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

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.

Figure 1

Genomic organization of LCO554202/miR-31 locus and their associated CpG island. A) Schematic representation of the genomic organization of the LOC554202 gene. BAC clones RP11-344A7 and RP11-354P17 are shown with horizontal open bars. A strong CpG island spans exon 1 of LOC554202. miR-31 which maps within intron 1 of LOC554202 is shown with a red horizontal bar. The size of each exon and intron is also shown. The arrows below Exon 1, Exon 3 and Exon 4 represent the location and orientation of the primers used for RT-PCR of LOC554202. B) The location of the predicted CpG island (blue line) and LOC554202 exon 1 (green line) are shown with respect to the sequence of Human BAC clone RP11-344A7 on chromosome 9p21.2-22.3, Accession number AL137022, which contains the 5' end of the LOC554202, the host Gene of microRNA miR-31. Each vertical bar represents one CpG dinucleotide. Exon 1 of LOC554201 is shown as a red line. C) Schematic representation of the predicted CpG island and the location of the MSP primers sets and the bisulfite sequencing primers.

Figure 2

The genomic structure of the LOC554202 miR-31 is preserved in the BC cell lines used in this study. (A) Schematic representation of the mapping of the LOC554202 exons, the CpG island and miR-31 with respect to BAC clones RP11-344A7 and RP11-354P17. The mapping was confirmed by genomic PCR analysis. The size of the PCR product of each amplicon is shown on the left. ERVK6 was used as a positive control for the total genomic DNA. (B) Genomic PCR analysis of each exon of LOC554202 and miR-31 in the BC cell lines used in this study. The size of the PCR product of each amplicon is shown on the left. ERVK6 was used a positive control for the total genomic DNA.

Figure 3

miR-31 and its host gene LOC554202 are downregulated in basal TNBC cell lines and highly expressed in luminal non-invasive BC cell lines. (A and B) Quantitative real-time RT-PCR of the mature miR-31 (A) and the pri-miR-31 (B) in non-malignant breast epithelial MCF10A, luminal MCF7, SKBr3 and T47D, and basal triple-negative MDA-MB-435S, MDA-MB-231 and BT549 BC cell lines. miR-16 and RNUB6 were used for normalization. (C) Semi-quantitative RT-PCR of the LOC554202 transcript in the same cell lines. GAPDH was used an internal control.

Figure 4

miR-31 and LOC554202 are epigenetically regulated in breast cancer. (A) Quantitative real-time RT-PCR of miR-31 in the indicated cell lines before and after treatment with 5 Aza-2dC and TSA. miR-16 was used for normalization. (B) Semi-quantitative RT-PCR of LOC554202 after treatment of MDA-MB-231 and BT549 with the de-methylating 5Aza2dC and the de-acetylating Trichostatin A (TSA) agents. Expression of LOC554202 in the untreated cell lines is also shown. GAPDH was used as an internal control.

Figure 5

miR-31 promoter is heavily methylated in the basal TN versus the luminal BC cell lines. (A) Methylation specific PCR [MSP] of bisulfite-modified DNA of the indicated cell lines using 2 sets of primers. (B) Ratio of overall methylated over unmethylated CpGs by sequencing of bisulfite-modified DNA from the indicated cell lines. The location of the MSP and bisulfite sequencing primers is shown in Figure 1C.