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Comparative analysis of ARE functioning in vivo and implication in hepatocellular carcinoma: the GPC3 mRNA as model – GPC3-POST-TRANS

Submission summary

In higher eukaryotes, the expression of many cellular genes is tightly controlled by the AU-rich element (ARE), a post-transcriptional cis-regulatory sequence located in the 3'-untranslated region (3'UTR) of certain mRNAs. This element is an inhibitor of gene expression as it induces the degradation and the translational repression of the mRNA. The ARE functioning is under the control of many trans-regulatory factors (the ARE-binding proteins, ARE-BPs). Whereas the binding of most of the ARE-BPs to the ARE activates the degradation and the translational silencing of the mRNA, that of HuR, a nuclear shutting protein, impede these effects. Consequently the half-life and the translation of the ARE-containing mRNA increase leading to a higher expression of the corresponding gene. Finally, small non coding RNAs, the miRNAs, are also involved in the post-transcriptional control of gene expression mediated by the ARE. The functional alteration of an ARE can have an incidence in human cancerology through the increased expression of particular genes. This alteration sometimes arises from a modification of the proteic nature of the ARE-associated complex because of the abnormal expression or atypical subcellular localization of an ARE-BP, as it has been shown for HuR. It can also arise from the down-regulated expression of a miRNA targeting the transcript of the overexpressed gene. In hepatocellular carcinoma (HCC) (a primitive liver cancer), many genes with a potential role in carcinogenesis and which contain an ARE in their transcript, are overexpressed. However, no investigation has been ever done in order to determine whether the abnormal expression of these ARE-containing genes in HCC is linked to an ARE dysfunction. The aim of this project is to comparatively study the in vivo functioning of various AREs in primary human hepatocytes and HCC tumoral cell lines. We therefore developed a simple EGFP-reporter system, based on lentiviral transduction, flow cytometry and quantitative PCR, allowing an evaluation of the in vivo functioning of various AREs deriving from genes overexpressed in HCC. Preliminary results showed that, at least for two genes (c-myc and Glypican-3 (GPC3)), the ability of the ARE to inhibit the expression of the EGFP reporter could be affected in HCC tumoral cell lines in comparison with the normal hepatocytes. In the case of GPC3, this is the first report of such post-transcriptional regulation. This is one of the reasons for which we chose to use the GPC3 3'UTR as model for our study. Complementary results showed that (i) HuR and TIA-1, two ARE-BPs with potential inhibitory effect on ARE function, bind the GPC3 3'UTR in vitro, (ii) TIA-1 is detectable only in tumoral cells, (iii) HuR is abnormally found in the cytoplasm of tumoral cells. The main objectives of the project presented herein are the following: 1- To map the functional cis-regulatory sequences in the GPC3 3'UTR. 2- To identify the other trans-regulatory proteins (other than HuR and TIA-1) binding the GP3 3'UTR. 3- To determine whether GPC3 3'UTR controls EGFP expression through mRNA stability. 4- To determine the role of HuR and TIA-1 (and other potent trans-regulatory factors) in the GPC3 3'UTR functioning and the GPC3 overexpression in HCC. 5- To determine whether the cytoplasmic localization of HuR plays a role in the positive control mediated by the GPC3 3'UTR on EGFP expression in tumoral cells. 6- To evaluate the pathophysiological relevance of TIA-1 overexpression and HuR cytoplasmic localization in HCC carcinogenesis. 7- To determine whether given miRNAs play a role in the post-transcriptional regulation mediated by the GPC3 3'UTR and the GPC3 overexpression in HCC. Indeed target sites of particular miRNAs are located in the GPC3 3'UTR. We will investigate the possibility that some miRNAs compete with HuR and TIA-1 for the binding to the GPC3 3'UTR. 8- Finally we will take advantage of having tumoral cells stably expressing the EGFP under the control of the GCP3 3'UTR to identify by high throughput screening new trans-regulatory molecules or factors. Altogether this work should help us to determine whether the functioning of the AREs is specifically or more broadly affected in HCC tumoral cells and whether this alteration plays a potential role in hepatic carcinogenesis through the overexpression of pro-oncogenic genes or genes involved in the tumour progression.

Project coordination

Christophe GROSSET (Organisme de recherche)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

Help of the ANR 150,000 euros
Beginning and duration of the scientific project: - 36 Months

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