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Pathology & Oncology Research

, Volume 25, Issue 1, pp 233–239 | Cite as

MicroRNA Expression in Laser Micro-dissected Breast Cancer Tissue Samples – a Pilot Study

  • Edward Seclaman
  • Diana Narita
  • Andrei Anghel
  • Natalia Cireap
  • Razvan Ilina
  • Ioan Ovidiu Sirbu
  • Catalin MarianEmail author
Original Article

Abstract

Breast cancer continues to represent a significant public health burden despite outstanding research advances regarding the molecular mechanisms of cancer biology, biomarkers for diagnostics and prognostic and therapeutic management of this disease. The studies of micro RNAs in breast cancer have underlined their potential as biomarkers and therapeutic targets; however most of these studies are still done on largely heterogeneous whole breast tissue samples. In this pilot study we have investigated the expression of four micro RNAs (miR-21, 145, 155, 92) known to be involved in breast cancer, in homogenous cell populations collected by laser capture microdissection from breast tissue section slides. Micro RNA expression was assessed by real time PCR, and associations with clinical and pathological characteristics were also explored. Our results have confirmed previous associations of miR-21 expression with poor prognosis characteristics of breast cancers such as high stage, large and highly proliferative tumors. No statistically significant associations were found with the other micro RNAs investigated, possibly due to the small sample size of our study. Our results also suggest that miR-484 could be a suitable endogenous control for data normalization in breast tissues, these results needing further confirmation by future studies. In summary, our pilot study showed the feasibility of detecting micro RNAs expression in homogenous laser captured microdissected invasive breast cancer samples, and confirmed some of the previously reported associations with poor prognostic characteristics of breast tumors.

Keywords

Breast cancer MicroRNA Laser capture microdissection (LCM) 

References

  1. 1.
    Al-Nakhle H, Burns PA, Cummings M, Hanby AM, Hughes TA, Satheesha S et al (2010) Estrogen receptor {beta}1 expression is regulated by miR-92 in breast cancer. Cancer Res 70:4778–4784CrossRefGoogle Scholar
  2. 2.
    Andersen CL, Jensen JL, Orntoft TF (2004) Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64:5245–5250CrossRefGoogle Scholar
  3. 3.
    Blenkiron C, Goldstein LD, Thorne NP, Spiteri I, Chin SF, Dunning MJ et al (2007) MicroRNA expression profiling of human breast cancer identifies new markers of tumor subtype. Genome Bio 8:R214CrossRefGoogle Scholar
  4. 4.
    Calin GA, Croce CM (2006) MicroRNA signatures in human cancers. Nat Rev Cancer 6:857–866CrossRefGoogle Scholar
  5. 5.
    Chen J, Wang X (2014) MicroRNA-21 in breast cancer: diagnostic and prognostic potential. Clin Transl Oncol 16:225–233CrossRefGoogle Scholar
  6. 6.
    Chen L, Li Y, Fu Y, Peng J, Mo MH, Stamatakos M et al (2013) Role of deregulated microRNAs in breast cancer progression using FFPE tissue. PLoS One 8:e54213CrossRefGoogle Scholar
  7. 7.
    Davoren PA, McNeill RE, Lowery AJ, Kerin MJ, Miller N (2008) Identification of suitable endogenous control genes for microRNA gene expression analysis in human breast cancer. BMC Mol Biol 9:76CrossRefGoogle Scholar
  8. 8.
    Ferlay J, Steliarova-Foucher E, Lortet-Tieulent J, Rosso S, Coebergh JW, Comber H et al (2013) Cancer incidence and mortality patterns in Europe: estimates for 40 countries in 2012. Eur J Cancer 49:1374–1403CrossRefGoogle Scholar
  9. 9.
    Fetica B, Balacescu O, Balacescu L, Rus M, Berindan-Neagoe I (2014) An alternative and sensitive method based on LCM and Q-PCR for HER2 testing in breast cancer. Cancer Biomark 14:129–135CrossRefGoogle Scholar
  10. 10.
    Foekens JA, Sieuwerts AM, Smid M, Look MP, de Weerd V, Boersma AW, et al (2008) Four miRNAs associated with aggressiveness of lymph node-negative, estrogen receptor-positive human breast cancer. Proc Natl Acad Sci U S A 105:13021–13026Google Scholar
  11. 11.
    Hagrass HA, Sharaf S, Pasha HF, Tantawy EA, Mohamed RH, Kassem R (2015) Circulating microRNAs - a new horizon in molecular diagnosis of breast cancer. Genes Cancer 6:281–287Google Scholar
  12. 12.
    Hannafon BN, Sebastiani P, De Las Morenas A, Lu J, Rosenberg CL (2011) Expression of microRNA and their gene targets are dysregulated in preinvasive breast cancer. Breast Cancer Res 13:R24CrossRefGoogle Scholar
  13. 13.
    Hasemeier B, Christgen M, Kreipe H, Lehmann U (2008) Reliable microRNA profiling in routinely processed formalin-fixed paraffin-embedded breast cancer specimens using fluorescence labelled bead technology. BMC Biotechnol 8:90CrossRefGoogle Scholar
  14. 14.
    Hu Z, Dong J, Wang LE, Ma H, Liu J, Zhao Y et al (2012) Serum microRNA profiling and breast cancer risk: the use of miR-484/191 as endogenous controls. Carcinogenesis 33:828–834CrossRefGoogle Scholar
  15. 15.
    Huang Q, Gumireddy K, Schrier M, le Sage C, Nagel R, Nair S, et al. (2008) The microRNAs miR-373 and miR-520c promote tumour invasion and metastasis. Nat Cell Biol 10:202–210Google Scholar
  16. 16.
    Hui AB, Shi W, Boutros PC, Miller N, Pintilie M, Fyles T et al (2009) Robust global micro-RNA profiling with formalin-fixed paraffin-embedded breast cancer tissues. Lab Investig 89:597–606CrossRefGoogle Scholar
  17. 17.
    Iorio MV, Ferracin M, Liu CG, Veronese A, Spizzo R, Sabbioni S et al (2005) MicroRNA gene expression deregulation in human breast cancer. Cancer Res 65:7065–7070CrossRefGoogle Scholar
  18. 18.
    Iorio MV, Casalini P, Tagliabue E, Menard S, Croce CM (2008) MicroRNA profiling as a tool to understand prognosis, therapy response and resistance in breast cancer. Eur J Cancer 44:2753–2759CrossRefGoogle Scholar
  19. 19.
    Khoshnaw SM, Powe DG, Ellis IO, Green AR (2011) Detection and quantification of microRNAs in laser-microdissected formalin-fixed paraffin-embedded breast cancer tissues. Methods Mol Biol 755:119–142CrossRefGoogle Scholar
  20. 20.
    Klopfleisch R, Weiss AT, Gruber AD (2011) Excavation of a buried treasure DNA, mRNA, miRNA and protein analysis in formalin fixed, paraffin embedded tissues. Histol Histopathol 26:797–810Google Scholar
  21. 21.
    Kodahl AR, Lyng MB, Binder H, Cold S, Gravgaard K, Knoop AS et al (2014) Novel circulating microRNA signature as a potential non-invasive multi-marker test in ER-positive early-stage breast cancer: a case control study. Mol Oncol 8:874–883CrossRefGoogle Scholar
  22. 22.
    Kong W, Zhao JJ, He L, Cheng JQ (2009) Strategies for profiling microRNA expression. J Cell Physiol 218:22–25CrossRefGoogle Scholar
  23. 23.
    Liu H, McDowell TL, Hanson NE, Tang X, Fujimoto J, Rodriguez-Canales J (2014) Laser capture microdissection for the investigative pathologist. Vet Pathol 51:257–269CrossRefGoogle Scholar
  24. 24.
    Lou G, Ma N, Xu Y, Jiang L, Yang J, Wang C et al (2015) Differential distribution of U6 (RNU6-1) expression in human carcinoma tissues demonstrates the requirement for caution in the internal control gene selection for microRNA quantification. Int J Mol Med 36(5):1400–1408CrossRefGoogle Scholar
  25. 25.
    Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D et al (2005) MicroRNA expression profiles classify human cancers. Nature 435:834–838CrossRefGoogle Scholar
  26. 26.
    Ma L, Teruya-Feldstein J, Weinberg RA (2007) Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature 449:682–688CrossRefGoogle Scholar
  27. 27.
    Mar-Aguilar F, Mendoza-Ramirez JA, Malagon-Santiago I, Espino-Silva PK, Santuario-Facio SK, Ruiz-Flores P et al (2013) Serum circulating microRNA profiling for identification of potential breast cancer biomarkers. Dis Markers 34:163–169CrossRefGoogle Scholar
  28. 28.
    Mattiske S, Suetani RJ, Neilsen PM, Callen DF (2012) The oncogenic role of miR-155 in breast cancer. Cancer Epidemiol Biomark Prev 21:1236–1243CrossRefGoogle Scholar
  29. 29.
    Min W, Wang B, Li J, Han J, Zhao Y, Su W et al (2014) The expression and significance of five types of miRNAs in breast cancer. Med Sci Monit Basic Res 20:97–104Google Scholar
  30. 30.
    Murray GI (2011) Laser Capture Microdissection: Methods and Protocols. Methods in molecular biology. SpringerGoogle Scholar
  31. 31.
    Narita D, Anghel A, Seclaman E, Ilina R, Cireap N, Ursoniu S (2010) Molecular profiling of ADAM12 gene in breast cancers. Romanian J Morphol Embryol 51:669–676Google Scholar
  32. 32.
    Ng EK, Li R, Shin VY, Jin HC, Leung CP, Ma ES et al (2013) Circulating microRNAs as specific biomarkers for breast cancer detection. PLoS One 8:e53141CrossRefGoogle Scholar
  33. 33.
    Nilsson S, Moller C, Jirstrom K, Lee A, Busch S, Lamb R et al (2012) Downregulation of miR-92a is associated with aggressive breast cancer features and increased tumour macrophage infiltration. PLoS One 7:e36051CrossRefGoogle Scholar
  34. 34.
    Pan F, Mao H, Deng L, Li G, Geng P (2014) Prognostic and clinicopathological significance of microRNA-21 overexpression in breast cancer: a meta-analysis. Int J Clin Exp Pathol 7:5622–5633Google Scholar
  35. 35.
    Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations. Biotechnol Lett 26:509–515CrossRefGoogle Scholar
  36. 36.
    Rice J, Roberts H, Rai SN, Galandiuk S (2015) Housekeeping genes for studies of plasma microRNA: A need for more precise standardization. Surgery 158(5):1345–1351CrossRefGoogle Scholar
  37. 37.
    Schmittgen TD, Lee EJ, Jiang J, Sarkar A, Yang L, Elton TS et al (2008) Real-time PCR quantification of precursor and mature microRNA. Methods 44:31–38CrossRefGoogle Scholar
  38. 38.
    Si ML, Zhu S, Wu H, Lu Z, Wu F, Mo YY (2007) miR-21-mediated tumor growth. Oncogene 26:2799–2803CrossRefGoogle Scholar
  39. 39.
    Si H, Sun X, Chen Y, Cao Y, Chen S, Wang H et al (2013) Circulating microRNA-92a and microRNA-21 as novel minimally invasive biomarkers for primary breast cancer. J Cancer Res Clin Oncol 139:223–229CrossRefGoogle Scholar
  40. 40.
    Siebolts U, Varnholt H, Drebber U, Dienes HP, Wickenhauser C, Odenthal M (2009) Tissues from routine pathology archives are suitable for microRNA analyses by quantitative PCR. J Clin Pathol 62:84–88CrossRefGoogle Scholar
  41. 41.
    Silver N, Best S, Jiang J, Thein SL (2006) Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol Biol 7:33CrossRefGoogle Scholar
  42. 42.
    Society AC. Cancer Facts & Figures 2015 (2015) Atlanta: American Cancer SocietyGoogle Scholar
  43. 43.
    Stahel R, Bogaerts J, Ciardiello F, de Ruysscher D, Dubsky P, Ducreux M, et al (2015) Optimising translational oncology in clinical practice: strategies to accelerate progress in drug development. Cancer Treat Rev 41:129–135Google Scholar
  44. 44.
    Stefani G, Slack FJ (2008) Small non-coding RNAs in animal development. Nat Rev Mol Cell Biol 9:219–230CrossRefGoogle Scholar
  45. 45.
    Sun EH, Zhou Q, Liu KS, Wei W, Wang CM, Liu XF et al (2014) Screening miRNAs related to different subtypes of breast cancer with miRNAs microarray. Eur Rev Med Pharmacol Sci 18:2783–2788Google Scholar
  46. 46.
    Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:RESEARCH0034Google Scholar
  47. 47.
    Wang J, Wu J (2012) Role of miR-155 in breast cancer. Front Biosci (Landmark Ed) 17:2350–2355CrossRefGoogle Scholar
  48. 48.
    Wang F, Hou J, Jin W, Li J, Yue Y, Jin H et al (2014) Increased circulating microRNA-155 as a potential biomarker for breast cancer screening: a meta-analysis. Molecules 19:6282–6293CrossRefGoogle Scholar
  49. 49.
    Xi Y, Nakajima G, Gavin E, Morris CG, Kudo K, Hayashi K et al (2007) Systematic analysis of microRNA expression of RNA extracted from fresh frozen and formalin-fixed paraffin-embedded samples. RNA 13:1668–1674CrossRefGoogle Scholar
  50. 50.
    Yan X, Chen X, Liang H, Deng T, Chen W, Zhang S et al (2014) miR-143 and miR-145 synergistically regulate ERBB3 to suppress cell proliferation and invasion in breast cancer. Mol Cancer 13:220CrossRefGoogle Scholar
  51. 51.
    Zhang L, Huang J, Yang N, Greshock J, Megraw MS, Giannakakis A et al (2006) microRNAs exhibit high frequency genomic alterations in human cancer. Proc Natl Acad Sci U S A 103:9136–9141CrossRefGoogle Scholar
  52. 52.
    Zou C, Xu Q, Mao F, Li D, Bian C, Liu LZ et al (2012) MiR-145 inhibits tumor angiogenesis and growth by N-RAS and VEGF. Cell Cycle 11:2137–2145CrossRefGoogle Scholar

Copyright information

© Arányi Lajos Foundation 2017

Authors and Affiliations

  1. 1.Department of Biochemistry and Pharmacology“Victor Babeş” University of Medicine and PharmacyTimişoaraRomania
  2. 2.Donauisar KlinikumInstitute for Laboratory Diagnostic and Transfusion MedicineDeggendorfGermany
  3. 3.Department of Surgical OncologyUniversity of Medicine and Pharmacy “Victor Babes”, and Municipal HospitalTimisoaraRomania

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