Expression profiling of cancerous and normal breast tissues identifies microRNAs that are differentially expressed in serum from patients with (metastatic) breast cancer and healthy volunteers

IntroductionMicroRNAs (miRNAs) are a group of small noncoding RNAs involved in the regulation of gene expression. As such, they regulate a large number of cellular pathways, and deregulation or altered expression of miRNAs is associated with tumorigenesis. In the current study, we evaluated the feasibility and clinical utility of circulating miRNAs as biomarkers for the detection and staging of breast cancer.MethodsmiRNAs were extracted from a set of 84 tissue samples from patients with breast cancer and eight normal tissue samples obtained after breast-reductive surgery. After reverse transcription and preamplification, 768 miRNAs were profiled by using the TaqMan low-density arrays. After data normalization, unsupervised hierarchical cluster analysis (UHCA) was used to investigate global differences in miRNA expression between cancerous and normal samples. With fold-change analysis, the most discriminating miRNAs between both tissue types were selected, and their expression was analyzed on serum samples from 20 healthy volunteers and 75 patients with breast cancer, including 16 patients with untreated metastatic breast cancer. miRNAs were extracted from 200 μl of serum, reverse transcribed, and analyzed in duplicate by using polymerase chain reaction (qRT-PCR).ResultsUHCA showed major differences in miRNA expression between tissue samples from patients with breast cancer and tissue samples from breast-reductive surgery (P < 0.0001). Generally, miRNA expression in cancerous samples tends to be repressed when compared with miRNA expression in healthy controls (P = 0.0685). The four most discriminating miRNAs by fold-change (miR-215, miR-299-5p, miR-411, and miR-452) were selected for further analysis on serum samples. All miRNAs at least tended to be differentially expressed between serum samples from patients with cancer and serum samples from healthy controls (miR-215, P = 0.094; miR-299-5P, P = 0.019; miR-411, P = 0.002; and miR-452, P = 0.092). For all these miRNAs, except for miR-452, the greatest difference in expression was observed between serum samples from healthy volunteers and serum samples from untreated patients with metastatic breast cancer.ConclusionsOur study provides a basis for the establishment of miRNAs as biomarkers for the detection and eventually staging of breast cancer through blood-borne testing. We identified and tested a set of putative biomarkers of breast cancer and demonstrated that altered levels of these miRNAs in serum from patients with breast cancer are particularly associated with the presence of metastatic disease.

[1]  C. Croce,et al.  MicroRNA gene expression deregulation in human breast cancer. , 2005, Cancer research.

[2]  Frank Speleman,et al.  A novel and universal method for microRNA RT-qPCR data normalization , 2009, Genome Biology.

[3]  Peizhang Xu,et al.  MicroRNAs and the regulation of cell death. , 2004, Trends in genetics : TIG.

[4]  C. Croce,et al.  A microRNA expression signature of human solid tumors defines cancer gene targets , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[5]  A. Nobel,et al.  Supervised risk predictor of breast cancer based on intrinsic subtypes. , 2009, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[6]  J. Lieberman,et al.  let-7 Regulates Self Renewal and Tumorigenicity of Breast Cancer Cells , 2007, Cell.

[7]  George A. Calin,et al.  Mammalian microRNAs: a small world for fine-tuning gene expression , 2006, Mammalian Genome.

[8]  G. Lutz,et al.  Nanopolymers improve delivery of exon skipping oligonucleotides and concomitant dystrophin expression in skeletal muscle of mdx mice , 2008, BMC biotechnology.

[9]  E. van Marck,et al.  The presence of circulating total DNA and methylated genes is associated with circulating tumour cells in blood from breast cancer patients , 2009, British Journal of Cancer.

[10]  F. Slack,et al.  RAS Is Regulated by the let-7 MicroRNA Family , 2005, Cell.

[11]  M Van Glabbeke,et al.  New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. , 2000, Journal of the National Cancer Institute.

[12]  H. Horvitz,et al.  MicroRNA expression profiles classify human cancers , 2005, Nature.

[13]  Michael L. Gatza,et al.  A pathway-based classification of human breast cancer , 2010, Proceedings of the National Academy of Sciences.

[14]  C. Morrison,et al.  MicroRNA-29 family reverts aberrant methylation in lung cancer by targeting DNA methyltransferases 3A and 3B , 2007, Proceedings of the National Academy of Sciences.

[15]  T. Siegal,et al.  Serum DNA can define tumor-specific genetic and epigenetic markers in gliomas of various grades. , 2010, Neuro-oncology.

[16]  Phillip D. Zamore,et al.  Ribo-gnome: The Big World of Small RNAs , 2005, Science.

[17]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[18]  Lin He,et al.  MicroRNAs: small RNAs with a big role in gene regulation , 2004, Nature Reviews Genetics.

[19]  Robert A. Weinberg,et al.  Tumour invasion and metastasis initiated by microRNA-10b in breast cancer (Nature (2007) 449, (682-688)) , 2008 .

[20]  J. Castle,et al.  Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs , 2005, Nature.

[21]  Leonard D. Goldstein,et al.  MicroRNA expression profiling of human breast cancer identifies new markers of tumor subtype , 2007, Genome Biology.

[22]  M. van Glabbeke,et al.  New guidelines to evaluate the response to treatment in solid tumors , 2000, Journal of the National Cancer Institute.

[23]  M. Byrom,et al.  Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis , 2005, Nucleic acids research.

[24]  Crislyn D'Souza-Schorey,et al.  Microvesicles: mediators of extracellular communication during cancer progression , 2010, Journal of Cell Science.

[25]  D. Sgroi,et al.  The molecular pathology of breast cancer progression , 2011, The Journal of pathology.

[26]  V. Ambros.  MicroRNA Pathways in Flies and Worms Growth, Death, Fat, Stress, and Timing , 2003, Cell.

[27]  Patrick Pauwels,et al.  Array-Based DNA Methylation Profiling for Breast Cancer Subtype Discrimination , 2010, PloS one.

[28]  Christophe Lemetre,et al.  MicroRNA signatures predict oestrogen receptor, progesterone receptor and HER2/neu receptor status in breast cancer , 2009, Breast Cancer Research.

[29]  M. F. Shannon,et al.  An autocrine TGF-β/ZEB/miR-200 signaling network regulates establishment and maintenance of epithelial-mesenchymal transition , 2011, Molecular biology of the cell.

[30]  J. Baak,et al.  Biologic profiling of lymph node negative breast cancers by means of microRNA expression , 2010, Modern Pathology.

[31]  Xantha Karp,et al.  Encountering MicroRNAs in Cell Fate Signaling , 2005, Science.

[32]  Lianbo Yu,et al.  Detection of microRNA Expression in Human Peripheral Blood Microvesicles , 2008, PloS one.

[33]  C. Burge,et al.  Conserved Seed Pairing, Often Flanked by Adenosines, Indicates that Thousands of Human Genes are MicroRNA Targets , 2005, Cell.

[34]  J. Lötvall,et al.  Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells , 2007, Nature Cell Biology.

[35]  P. V. van Dam,et al.  Circulating tumour cell detection: a direct comparison between the CellSearch System, the AdnaTest and CK-19/mammaglobin RT–PCR in patients with metastatic breast cancer , 2009, British Journal of Cancer.

[36]  Zhijun Duan,et al.  Epigenetic Regulation of Protein-Coding and MicroRNA Genes by the Gfi1-Interacting Tumor Suppressor PRDM5 , 2007, Molecular and Cellular Biology.

[37]  Jason I. Herschkowitz,et al.  Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer , 2010, Breast Cancer Research.

[38]  Sijin Liu,et al.  Inhibition of rho-associated kinase signaling prevents breast cancer metastasis to human bone. , 2009, Cancer research.

[39]  Matthew A. Titmus,et al.  Molecular mechanism of chemoresistance by miR-215 in osteosarcoma and colon cancer cells , 2010, Molecular Cancer.

[40]  Ulrich Lehmann,et al.  MicroRNA profiles of healthy basal and luminal mammary epithelial cells are distinct and reflected in different breast cancer subtypes , 2011, Breast Cancer Research and Treatment.

[41]  T. Ihalainen,et al.  Internalization of novel non-viral vector TAT-streptavidin into human cells , 2007, BMC biotechnology.

[42]  Danish Sayed,et al.  MicroRNAs in development and disease. , 2011, Physiological reviews.

[43]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[44]  Stefano Volinia,et al.  MicroRNA expression profiling of human metastatic cancers identifies cancer gene targets , 2009, The Journal of pathology.

[45]  E. van Marck,et al.  Distinct molecular phenotype of inflammatory breast cancer compared to non-inflammatory breast cancer using Affymetrix-based genome-wide gene-expression analysis , 2007, British Journal of Cancer.

[46]  E. Kroh,et al.  Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma , 2011, Proceedings of the National Academy of Sciences.

[47]  Daniel B. Martin,et al.  Circulating microRNAs as stable blood-based markers for cancer detection , 2008, Proceedings of the National Academy of Sciences.

[48]  Mark Gerstein,et al.  mRNA expression profiles show differential regulatory effects of microRNAs between estrogen receptor-positive and estrogen receptor-negative breast cancer , 2009, Genome Biology.

[49]  Michael J Kerin,et al.  Circulating microRNAs as Novel Minimally Invasive Biomarkers for Breast Cancer , 2010, Annals of surgery.

[50]  G. Hannon,et al.  The estrogen receptor-α-induced microRNA signature regulates itself and its transcriptional response , 2009, Proceedings of the National Academy of Sciences.

[51]  Christian A. Rees,et al.  Molecular portraits of human breast tumours , 2000, Nature.

[52]  Robin L. Jones,et al.  Down-regulation of the miRNA master regulators Drosha and Dicer is associated with specific subgroups of breast cancer. , 2011, European journal of cancer.

[53]  Wei Yu,et al.  The SOX2 response program in glioblastoma multiforme: an integrated ChIP-seq, expression microarray, and microRNA analysis , 2011, BMC Genomics.

[54]  M. L. Hastings,et al.  Selective Release of MicroRNA Species from Normal and Malignant Mammary Epithelial Cells , 2010, PloS one.

[55]  F. Ferrari,et al.  A MicroRNA Targeting Dicer for Metastasis Control , 2010, Cell.

[56]  John W M Martens,et al.  mRNA and microRNA Expression Profiles in Circulating Tumor Cells and Primary Tumors of Metastatic Breast Cancer Patients , 2011, Clinical Cancer Research.

[57]  Hongling Li,et al.  miR-17-5p promotes human breast cancer cell migration and invasion through suppression of HBP1 , 2011, Breast Cancer Research and Treatment.

[58]  K. Vickers,et al.  MicroRNAs are Transported in Plasma and Delivered to Recipient Cells by High-Density Lipoproteins , 2011, Nature Cell Biology.

[59]  Yong Li,et al.  MicroRNAs in NF-kappaB signaling. , 2011, Journal of molecular cell biology.

[60]  I. Van der Auwera,et al.  Integrated miRNA and mRNA expression profiling of the inflammatory breast cancer subtype , 2010, British Journal of Cancer.

[61]  Y. Shoshan,et al.  Gliomas display a microRNA expression profile reminiscent of neural precursor cells. , 2010, Neuro-oncology.

[62]  Kazuhiko Hayashi,et al.  Systematic analysis of microRNA expression of RNA extracted from fresh frozen and formalin-fixed paraffin-embedded samples. , 2007, RNA.

[63]  R. Pillai.  MicroRNA function: multiple mechanisms for a tiny RNA? , 2005, RNA.

[64]  Yariv Yogev,et al.  Serum MicroRNAs Are Promising Novel Biomarkers , 2008, PloS one.

[65]  G. Goodall,et al.  The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1 , 2008, Nature Cell Biology.