title: [Dectection and analysis of miRNA expression in breast cancer-associated fibroblasts].
abstract: OBJECTIVE
  To investigate the difference of miRNA expression levels of cancer-associated fibroblasts (CAFs) and normal fibroblasts (NFs) in human breast cancer microenvironment and its effect on the biological features of CAFs.
  
  
  METHODS
  Collagenase-1 was used to digest the cancer and adjacent tissues to isolate CAFs and NFs. The isolated cells were cultured and characterized in purity and biological features. The expression of fibroblast secretory protein (FSP) in CAFs and NFs was detected by immunofluorescence staining and Western blotting. Transwell(TM) assay was adopted to compare the invasion ability of CAFs and NFs. The different expressions of miRNAs in CAFs versus NFs were detected by miRNA microarray and analyzed by Significance Analysis of Microarrays (SAM). The differences in miR-205 and miR-221 expressions were verified by real-time quantitative PCR (qRT-PCR). The common target genes of the miRNAs were predicted using multi-bioinformatics tools. The pathway analysis was conducted through the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7. The secreting products of TGF-β or IL-6 signaling pathway, matrix metalloproteinase (MMP)-1, MMP-2 and MMP-9 were analyzed by ELISA.
  
  
  RESULTS
  The primary CAFs and NFs were isolated from breast cancer patients with a purity of over 95%. Compared with NFs, the expression of FSP was obviously elevated in CAFs, and the invasion ability of CAFs was enhanced. The miRNA microarray results showed that there were 10 miRNA genes dysregulated in CAFs, including 3 up-regulated (miR-221-5p, miR-31-3p, miR-221-3p) and 7 down-regulated genes (miR-205, miR-200b , miR-200c, miR-141, miR-101, miR-342-3p, let-7g). The common targets genes of the dysregulated miRNAs were mainly focused on HGF, chemokine signaling, insulin signaling, MAPK signaling, tight junction signaling, adherence junction signaling, EGF1 signaling, androgen-receptor signaling, Wnt and IL-7 signaling. In addition, dysregulated miR-200b/c and miR-141 et al. affect TGF-β and IL-6 signaling through inhibiting their target genes in CAFs, thus promoting invasion and migration of CAFs.
  
  
  CONCLUSIONS
  The miRNA expression profile was markedly dysregulated in CAFs. Those dysregulated miRNAs may take part in the transformation from NFs to CAFs, and also have a close relationship with adhesion, migration, proliferation, secretion and cell-cell interaction of CAFs.
authors.affiliation: College of Laboratory Medicine, Ministry of Education Key Laboratory of Laboratory Medical Diagnostics, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Zongyue
authors.initials: Z
authors.lastname: Zeng
authors.affiliation: College of Laboratory Medicine, Ministry of Education Key Laboratory of Laboratory Medical Diagnostics, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Hu
authors.affiliation: College of Laboratory Medicine, Ministry of Education Key Laboratory of Laboratory Medical Diagnostics, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Xi
authors.initials: X
authors.lastname: Tang
authors.affiliation: College of Laboratory Medicine, Ministry of Education Key Laboratory of Laboratory Medical Diagnostics, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Hailong
authors.initials: H
authors.lastname: Zhang
authors.affiliation: College of Laboratory Medicine, Ministry of Education Key Laboratory of Laboratory Medical Diagnostics, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Yane
authors.initials: Y
authors.lastname: Du
authors.affiliation: College of Laboratory Medicine, Ministry of Education Key Laboratory of Laboratory Medical Diagnostics, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Siyang
authors.initials: S
authors.lastname: Wen
authors.affiliation: College of Laboratory Medicine, Ministry of Education Key Laboratory of Laboratory Medical Diagnostics, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Manran
authors.initials: M
authors.lastname: Liu
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002448:Cell Adhesion / Q000235:genetics; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D020782:Matrix Metalloproteinases / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D035683:MicroRNAs; D020782:Matrix Metalloproteinases