ABSTRACT
It is well established that transcriptional silencing of critical tumor-suppressor genes by DNA methylation is a fundamental component in the initiation of breast cancer. However, the involvement of microRNAs (miRNAs) in restoring abnormal DNA methylation patterns in breast cancer is not well understood. Therefore, we investigated whether miRNA-29b, due to its complimentarity to the 3'- untranslated region of DNA methyltransferase 3A (DNMT3A) and DNMT3B, could restore normal DNA methylation patterns in human breast cancers and breast cancer cell lines. We demonstrated that transfection of pre-miRNA-29b into less aggressive MCF-7 cells, but not MDA-MB-231 mesenchymal cells, inhibited cell proliferation, decreased DNMT3A and DNMT3B messenger RNA (mRNA), and decreased promoter methylation status of ADAM23 , CCNA1, CCND2, CDH1, CDKN1C, CDKN2A, HIC1, RASSF1, SLIT2, TNFRSF10D, and TP73 tumor-suppressor genes. Using methylation polymerase chain reaction (PCR) arrays and real-time PCR, we also demonstrated that the methylation status of several critical tumor-suppressor genes increased as stage of breast disease increased, while miRNA-29b mRNA levels were significantly decreased in breast cancers versus normal breast. This increase in methylation status was accompanied by an increase in DNMT1 and DNMT3B mRNA in advanced stage of human breast cancers and in MCF-7, MDA-MB-361, HCC70, Hs-578T, and MDA-MB-231 breast cancer cells as compared to normal breast specimens and MCF-10-2A, a non-tumorigenic breast cell line, respectively. Our findings highlight the potential for a new epigenetic approach in improving breast cancer therapy by targeting DNMT3A and DNMT3B through miRNA-29b in non-invasive epithelial breast cancer cells.
ABSTRACT
Hepatocellular carcinoma (HCC) mostly develops in patients with advanced fibrosis; however, the mechanisms of interaction between a genotoxic insult and fibrogenesis are not well understood. This study tested a hypothesis that fibrosis promotes HCC via a mechanism that involves activation of liver stem cells. First, B6C3F1 mice were administered diethylnitrosamine (DEN; single ip injection of 1mg/kg at 14 days of age). Second, carbon tetrachloride (CCl(4); 0.2ml/kg, 2/week ip starting at 8 weeks of age) was administered for 9 or 14 weeks to develop advanced liver fibrosis. In animals treated with DEN as neonates, presence of liver fibrosis led to more than doubling (to 100%) of the liver tumor incidence as early as 5 months of age. This effect was associated with activation of cells with progenitor features in noncancerous liver tissue, including markers of replicative senescence (p16), oncofetal transformation (Afp, H19, and Bex1), and increased "stemness" (Prom1 and Epcam). In contrast, the dose of DEN used did not modify the extent of liver inflammation, fibrogenesis, oxidative stress, proliferation, or apoptosis induced by subchronic CCl(4) administration. This study demonstrates the potential role of liver stem-like cells in the mechanisms of chemical-induced, fibrosis-promoted HCC. We posit that the combination of genotoxic and fibrogenic insults is a sensible approach to model liver carcinogenesis in experimental animals. These results may contribute to identification of cirrhotic patients predisposed to HCC by analyzing the expression of hepatic progenitor cell markers in the noncancerous liver tissue.
