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. 2012;7(4):e36051.
doi: 10.1371/journal.pone.0036051. Epub 2012 Apr 26.

Downregulation of miR-92a is associated with aggressive breast cancer features and increased tumour macrophage infiltration

Sofie Nilsson  1 , Christina Möller, Karin Jirström, Alexander Lee, Susann Busch, Rebecca Lamb, Göran Landberg
Affiliations

Downregulation of miR-92a is associated with aggressive breast cancer features and increased tumour macrophage infiltration

Sofie Nilsson et al. PLoS One. 2012.

Abstract

Background: MicroRNAs are small non-coding RNAs involved in the regulation of gene expression on a posttranscriptional level. These regulatory RNAs have been implicated in numerous cellular processes and are further deregulated in different cancer types, including breast cancer. MiR-92a is part of the miR-17∼92 cluster, which was first reported to be linked to tumourigenesis. However, little is known about the expression of miR-92a in breast cancer and potential associations to tumour properties. The expression of miR-92a was therefore characterized in 144 invasive breast cancer samples using in situ hybridization and related to clinico-pathological data as well as to selected key properties of the tumour stroma, including the presence of macrophages (CD68) and cancer activated fibroblasts (alpha-SMA).

Methodology/principal findings: To measure miR-92a levels, an in situ hybridisation protocol was developed and validated using cell lines and miR-92a inhibitors. The expression in the tumour samples was objectively evaluated using digital image analysis program subtracting background activities. We found that the miR-92a expression varied between tumours and was inversely correlated to tumour grade (r = -0.276, p = 0.003) and recurrence-free survival (p = 0.008) and provided independent prognostic information in multivariate Cox analysis (HR: 0.375, CI: 0.145-0.972, p = 0.043). MiR-92a was moreover inversely correlated to the number of infiltrating macrophages in the tumour stroma (r = -0.357, p<0.001), and downregulation of miR-92a promoted cell migration (p<0.01).

Conclusions/significance: This study demonstrates that downregulation of miR-92a in breast cancer is linked to key epithelial and stromal properties as well as clinical outcome.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Validation of miR-92a in situ hybridization probe.
A) Downregulated of miR-92a in MDA-231 cells was validated and confirmed by qRT-PCR. B) Detection of miR-92a using in situ hybridization in MDA-231 cells after downregulation of miR-92a. Lower panel shows the definition of positive pixels identified by the digital image analysis program. C) Quantification of positive pixels by the image analysis program.
Figure 2
Figure 2. MiR-92a expression in normal breast and breast cancer samples.
A) and B) Two examples of miR-92a expression in normal breast tissue compared to tumour areas.
Figure 3
Figure 3. MiR-92a expression in breast cancer samples.
A) In situ hybridization was performed on a breast cancer tissue microarray using a detection probe for miR-92a and a negative control probe. Tumours were divided into quartiles 1–4 (Q1–Q4) based on the staining intensity (Q1 = weakest staining, Q4 = strongest staining). 20× magnification. B) Example of positive pixel definition of representative Q4 tumour stained with miR-92a probe and negative control probe. 40× magnification.
Figure 4
Figure 4. Recurrence-free survival among breast cancer patients according to miR-92a expression.
A) Tumours were divided into four groups based on the staining intensity of miR-92a and recurrence-free survival for the patients was determined, p = 0.065. B) Recurrence-free survival analysis after merging of tumours in Q1–Q2 and Q3–Q4, p = 0.008.
Figure 5
Figure 5. Cellular properties of miR-92a in breast cancer cell line MDA-231.
Downregulation of miR-92a in MDA-231 cells led to in increased migration (A) but did not affect the proliferation of the cells (B).
Figure 6
Figure 6. Inverse link between CD68-positive macrophages and miR-92a expression.
Tumours were divided into quartiles based on number of CD68-positive cells (macrophages) and compared to miR-92a staining intensity.
See this image and copyright information in PMC

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