Epigenetic inactivation of microRNA gene hsa‐mir‐9‐1 in human breast cancer

Abstract

MicroRNAs (miRNAs) represent a new class of small non-coding RNAs regulating gene expression by inducing RNA degradation or interfering with translation. Aberrant miRNA expression has been described for several human malignancies and tumour suppressor functions have been ascribed to this new class of small regulatory RNAs. Accordingly, inactivation due to deletion or mutation has been found in human malignancies. Here, we describe the role of aberrant hypermethylation as an additional mechanism for miRNA gene inactivation in human breast cancer. Aberrant hypermethylation was shown for mir-9-1, mir-124a3, mir-148, mir-152, and mir-663 in 34-86% of cases in a series of 71 primary human breast cancer specimens. For comprehensive methylation analysis, combined bisulphite restriction analysis, bisulphite sequencing, and Pyrosequencing were employed. miRNA gene hypermethylation correlated strongly with methylation of known tumour suppressor genes (p = 0.003). After treatment of various breast cancer cell lines with the demethylating agent 5-aza-2'-deoxycytidine, reduction of mir-9-1 gene methylation and concomitant reactivation of expression could be observed. For the mir-9-1 gene, which is already hypermethylated in pre-invasive intraductal lesions, a good correlation between quantitative methylation level and reduction of expression could be demonstrated in a subset of primary human breast cancer specimen (r = 0.8). In conclusion, this study demonstrates that various microRNA genes are also affected by epigenetic inactivation due to aberrant hypermethylation and that this is an early and frequent event in breast cancer development.

Hsa-miR-9 methylation status is associated with cancer development and metastatic recurrence in patients with clear cell renal cell carcinoma

“…Previous studies have shown extensive epigenetic silencing of one or several of the miR-9 genes in cancer (Lehmann et al, 2008;Lujambio et al, 2008;Omura et al, 2008;Bandres et al, 2009;Hsu et al, 2009). This silencing was common in tumor tissue, whereas nearly absent in normal tissue.…”

“…In addition to analysis of hsa-miR-9 methylation status, microRNA expression studies have shown that hsa-miR-9 expression is downregulated for several cancer sites, including breast (Lehmann et al, 2008;Hsu et al, 2009) Hsa-miR-9 methylation and renal cell carcinoma MAT Hildebrandt et al gastric (Luo et al, 2009) and ovarian (Laios et al, 2008). Furthermore, studies of liver (Budhu et al, 2008), breast (Iorio et al, 2005) and colorectal (Bandres et al, 2009) cancers have shown that altered hsa-miR-9 expression was associated with metastatic tumors.…”

“…The mature hsa-miR-9 transcript is produced by three independent genes: miR-9-1, miR-9-2 and miR-9-3, located on chromosomes 1, 5 and 15, respectively. Previous studies have shown that these microRNA genes are commonly hypermethylated in tumor tissue (Lehmann et al, 2008;Lujambio et al, 2008;Omura et al, 2008;Bandres et al, 2009;Hsu et al, 2009). Furthermore, hsa-miR-9 methylation was shown to be associated with the development of metastasis (Lujambio et al, 2008;Bandres et al, 2009).…”

The microRNA network and tumor metastasis

“…The epigenetic inactivation of miR-148 has been found in several cancer cells, for example, in melanoma and breast cancer (Lehmann et al, 2008;Lujambio et al, 2008). DNA methylation-associated silencing of miR-148 contributes to the development of cancer metastasis by upregulating its target gene, TGIF2.…”

Comprehensive genetic and functional characterization of IPH‐926: a novel CDH1‐null tumour cell line from human lobular breast cancer

“…Oligonucleotide primers designed to amplify the CDH1 mutation site in IPH-926 cells were forward, 5 -CTCGACACCCGATTCAAAGT-3 ; reverse, 5 -CCG-TAGAGGCCTTTTGACTG-3 . Methylation-specific PCR was done with bisulphite-treated genomic DNA [31] and CDH1 methylation-sensitive PCR primers described elsewhere [32].…”