Identification of Dlk1-Dio3 Imprinted Gene Cluster Noncoding RNAs as Novel Candidate Biomarkers for Liver Tumor Promotion

Abstract

The molecular events during nongenotoxic carcinogenesis and their temporal order are poorly understood but thought to include long-lasting perturbations of gene expression. Here, we have investigated the temporal sequence of molecular and pathological perturbations at early stages of phenobarbital (PB) mediated liver tumor promotion in vivo. Molecular profiling (mRNA, microRNA [miRNA], DNA methylation, and proteins) of mouse liver during 13 weeks of PB treatment revealed progressive increases in hepatic expression of long noncoding RNAs and miRNAs originating from the Dlk1-Dio3 imprinted gene cluster, a locus that has recently been associated with stem cell pluripotency in mice and various neoplasms in humans. PB induction of the Dlk1-Dio3 cluster noncoding RNA (ncRNA) Meg3 was localized to glutamine synthetase-positive hypertrophic perivenous hepatocytes, suggesting a role for β-catenin signaling in the dysregulation of Dlk1-Dio3 ncRNAs. The carcinogenic relevance of Dlk1-Dio3 locus ncRNA induction was further supported by in vivo genetic dependence on constitutive androstane receptor and β-catenin pathways. Our data identify Dlk1-Dio3 ncRNAs as novel candidate early biomarkers for mouse liver tumor promotion and provide new opportunities for assessing the carcinogenic potential of novel compounds.

Transdifferentiated rat pancreatic progenitor cells (AR42J-B13/H) respond to phenobarbital in a rat hepatocyte-specific manner.

“…We chose 3 and 9 days PB treatment, as these time points reflect PB-induced miRNA expression changes seen in vivo in male Fisher rats (Koufaris et al 2013). MiR-182 and its cluster partner miR-96 are both reported to be progressively induced by PB treatment in vivo in the rat (Koufaris et al 2012;Koufaris et al 2013); similar changes are not seen in the mouse (Lempiäinen et al 2013b). As previously described, B13/H cells treated with PB (2mM) for up to 9 days showed a significant increase in Cyp2b enzyme activity compared to control (Figure 2 B).…”

“…The PB-mediated induction of these miRNAs are specific to the rat, as they are not shown to be up-regulated in the mouse following PB treatment (Lempiäinen et al 2013b). We go on to confirm that the B13/H cells behave in a rat hepatic-specific manner, by demonstrating that the activity of the PB marker, Cyp2b, and the expression of the PB-sensitive miRNAs, miR-182/96, are not affected by the mouse Car activator, TCPOBOP, under the conditions used here.…”

“…Lempiäinen et al, (Lempiäinen et al 2013a) demonstrated that PB (0.05% w/v in drinking water) administered to B6C3F1/Cr1 mice significantly increased the expression of the pluripotent-associated noncoding RNA cluster, Dlk1-Dio3, which the authors suggested may be linked to the promotion of PB-mediated tumours in the mouse. However, we have shown in the rat that PB induced a different set of miRNAs, including the miR-200a/b/429 and miR-96/182 clusters, in a time-and dose-dependent fashion (Koufaris et al 2012).…”

Downregulation of IL6 Targeted MiR-376b May Contribute to a Positive IL6 Feedback Loop During Early Liver Regeneration in Mice

“…Interestingly, the three miRNAs are located in the imprinted Dlk1-Gtl2 imprinted region 12qF1 [15], map to the human Dlk1-Dio3 region on chromosome 14q32.2, which exert inhibitory effect in HCC malignancy in vitro [16]. The Dlk1-Dio3 region is also a cancer susceptibility locus and dysregulation of the miRNAs in this region has been found in liver tumors [17][18][19]. The above researches, to date, did not focus on the potential role of the Dlk1-Dio3 miRNA clusters during liver regeneration.…”

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“…In this context, the MARCAR project (2010-2015, http://www.imi-marcar.eu/) generated a wealth of data to increase insights into mechanisms of NGC action and to detect carcinogenic effects of drug candidates earlier (see for example (Lempiainen et al, 2013;Unterberger et al, 2014;Thomson et al, 2014;Römer et al, 2014)). The MARCAR project explored primarily the liver, which is the major target organ of NGC induced tumors in rodents (Knight et al, 2006).…”