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01.12.2012 | Research | Ausgabe 1/2012 Open Access

Molecular Cancer 1/2012

miR-31 and its host gene lncRNA LOC554202 are regulated by promoter hypermethylation in triple-negative breast cancer

Zeitschrift:
Molecular Cancer > Ausgabe 1/2012
Autoren:
Katarzyna Augoff, Brian McCue, Edward F Plow, Khalid Sossey-Alaoui
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1476-4598-11-5) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

KA carried out most of the experiments including the quantitative real-time RT-PCR for miR-31 and LOC445202 as well as the methylation specific PCR and assisted in drafting the manuscript. BM performed the bisufite sequencing experiments and was responsible for the tissue culture of cell lines and their treatments as well as DNA and RNA isolation. EFP discussed the design of the experiments, the results and assisted in writing. KSA supervised the overall design of the experiments and wrote the manuscript.

Grant support

DOD Breast Cancer Idea Award grant W81XWH0810236 (K. Sossey-Alaoui), NIH grants P01HL073311 and P50HL077107 (E.F. Plow). K. Augoff is funded in part by a research fellowship "Development program of Wroclaw Medical University" from the European Social Fund, Human Capital, national Cohesion Strategy" (contract # UDA-POKL.04.01.01-00-010/08-00)".

Abstract

Background

microRNAs have been established as powerful regulators of gene expression in normal physiological as well as in pathological conditions, including cancer progression and metastasis. Recent studies have demonstrated a key role of miR-31 in the progression and metastasis of breast cancer. Downregulation of miR-31 enhances several steps of the invasion-metastasis cascade in breast cancer, i.e., local invasion, extravasation and survival in the circulation system, and metastatic colonization of distant sites. miR-31 exerts its metastasis-suppressor activity by targeting a cohort of pro-metastatic genes, including RhoA and WAVE3. The molecular mechanisms that lead to the loss of miR-31 and the activation of its pro-metastatic target genes during these specific steps of the invasion-metastasis cascade are however unknown.

Results

In the present report, we identify promoter hypermethylation as one of the major mechanisms for silencing miR-31 in breast cancer, and in the triple-negative breast cancer (TNBC) cell lines of basal subtype, in particular. miR-31 maps to the intronic sequence of a novel long non-coding (lnc)RNA, LOC554202 and the regulation of its transcriptional activity is under control of LOC554202. Both miR-31 and the host gene LOC554202 are down-regulated in the TNBC cell lines of basal subtype and over-expressed in the luminal counterparts. Treatment of the TNBC cell lines with either a de-methylating agent alone or in combination with a de-acetylating agent resulted in a significant increase of both miR-31 and its host gene, suggesting an epigenetic mechanism for the silencing of these two genes by promoter hypermethylation. Finally, both methylation-specific PCR and sequencing of bisulfite-converted DNA demonstrated that the LOC554202 promoter-associated CpG island is heavily methylated in the TNBC cell lines and hypomethylated in the luminal subtypes.

Conclusion

Loss of miR-31 expression in TNBC cell lines is attributed to hypermethylation of its promoter-associated CpG island. Together, our results provide the initial evidence for a mechanism by which miR-31, an important determinant of the invasion metastasis cascade, is regulated in breast cancer.
Zusatzmaterial
Additional file 1: Quantitative real-time RT-PCR of miR-16 (A and B) and RNU6B (C and D) in the indicated cell lines as well as before and after treatment with 5Aza-2dC and TSA. GAPDH was used as an internal control for normalization. (PDF 344 KB)
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Additional file 2: List of oligonucleotide primers and microRNA assays used in this study. (PDF 31 KB)
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Authors’ original file for figure 1
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Authors’ original file for figure 2
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Authors’ original file for figure 3
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Authors’ original file for figure 4
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Authors’ original file for figure 5
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Literatur
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