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

Research output: Contribution to journalArticle

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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

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Fibroblasts
MicroRNAs
microRNA
carcinogenesis
breasts
fibroblasts
Tumors
Carcinogenesis
Breast
epithelial cells
Down-Regulation
Epithelial Cells
breast neoplasms
neoplasms
Breast Neoplasms
Neoplasms
Carcinoma, Intraductal, Noninfiltrating
Microdissection
epithelium
Epithelium

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

Cite this

Down-Regulation of miR-92 in Breast Epithelial Cells and in Normal but Not Tumour Fibroblasts Contributes to Breast Carcinogenesis. / Smith, Laura; Baxter, Euan W; Chambers, Philip A; Green, Caroline A; Hanby, Andrew M; Hughes, Thomas A; Nash, Claire E; Millican-Slater, Rebecca A; Stead, Lucy F; Verghese, Eldo T; Speirs, Valerie.

In: PloS ONE, Vol. 10, No. 10, e0139698, 05.10.2015.

Research output: Contribution to journalArticle

Smith, L, Baxter, EW, Chambers, PA, Green, CA, Hanby, AM, Hughes, TA, Nash, CE, Millican-Slater, RA, Stead, LF, Verghese, ET & 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, vol. 10, no. 10, e0139698. https://doi.org/10.1371/journal.pone.0139698
Smith, Laura ; Baxter, Euan W ; Chambers, Philip A ; Green, Caroline A ; Hanby, Andrew M ; Hughes, Thomas A ; Nash, Claire E ; Millican-Slater, Rebecca A ; Stead, Lucy F ; Verghese, Eldo T ; Speirs, Valerie. / Down-Regulation of miR-92 in Breast Epithelial Cells and in Normal but Not Tumour Fibroblasts Contributes to Breast Carcinogenesis. In: PloS ONE. 2015 ; Vol. 10, No. 10.
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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.",
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author = "Laura Smith and Baxter, {Euan W} and Chambers, {Philip A} and Green, {Caroline A} and Hanby, {Andrew M} and Hughes, {Thomas A} and Nash, {Claire E} and Millican-Slater, {Rebecca A} and Stead, {Lucy F} and Verghese, {Eldo T} and Valerie Speirs",
note = "Funded by Breast Cancer Now (formerly Breast Cancer Campaign) Grant numbers 2010NovPR56, TB2009LEE (http://breastcancernow.org/) to VS, Medical Research Council Grant number G0902032 (http://www.mrc.ac.uk/) to ETV, and Yorkshire Cancer Research Grant number L316 (http://yorkshirecancerresearch.org.uk/) to VS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.",
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TY - JOUR

T1 - Down-Regulation of miR-92 in Breast Epithelial Cells and in Normal but Not Tumour Fibroblasts Contributes to Breast Carcinogenesis

AU - Smith, Laura

AU - Baxter, Euan W

AU - Chambers, Philip A

AU - Green, Caroline A

AU - Hanby, Andrew M

AU - Hughes, Thomas A

AU - Nash, Claire E

AU - Millican-Slater, Rebecca A

AU - Stead, Lucy F

AU - Verghese, Eldo T

AU - Speirs, Valerie

N1 - Funded by Breast Cancer Now (formerly Breast Cancer Campaign) Grant numbers 2010NovPR56, TB2009LEE (http://breastcancernow.org/) to VS, Medical Research Council Grant number G0902032 (http://www.mrc.ac.uk/) to ETV, and Yorkshire Cancer Research Grant number L316 (http://yorkshirecancerresearch.org.uk/) to VS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

PY - 2015/10/5

Y1 - 2015/10/5

N2 - 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.

AB - 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.

KW - Adult

KW - Aged

KW - Aged, 80 and over

KW - Breast Neoplasms

KW - Carcinogenesis

KW - Carcinoma, Intraductal, Noninfiltrating

KW - Down-Regulation

KW - Epithelial Cells

KW - Estrogen Receptor beta

KW - Female

KW - Fibroblasts

KW - Gene Expression Profiling

KW - Gene Expression Regulation, Neoplastic

KW - Humans

KW - Laser Capture Microdissection

KW - MicroRNAs

KW - Middle Aged

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1371/journal.pone.0139698

DO - 10.1371/journal.pone.0139698

M3 - Article

C2 - 26437339

VL - 10

JO - PloS ONE

JF - PloS ONE

SN - 1932-6203

IS - 10

M1 - e0139698

ER -