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ALCAM is indirectly modulated by miR-125b in MCF7 cells

Abstract

MicroRNA (miRNA) deregulation is associated with various cancers. Among an expanding list of cancer-related miRNAs, deregulation of miR-125b has been well documented in many cancers including breast. Based on current knowledge, miR-125b is considered to be a tumor suppressor in breast cancers. While important messenger RNA (mRNA) targets have been defined for miR-125b, here, we aimed to further investigate direct/indirect consequences of miR-125b expression in breast cancer cells by using a transcriptome approach. Upon miR-125b expression, a total of 138 cancer-related genes were found to be differentially expressed in breast cancer cells. While only a few of these were predicted to be direct mRNA targets, majority of the gene expression changes were potentially downstream and indirect effects of miR-125b expression. Among these, activated leukocyte antigen molecule (ALCAM) mRNA and protein levels were found to be highly significantly increased upon miR-125b expression. Given the tumor suppressor role of miR-125b in our model system, upon silencing of ALCAM expression, cell proliferation rate re-increased in miR-125b-expressing cells. While ALCAM’s possible context-dependent roles are not clear in breast cancer, a diverse expression pattern of ALCAM mRNA was detected in a panel of breast cancer patient samples. Differentially expressed/regulated cancer-related genes upon miR-125b expression along with the significant increase of ALCAM are of future interest to understand how deregulated expression of miR-125b may have a tumor suppressor role in breast and other cancers.

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Acknowledgments

We would like to thank Dr. Q. Fan and Prof. Ofori-Acquah for sharing ALCAM promoter constructs, Dr. J. Weiner for ALCAM shRNA construct, and Serkan Tuna and Cansaran Saygılı for technical assistance. CS, MTA, and DS were supported by funding from Science Foundation Ireland, IRCSET, and Cancer Research Ireland. This research was funded by METU internal funds and partially by TUBITAK 108S381.

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Correspondence to A. Elif Erson-Bensan.

Electronic supplementary material

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Supp. Table 1

Breast cancer patient panel information. (PDF 126 kb)

Supp. Table 2, 3

Significantly up-regulated (n=68) and down-regulated genes (n=70) in MCF7-125b cells. (PDF 265 kb)

Supp. Fig. 1

Volcano plot represents the relationship between fold change and statistical significance. X-axis represents the log2 Fold Change between MCF7-EV and MCF7-miR-125b cells. The vertical axis represents the Log Odds (B-Statistic). (PDF 810 kb)

Supp. Fig. 2

A. T47D cells stably transfected with miR-125b or with EV (empty vector, pSUPER). miR-125b expression levels were normalized to U6 expression. Two independent experiments were performed with triplicate replicates. The Student t-test was used for analyzing the data. Transfected cells were polyclonally selected. B. Cell proliferation assay (MTT) was performed in T47D cells, transfected with precursor miR-125b and Empty Vector (EV) as control. Experiment was performed with 4 replicates for each sample. All OD570 values were divided into that of day 1 for both miR-125b and EV cells. 10,000 cells were initially seeded into each well of 96-well cell culture plates and four replicates were used for each sample. C. Seven fold induction of ALCAM expression in MCF7-EV and MCF7-125b cells. Bands were quantified using ImageJ. D. ALCAM protein levels were detectable in T47D-EV cells and no apparent increase was detected upon miR-125b expression. (PDF 256 kb)

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Akman, H.B., Selcuklu, S.D., Donoghue, M.T.A. et al. ALCAM is indirectly modulated by miR-125b in MCF7 cells. Tumor Biol. 36, 3511–3520 (2015). https://doi.org/10.1007/s13277-014-2987-5

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  • DOI: https://doi.org/10.1007/s13277-014-2987-5

Keywords

  • miR-125b
  • ALCAM
  • Breast cancer
  • PERP
  • CTGF
  • Microarray