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

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.