The BMP-6 promoter is hypermethylated, and BMP-6 is expressed at a low level in HCC tissues.

<p>(A) An MSP-based assay was used to detect BMP-6 promoter methylation in 6 HCC cell lines and normal liver tissues. No methylation of the BMP-6 promoter was observed in 5 normal liver tissues. (B) An MSP-based assay was used to detect BMP-6 promoter methylation in 60 pairs of clinical HCC tissues. BMP-6 promoter methylation was demonstrated in 65% of HCC tissues, whereas this rate in the adjacent non-cancerous tissue was 50%. (C) The Taqman probe-based MethyLight assay was used to detect BMP-6 promoter methylation in 114 pairs of clinical HCC tissues, and the level of DNA methylation was upregulated by 23.44 fold in HCC tissues compared to adjacent non-cancerous tissues. (D) qRT-PCR was used to detect BMP-6 mRNA expression in normal liver tissue and HCC cells. The HCC cell lines used are shown, and the normal liver tissue was the NL1 sample shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087994#pone-0087994-g004" target="_blank">Figure 4A</a>. All BMP-6 expression levels in HCC cell lines were lower than those in normal liver tissue. (E) qRT-PCR was used to detect the BMP-6 mRNA expression in 60 pairs of HCC clinical samples, containing both DNA and RNA samples. We obtained similar results as those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087994#pone-0087994-g001" target="_blank">Figure 1A</a>. (F) The expression level of BMP-6 was correlated with BMP-6 promoter methylation. The level of BMP-6 DNA methylation obtained according to the MethyLight assay in 60 pairs HCC tissues is listed from lowest to highest. The 60 tissues with the lowest DNA methylation levels were placed into the hypomethylation group, whereas the 60 tissues with the highest DNA methylation levels were placed into the hypermethylation group. The HCC patients with BMP-6 hypermethylation had low levels of BMP-6 mRNA. The results were analysed using the Student's <i>t</i>-test.