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Profiling differential microRNA expression between in situ, infiltrative and lympho-vascular space invasive breast cancer: a pilot study

Abstract

Ductal carcinoma in situ (DCIS), invasive breast cancer (IBC) and lympho-vascular invasion (LVI) represent distinct stages in breast cancer progression with different clinical implications. Altered microRNA (miRNA) expression may play a role in mediating the progression of DCIS to IBC and LVI. The aim of this pilot study was to investigate whether differential miRNA expression could play a role in breast cancer progression. Cancer cells from DCIS, IBC and LVI were microdissected from formalin fixed paraffin embedded (FFPE) tissue of five breast cancer samples. MiRNA profiling of extracted RNA was performed using the TaqMan® Array Human MicroRNA Cards A and B v3.0. Candidate miRNAs and gene targets were validated by qPCR. 3D culture of MCF10A, MCF10DCIS.com and T47D cells were used as models for normal, DCIS and IBC. Immunohistochemistry of candidate genes was performed on FFPE 3D cell cultures as well as on tissue microarray which included cores of DCIS and IBC samples. MiR-150, miR-126 and miR-155 were found to be more highly expressed in IBC and LVI compared to DCIS. Gene targets of these miRNAs, RhoA, PEG10 and MYB, were found to be more highly expressed in DCIS compared to IBC by qPCR and in MCF10A and MCF10DCIS.com cells compared to T47D cells by immunohistochemistry. There was no difference in intensity of staining of RhoA by immunohistochemistry in DCIS versus IBC samples on tissue microarray. In this pilot study, we found evidence to support a potential role for variation in miRNA levels in the transition from DCIS to IBC.

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Acknowledgements

This work was funded by Cure Cancer Australia Foundation/Cancer Australia (APP632547) and National Breast Cancer Foundation Novel Concept Award/Pilot Study Grant. RLB was a CINSW Fellow. Tissue samples were received from the Australian Breast Cancer Tissue Bank (http://www.abctb.org) which is generously supported by the National Health and Medical Research Council of Australia, The Cancer Institute NSW (CINSW) and the National Breast Cancer Foundation. The tissues and samples are made available to researchers on a non-exclusive basis. The assistance of Virginia James is gratefully acknowledged.

Author information

Correspondence to Patsy S. Soon.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1—Relative expression of 102 miRNAs in the TaqMan® Array Human MicroRNA cards A (97 miRNAs) and B (5 miRNAs) and 3 housekeepers (MammU6, RNU44 and RNU48) in 5 matched samples of DCIS, IBC and LVI analysed using DataAssist (XLSX 52 KB)

Supplementary material 2—Differentially expressed miRNAs in IBC compared to DCIS samples as analysed with DataAssist. Twenty-nine miRNAs were overexpressed and 3 miRNAs were underexpressed in at least 4 of the 5 IBC samples compared to DCIS. List of 24 genes, potential targets of candidate miRNAs, and 4 housekeeper genes assessed by qPCR using TaqMan® Custom 96-well Plates (DOCX 65 KB)

Supplementary material 3—Comparison of eight miRNA expression results using Taqman TLDA card and single well qPCR data. Greater variability was noted in qPCR data compared to TLDA data (DOCX 373 KB)

Supplementary material 4—Intensity of staining for RhoA in matched samples of DCIS and IBC in 27 cases (p=0.08) (DOCX 70 KB)

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Soon, P.S., Provan, P.J., Kim, E. et al. Profiling differential microRNA expression between in situ, infiltrative and lympho-vascular space invasive breast cancer: a pilot study. Clin Exp Metastasis 35, 3–13 (2018). https://doi.org/10.1007/s10585-017-9868-4

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Keywords

  • Breast cancer
  • Ductal carcinoma in situ
  • Lymphovascular invasion
  • MicroRNA