Abstract 537: Toll-like receptor (TLR3) signaling in human hepatocellular carcinoma (HCC) depends on the tumor-associated long-noncoding RNA H19
Authors: Paulus Patrick , Urbschat Anja , Reissig Christin , Zacharowski Kai , Dröse Stefan , Scheller Bertram , Tybl Elisabeth 
Published: May 2015

HCC is the third leading cause of cancer-related mortality worldwide. Metabolic syndrome diseases are shown to facilitate the progression of HCC in industrial countries. Recent studies unraveled pivotal roles of long non-coding RNAs (lncRNAs) in carcinogenesis and cancer metastasis. The controversy that the non-translated H19 mRNA harbors pro- as well as anti-tumorigenic potential shows that its function is poorly characterized. TLR3 plays a key role in the recognition of exogenous pathogen-associated dsRNA and even more importantly TLR3 is activated by endogenous injury-associated ligands. There is increasing evidence that the implication of TLR3 in HCC development might be associated with tumor size, aggressiveness and survival.

To study the relationship between the H19 mRNA and TLR3 we first looked at the TLR3 signaling in livers from TLR3-/- mice in comparison to WT mice. By performing real-time RT-PCR experiments, we could demonstrate that TLR3-/- mice showed a significantly enhanced H19 mRNA expression. In order to find out whether H19 mRNA acts as a regulator of TLR3 signaling, we used RNA interference (RNAi) and added the synthetic TLR3 ligand poly I:C on the human hepatoma cell line HepG2. TLR3 knockdown was followed by a significant increase in H19 mRNA expression levels, whereas poly I:C treatment resulted in significantly decreased H19 levels. Vice versa, H19 mRNA knockdown verified a significant reduction in TLR3 mRNA expression. Determination of free RNA in the cell culture supernatants revealed a significant increase of free RNA only after hypoxic incubation whereas no changes could be detected after poly I:C nor RNAi application. Treatment based on H19 inhibition may be a specific approach to target tumor cells, as it is already known that silencing of H19 significantly reduces cell viability. Due to the implication of TLR3 in mRNA stabilization, we addressed to question of a reciprocal relation between both genes by actinomycin D treatment. Indeed, H19 expression was shown to depend on TLR3 expression and a reduced H19 stability was detected after RNAi of TLR3. Since caspase-3 is a key executor of apoptosis and due to the fact that TLRs are known to be involved in the regulation of the apoptotic machinery, we performed caspase-3-like activity assay. Whereas no significant changes in basal caspase-3 like activity could be detected, a strong increase in caspase-3 like activity was seen after siRNA-mediated TLR3 knockdown under normoxic and hypoxic conditions.

This indicates that TLR3 may play a crucial role in mediating the signaling through non-coding RNAs, which function is so far poorly understood. We show for the first time the direct relation between H19 and TLR3. Changes in expressions of either of these molecules results in an altered cell response. The knowledge of this interplay might lead to novel strategies for treating HCC.

Citation Format: Patrick Paulus, Anja Urbschat, Christin Reissig, Kai Zacharowski, Stefan Dröse, Bertram Scheller, Elisabeth Tybl. Toll-like receptor (TLR3) signaling in human hepatocellular carcinoma (HCC) depends on the tumor-associated long-noncoding RNA H19. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 537. doi:10.1158/1538-7445.AM2014-537