inoue I. Conditional expression of MCM7 increases tumor growth without altering DNA replication activity. FEBS Lett. 2003;553:213–7.PubMedCrossRefGoogle Scholar
  50. 50.
    Yeung ML, Yasunaga J, Bennasser Y, Dusetti N, Harris D, Ahmad N, et al. Roles for microRNAs, miR-93 and miR-130b, and tumor protein 53-induced nuclear protein 1 tumor suppressor in cell growth dysregulation by human T-cell lymphotrophic virus 1. Cancer Res. 2008;68:8976–85.PubMedCrossRefGoogle Scholar
  51. 51.
    Luedde T. MicroRNA-151 and its hosting gene FAK (focal adhesion kinase) regulate tumor cell migration and spreading of hepatocellular carcinoma. Hepatology. 2010;52:1164–6.PubMedCrossRefGoogle Scholar
  52. 52.
    Duxbury MS, Ito H, Zinner MJ, Ashley SW, Whang EE. Focal adhesion kinase gene silencing promotes anoikis and suppresses metastasis of human pancreatic adenocarcinoma cells. Surgery. 2004;135:555–62.PubMedCrossRefGoogle Scholar
  53. 53.
    Miyazaki T, Kato H, Nakajima M, Sohda M, Fukai Y, Masuda N, et al. FAK overexpression is correlated with tumour invasiveness and lymph node metastasis in oesophageal squamous cell carcinoma. Br J Cancer. 2003;89:140–5.PubMedCrossRefGoogle Scholar
  54. 54.
    Weiner TM, Liu ET, Craven RJ, Cance WG. Expression of focal adhesion kinase gene and invasive cancer. Lancet. 1993;342:1024–5.PubMedCrossRefGoogle Scholar
  55. 55.
    Schimanski CC, Frerichs K, Rahman F, Berger M, Lang H, Galle PR, et al. High miR-196a levels promote the oncogenic phenotype of colorectal cancer cells. World J Gastroenterol. 2009;15:2089–96.PubMedCrossRefGoogle Scholar
  56. 56.
    De Souza Setubal Destro MF, Bitu CC, Zecchin KG, Graner E, Lopes MA, Kowalski LP, et al. Overexpression of HOXB7 homeobox gene in oral cancer induces cellular proliferation and is associated with poor prognosis. Int J Oncol. 2010;36:141–9.PubMedGoogle Scholar
  57. 57.
    Ma L, Teruya-Feldstein J, Weinberg RA. Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature. 2007;449:682–8.PubMedCrossRefGoogle Scholar
  58. 58.
    Miyazaki YJ, Hamada J, Tada M, Furuuchi K, Takahashi Y, Kondo S, et al. HOXD3 enhances motility and invasiveness through the TGF-beta-dependent and -independent pathways in A549 cells. Oncogene. 2002;21:798–808.PubMedCrossRefGoogle Scholar
  59. 59.
    Guo C, Sah JF, Beard L, Willson JK, Markowitz SD, Guda K. The noncoding RNA, miR-126, suppresses the growth of neoplastic cells by targeting phosphatidylinositol 3-kinase signaling and is frequently lost in colon cancers. Gene Chromosome Cancer. 2008;47:939–46.CrossRefGoogle Scholar
  60. 60.
    Saito Y, Friedman JM, Chihara Y, Egger G, Chuang JC, Liang G. Epigenetic therapy upregulates the tumor suppressor microRNA-126 and its host gene EGFL7 in human cancer cells. Biochem Biophys Res Commun. 2009;379:726–31.PubMedCrossRefGoogle Scholar
  61. 61.
    Wong TS, Liu XB, Wong BY, Ng RW, Yuen AP, Wei WI. Mature miR-184 as potential oncogenic microRNA of squamous cell carcinoma of tongue. Clin Cancer Res. 2008;14:2588–92.PubMedCrossRefGoogle Scholar
  62. 62.
    Aikawa T, Whipple CA, Lopez ME, Gunn J, Young A, Lander AD, et al. Glypican-1 modulates the angiogenic and metastatic potential of human and mouse cancer cells. J Clin Invest. 2008;118:89–99.PubMedCrossRefGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2011

Authors and Affiliations

  • Heba W. Z. Khella
    • 1
    • 2
  • Nicole M. A. White
    • 1
    • 3
  • Hala Faragalla
    • 1
    • 3
  • Manal Gabril
    • 4
  • Mina Boazak
    • 1
  • David Dorian
    • 1
  • Bishoy Khalil
    • 1
  • Hany Antonios
    • 1
  • Tian Tian Bao
    • 1
  • Maria D. Pasic
    • 3
  • R. John Honey
    • 5
  • Robert Stewart
    • 5
  • Kenneth T. Pace
    • 5
  • Georg A. Bjarnason
    • 6
  • Michael A. S. Jewett
    • 7
  • George M. Yousef
    • 1
    • 3
  1. 1.Department of Laboratory Medicine and the Keenan Research CentreLi Ka Shing Knowledge Institute of St. Michael’s HospitalTorontoCanada
  2. 2.Institute of Medical Science, University of TorontoTorontoCanada
  3. 3.Department of Laboratory Medicine and PathobiologyUniversity of TorontoTorontoCanada
  4. 4.Department of PathologyLondon Health Sciences CentreLondonCanada
  5. 5.Division of Urology, St. Michael’s HospitalTorontoCanada
  6. 6.Division of Medical Oncology and Hematology, Sunnybrook Odette Cancer CenterTorontoCanada
  7. 7.Department of Surgery, Division of Urologic Oncology, Princess Margaret Hospital, University Health NetworkUniversity of TorontoTorontoCanada

Personalised recommendations