Down-Regulation of miR-92 in Breast Epithelial Cells and in Normal but Not Tumour Fibroblasts Contributes to Breast Carcinogenesis

Laura Smith, Euan W Baxter, Philip A Chambers, Caroline A Green, Andrew M Hanby, Thomas A Hughes, Claire E Nash, Rebecca A Millican-Slater, Lucy F Stead, Eldo T Verghese, Valerie Speirs

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Abstract

BACKGROUND: MicroRNA (miR) expression is commonly dysregulated in many cancers, including breast. MiR-92 is one of six miRs encoded by the miR-17-92 cluster, one of the best-characterised oncogenic miR clusters. We examined expression of miR-92 in the breast epithelium and stroma during breast cancer progression. We also investigated the role of miR-92 in fibroblasts in vitro and showed that down-regulation in normal fibroblasts enhances the invasion of breast cancer epithelial cells.

METHODOLOGY/PRINCIPAL FINDINGS: We used laser microdissection (LMD) to isolate epithelial cells from matched normal, DCIS and invasive tissue from 9 breast cancer patients and analysed miR-92 expression by qRT-PCR. Expression of ERβ1, a direct miR-92 target, was concurrently analysed for each case by immunohistochemistry. LMD was also used to isolate matched normal (NFs) and cancer-associated fibroblasts (CAFs) from 14 further cases. Effects of miR-92 inhibition in fibroblasts on epithelial cell invasion in vitro was examined using a Matrigel™ assay. miR-92 levels decreased in microdissected epithelial cells during breast cancer progression with highest levels in normal breast epithelium, decreasing in DCIS (p<0.01) and being lowest in invasive breast tissue (p<0.01). This was accompanied by a shift in cell localisation of ERβ1 from nuclear expression in normal breast epithelium to increased cytoplasmic expression during progression to DCIS (p = 0.0078) and invasive breast cancer (p = 0.031). ERβ1 immunoreactivity was also seen in stromal fibroblasts in tissues. Where miR-92 expression was low in microdissected NFs this increased in matched CAFs; a trend also seen in cultured primary fibroblasts. Down-regulation of miR-92 levels in NFs but not CAFs enhanced invasion of both MCF-7 and MDA-MB-231 breast cancer epithelial cells.

CONCLUSIONS: miR-92 is gradually lost in breast epithelial cells during cancer progression correlating with a shift in ERβ1 immunoreactivity from nuclei to the cytoplasm. Our data support a functional role in fibroblasts where modification of miR-92 expression can influence the invasive capacity of breast cancer epithelial cells. However in silico analysis suggests that ERβ1 may not be the most important miR-92 target in breast cancer.

Original languageEnglish
Article numbere0139698
JournalPloS ONE
Volume10
Issue number10
DOIs
Publication statusPublished - 5 Oct 2015

Keywords

  • Adult
  • Aged
  • Aged, 80 and over
  • Breast Neoplasms
  • Carcinogenesis
  • Carcinoma, Intraductal, Noninfiltrating
  • Down-Regulation
  • Epithelial Cells
  • Estrogen Receptor beta
  • Female
  • Fibroblasts
  • Gene Expression Profiling
  • Gene Expression Regulation, Neoplastic
  • Humans
  • Laser Capture Microdissection
  • MicroRNAs
  • Middle Aged
  • Journal Article
  • Research Support, Non-U.S. Gov't

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    Smith, L., Baxter, E. W., Chambers, P. A., Green, C. A., Hanby, A. M., Hughes, T. A., Nash, C. E., Millican-Slater, R. A., Stead, L. F., Verghese, E. T., & Speirs, V. (2015). Down-Regulation of miR-92 in Breast Epithelial Cells and in Normal but Not Tumour Fibroblasts Contributes to Breast Carcinogenesis. PloS ONE, 10(10), [e0139698]. https://doi.org/10.1371/journal.pone.0139698