Hypermethylation leads to bone morphogenetic protein 6 downregulation in hepatocellular carcinoma.
BACKGROUND
In the liver, bone morphogenetic protein 6 (BMP-6) maintains balanced iron metabolism. However, the mechanism that underlies greater BMP-6 expression in hepatocellular carcinoma (HCC) tissue than adjacent non-cancerous tissue is unclear. This study sought to investigate the epigenetic mechanisms of BMP-6 expression by analysing the relationship between the DNA methylation status of BMP-6 and the expression of BMP-6.


METHODS
Methylation-specific polymerase chain reaction (PCR), bisulphite sequencing PCR, the MethyLight assay, and quantitative real-time PCR were performed to examine BMP-6 methylation and mRNA expression levels. Immunohistochemistry (IHC) was performed on tissue arrays to evaluate the BMP-6 protein level.


RESULTS
BMP-6 mRNA expression was approximately 84.09% lower in HCC tissues than in adjacent non-cancerous tissues, and this low level of expression was associated with a poor prognosis. Moreover, the hypermethylation observed in HCC cell lines and HCC tissues was correlated with the BMP-6 mRNA expression level, and this correlation was validated following treatment with 5-aza-CdR, a demethylation agent. In addition, BMP-6 DNA methylation was upregulated by 68.42% in 114 clinical HCC tissue samples compared to adjacent normal tissues, whereas the BMP-6 staining intensity was downregulated by 77.03% in 75 clinical HCC tissue samples in comparison to adjacent normal tissues. Furthermore, elevated expression of BMP-6 in HCC cell lines inhibited cell colony formation.


CONCLUSIONS
Our results suggest that BMP-6 CpG island hypermethylation leads to decreased BMP-6 expression in HCC tissues.
State Key Laboratory of Oncogenes & Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China.
Yinghua
Y
He
Guangxi Cancer Institute, Nanning, Guangxi, China.
Ying
Y
Cui
Shanghai No. 6 People's Hospital, Medical School of Shanghai Jiaotong University, Shanghai, China.
Baiying
B
Xu
State Key Laboratory of Oncogenes & Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China.
Jun
J
Gu
State Key Laboratory of Oncogenes & Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China.
Wei
W
Wang
State Key Laboratory of Oncogenes & Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China.
Xiaoying
X
Luo
D055418:Bone Morphogenetic Protein 6 / Q000235:genetics*; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation / Q000235:genetics*; D015536:Down-Regulation / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D006801:Humans; D008099:Liver / Q000473:pathology; D008113:Liver Neoplasms / Q000235:genetics* / Q000473:pathology; D011379:Prognosis; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D015854:Up-Regulation / Q000235:genetics
C525816:BMP6 protein, human; D055418:Bone Morphogenetic Protein 6; D012333:RNA, Messenger