ABSTRACT
Expression of the imprinted H19 gene is remarkably elevated in a large number of human cancers. Recently, we reported that H19 RNA is up-regulated in hypoxic stress and furthermore, it possesses oncogenic properties. However, the underlying mechanism(s) of these phenomena remain(s) unknown. Here we demonstrate a tight correlation between H19 RNA elevation by hypoxia and the status of the p53 tumor suppressor. Wild type p53 (p53(wt)) prevents the induction of H19 upon hypoxia, and upon its reconstitution in p53(null) cells. The last case is accompanied by a decrease in cell viability. The p53 effect is nuclear and seems independent of its tetramerization. Furthermore, using knockdown and over-expression approaches we identified HIF1-alpha as a critical factor that is responsible for H19 induction upon hypoxia. Knocking down HIF1-alpha abolishes H19 RNA induction, while its over-expression significantly enhances the H19 elevation in p53(null) hypoxic cells. In p53(wt) hypoxic cells simultaneous suppression of p53 and over-expression of HIF1-alpha are needed to induce H19 significantly, while each treatment separately resulting in a mild induction, indicating that the molecular mechanism of p53 suppression effect on H19 may at least in part involve interfering with HIF1-alpha activity. In vivo a significant increase in H19 expression occurred in tumors derived from p53(null) cells but not in p53(wt) cells. Taken together, our results indicate that a functional link exists between p53, HIF1-alpha and H19 that determines H19 elevation in hypoxic cancer cells. We suggest that this linkage plays a role in tumor development.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia/metabolism , Neoplasms/metabolism , RNA, Untranslated/genetics , Tumor Suppressor Protein p53/physiology , Animals , Blotting, Western , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , Humans , Hypoxia/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Mice , Mice, Nude , Neoplasms/genetics , Neoplasms/pathology , RNA, Long Noncoding , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/pharmacology , RNA, Untranslated/antagonists & inhibitors , RNA, Untranslated/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
BACKGROUND: Mutations and epigenetic aberrant signaling of growth factors pathways contribute to carcinogenesis. Recent studies reveal that non-coding RNAs are controllers of gene expression. H19 is an imprinted gene that demonstrates maternal monoallelic expression without a protein product; although its expression is shut off in most tissues postnatally, it is re-activated during adult tissue regeneration and tumorigenesis. Moreover, H19 is highly expressed in liver metastasis derived from a range of carcinomas. The objective of this study is to explore the role of H19 in carcinogenesis, and to determine its identification as an anti-tumor target. METHODOLOGY/PRINCIPLE FINDINGS: By controlling oxygen pressure during tumor cell growth and H19 expression levels, we investigated the role of H19 expression in vitro and in vivo in hepatocellular (HCC) and bladder carcinoma. Hypoxia upregulates the level of H19 RNA. Ablations of tumorigenicity of HCC and bladder carcinomas in vivo are seen by H19 knockdown which also significantly abrogates anchorage-independent growth after hypoxia recovery, while ectopic H19 expression enhances tumorigenic potential of carcinoma cells in vivo. Knocking-down H19 message in hypoxic stress severely diminishes p57(kip2) induction. We identified a number of potential downstream targets of H19 RNA, including angiogenin and FGF18. CONCLUSIONS: H19 RNA harbors pro-tumorigenic properties, thus the H19 gene behaves as an oncogene and may serve as a potential new target for anti-tumor therapy.
