Global, cancer-specific microRNA cluster hypomethylation was functionally associated with the development of non-B non-C hepatocellular carcinoma

Masanori Nojima, Takeshi Matsui, Akihiro Tamori, Shoji Kubo, Ken Shirabe, Koichi Kimura, Mitsuo Shimada, Tohru Utsunomiya, Yasuteru Kondo, Etsuko Iio, Yutaka Naito, Takahiro Ochiya, Yasuhito Tanaka

While hepatitis B and C viral infection have been suppressed, non-B non-C hepatocellular carcinoma (NBNC-HCC) is considered to be rising in incidence terms in some developed countries where prevalence of those viral infections among HCC patients had been very high (such as Japan, Korea, and Italy). To elucidate critical molecular changes in NBNC-HCC, we integrated three large datasets relating to comprehensive array-based analysis of genome-wide DNA methylation (N = 43 pairs) and mRNA/miRNA expression (N = 15, and 24 pairs, respectively) via statistical modeling. Hierarchical clustering of DNA methylation in miRNA coding regions clearly distinguished NBNC-HCC tissue samples from relevant background tissues, revealing a remarkable tumor-specific hypomethylation cluster. In addition, miRNA clusters were extremely hypomethylated in tumor samples (median methylation change for non-clustered miRNAs: -2.3%, clustered miRNAs: -24.6%). The proportion of CpGs hypomethylated in more than 90% of the samples was 55.9% of all CpGs within miRNA clusters, and the peak methylation level was drastically shifted from 84% to 39%. Following statistical adjustment, the difference in methylation levels within miRNA coding regions was positively associated with their expression change. Receiver operating characteristic (ROC) analysis revealed a great discriminatory ability in respect to cluster-miRNA methylation. Moreover, miRNA methylation change was negatively correlated with corresponding target gene expression amongst conserved and highly matched miRNA sites. We observed a drastic negative shift of methylation levels in miRNA cluster regions. Changes in methylation status of miRNAs were more indicative of target gene expression and pathological diagnosis than respective miRNA expression changes, suggesting the importance of genome-wide miRNA methylation for tumor development. Our study dynamically summarized global miRNA hypomethylation and its genome-wide scale consequence in NBNC-HCC.