The miR-200 family is comprised of five miRNAs that are encoded within two clusters. Each cluster encodes a polycistronic gene. One cluster resides on human chromosome 1 and encodes miR-200b, miR-200a, and miR-429, while the other cluster is located on human chromosome 12, and encodes miR-200c and miR-141. Our small RNA library sequencing data () show that the miR-200 family is highly expressed in cultured normal human mammary epithelial cells (HMEC) derived from three different individuals, whereas the isogenic human mammary fibroblast cells (FB) lack miR-200 family expression (). It is apparent from the small RNA library sequencing data () that the most highly expressed members of the miR-200 family in HMEC are miR-200c and miR-141. We corroborated the expression of miR-200c and miR-141 in the same set of normal mammary samples by real-time PCR, and then expanded these results to pairs of epithelial cells and fibroblasts from prostate and skin, as well (; Figure S1
). In all cases, miR-200c and miR-141 were highly expressed in epithelial cells, but were not expressed in fibroblasts.
miR-200c is expressed in an epithelial selective fashion.
The mir-200c hairpin coding sequence and approximately 300 bp of upstream genomic sequence is CpG rich. According to our calculations using the program CpG Cluster 
this region is a highly statistically significant CpG cluster (). This CpG cluster (length 334 bp, GC% 68.56, O/E ratio 0.58, 21CpGs, p-value 8.44×10−11
) has the characteristics close to a CpG island definition based on size, GC content and CpG dinucleotide frequency 
, as well as its location with respect to the transcriptional unit 
. In addition, this region is considered a CpG island based on a recently published probabilistic definition 
. We analyzed this region as a possible target of epigenetic control.
The mir-200c CpG island shows differential cytosine methylation between miR-200c-positive and miR-200c-negative normal human tissues.
The epigenetic state of the miR-200c/141 CpG island shows clear and extensive cell type specific differences between normal miR-200c/141-positive and miR-200c/141-negative cells. We used MassARRAY technology to analyze the DNA methylation state of the mir-200c cluster CpG island (). Results show that the CpG sites are unmethylated in three separate strains of miR-200c/miR-141-positive HMEC. In contrast, all the CpG sites are highly methylated in the isogenic miR-200c/miR-141-negative fibroblast strains. The inverse correlation between miRNA expression and DNA methylation extends to other miR-200c/miR-141-positive/negative pairs of normal cells, such as prostate epithelial cells and skin keratinocytes, and their mesenchymal cell type counterparts, prostate and skin fibroblasts. Thus, the miR-200c cluster CpG island is unmethylated in normal miR-200c/miR-141-positive epithelial cells, while being densely methylated in the paired normal miR-200c/miR-141-negative fibroblasts (, , Figure S2
miR-200c and miR-141 expression is lost in different types of cancer cells 
, and we sought to determine if this loss of expression was linked to epigenetic changes in the miR-200c/miR-141 CpG island. We analyzed 11 breast cancer cell lines, and in each case, miR-200c and miR-141 expression was closely linked to the DNA methylation state of the CpG island (; Figure S3
). Seven of the breast cancer cell lines tested express miR-200c and miR-141 and each has an unmethylated mir-200c CpG island. The other four breast cancer cell lines tested do not express miR-200c and miR-141 and exhibit a densely methylated mir-200c CpG island. A similar picture emerges with respect to prostate cancer cells. We show two prostate cancer cell lines (PC3 and PC3 B1) where loss of miR-200c and miR-141 expression is linked with aberrant DNA methylation of the mir-200c/141 CpG island (; Figure S3
), and two prostate cancer cell lines (LNCaP and DU145) that retain miR-200c/miR-141 expression and an unmethylated mir-200c/141 CpG island. Together these results indicate that cancer cells derived from normal miR-200c/miR-141-positive epithelial cells can replicate the cell type-specific DNA methylation pattern of the miR-200c/141 CpG island seen in normal miR-200c/miR-141-negative cells, and that the aberrant DNA methylation of the miR200c/141 CpG island in these cancer cells is associated with its transcriptional silencing in carcinoma cells.
DNA methylation of mir-200c CpG island in breast and prostate cancer cell lines.
To demonstrate the functional significance of the epigenetic state of the miR-200c/mir-141 CpG island in cancer cells, we exposed cancer cells to the epigenetic modifier and DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-AdC). The miR-200c/miR-141-negative breast cancer cell lines MDA-MB-231 and BT549 and prostate cancer cell line PC3 were treated with 3 µM 5-AdC for 96 h and miR-200c/141 expression was assessed by real-time PCR. shows 5-AdC reactivated miR-200c expression in all three cancer cell lines. The level of miR-200c increased 4.3-fold in MDA-MB-231 (p-value
0.0004), 6.4-fold in BT549 (p-value
0.0107) and 4.2-fold in PC3 cells (p-value
0.0072). A similar reactivation of miR-141 expression (p-value<0.01) was also observed in these cancer cell lines after 5-AdC treatment (Figure S4
). These data suggest that epigenetic mechanisms participate in the inappropriate repression of miR-200c/miR-141 expression in cancer cells.
5-aza-2′-deoxycytidine treatment reactivates miR-200c expression in breast and prostate cancer cell lines.
The histone modification state of the mir-200c cluster CpG island also shows cell type-specific differences that are closely linked to the expression state of miR-200c/141 in normal and cancer cells. shows the results of chromatin immunoprecipitations coupled to quantitative real-time PCR analysis that were used to examine the histone modification state of the miR-200c/141 CpG island in normal and cancer cells. The CpG island of mir-200c/141 in the three different strains of miR-200c/miR-141-positive HMEC exists in a transcriptionally competent state; it is enriched for the transcriptionally permissive modifications of histone H3 acetylation (H3Ac) and lysine 4 trimethylation (H3TriMeK4), while the transcriptionally repressive histone mark of histone H3 lysine 9 dimethylation (H3DiMeK9) is underrepresented (). In contrast, in the isogenic miR-200c/miR-141-negative mammary fibroblasts permissive histone modifications are absent, and the repressive H3 lysine 9 dimethylation mark is present (). Similarly, the breast cancer cell lines that had lost miR-200c/141 expression lost histone H3 acetylation and K4 trimethylation and acquired a repressive histone state, enriched for the H3 lysine 9 dimethylation mark (). No enrichment of trimethylation of histone H3 lysine 27 was detected in the miR-200c/141 CpG island in the samples analyzed (Figure S5
). Taken together, the results from the analyses of miR-200c/141 expression, DNA methylation, and histone modification states across a variety of normal and cancer cell types demonstrate a close link between the expression of mir-200c/141 and the epigenetic state of their associated CpG island.
The histone modification state of the mir-200c CpG island.
Finally, we sought to determine if the epigenetic regulation of miR-200c expression in normal cells is conserved evolutionarily, reasoning that DNA methylation-linked control of miR-200c expression across mammalian species would provide further experimental support for epigenetic control of cell-type specific expression of miR-200c. The whole genomic cluster containing mir-200c and mir-141 is well conserved between the human and mouse genome. Similar to the human mir-200c/141 genomic region, the mouse mir-200c/141 genomic region also contains a CpG island (; length 325 bp, GC% 66.77, O/E ratio 0.58, 19 CpGs, p-value 1.06×10−10
). To evaluate a potential role for DNA methylation in the control of miR-200c/141 in mice, CpG methylation and miRNA expression were analyzed in mouse epithelial cells (epidermis of SKH-1 mouse and keratinocyte cell lines 308 and 6R90) and mouse fibroblasts (cell lines NIH 3T3, NIH 3T6, and NR6). A MassARRAY amplicon was designed to analyze the DNA methylation state of the mouse mir-200c/141 region homologous to that analyzed in human (). Strikingly similar results for miR-200c were found between the human cells and mouse cells. Mouse keratinocytes expressed significant levels of miR-200c, while the mouse fibroblasts did not express detectable levels of miR-200c (). DNA methylation analysis by MassARRAY revealed that the miR-200c-positive keratinocytes showed minimal DNA methylation in the mir-200c CpG island, while the miR-200c-negative mouse fibroblasts showed extensive DNA methylation of all CpG sites in the region (). The significant conservation in DNA sequence, patterns of cell type-specific DNA methylation, and the associated miR-200c expression patterns between the human and mouse genomes, which are separated by 75 million years of evolution 
, provides evidence that epigenetic mechanisms play a functional role in the control of miR-200c expression.
Epigenetic control of miR-200c expression is evolutionarily conserved.
In summary, our findings provide multiple lines of evidence that epigenetic mechanisms are involved in the regulation of miR-200c/141 expression in both normal and cancer cells. First, there is a consistent inverse correlation between expression and DNA methylation states in normal human and mouse cell types, as well as human breast and prostate cancer cell lines. Second, different histone codes exist between miR-200c/141 expressing and non-expressing cells that accurately mirror the expression and DNA methylation states. Third, the epigenetic modifier 5-aza-2′-deoxycytidine relieves the repression of miR-200c/miR-141 in cancer cell lines. Fourth, the link between DNA methylation and expression states occurs across mammalian species, since it is seen in human and mouse. Taken together, these findings indicate that miR-200c/141 is an evolutionarily conserved epigenetically labile miRNA cluster.
Dysregulation of miR-200c and miR-141 occurs in multiple cancer types 
, and this dysregulation involves a compromise of the epigenetic state of the CpG island associated with miR-200c and miR141. Results suggest that these carcinoma cells may co-opt de novo
DNA methylation pathways involved in the epigenetic control of normal cell type-specific genes, such as those that govern the epigenetic state of miR-200c/miR-141. A similar apparent co-option of cell type specific DNA methylation pathways by cancer cells is also seen in protein-coding genes, such as maspin
and 14-3-3 sigma 
. Together these results suggest that pathways responsible for the establishment or maintenance of normal cell type-specific DNA methylation states may be disrupted during carcinogenesis.
Since miR-200c and miR141 play an important role in EMT and therefore cell identity, disruption of mechanisms that govern cell type specific DNA methylation patterns during carcinogenesis could likely effect expression of miR-200c and miR141 and provide phenotypic plasticity to cancer cells. Support of this possibility comes from the phenotypes of the cancer cells analyzed in this study. All four of the breast cancer cell lines that lost miR-200c and miR-141 expression have an aberrantly methylated mir-200c/141 CpG island, and each of these cell lines displays a mesenchymal phenotype 
. In contrast, those breast cancer cell lines that express miR-200c and miR-141 and have an unmethylated CpG island display an epithelial phenotype 
. A similar picture emerges in the prostate cancer cell lines. The PC3 cells that have lost miR-200c and miR-141 expression, display an aberrantly methylated CpG island and a mesenchymal phenotype, whereas LnCaP and Du145 retain miR-200c and miR-141 expression and an epithelial phenotype 
. These results suggest that DNA methylation may control the phenotypic changes observed in cancer cells.