July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Analysis of the promoter and 5’-flanking region of the human serotonin receptor gene HTR2B and its expression in uveal melanoma
Author Affiliations & Notes
    Biologie cellulaire et moléculaire, Université Laval, Québec, Quebec, Canada
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    Commercial Relationships   Manel BENHASSINE, None
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Investigative Ophthalmology & Visual Science July 2018, Vol.59, 3171. doi:
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      Manel BENHASSINE; Analysis of the promoter and 5’-flanking region of the human serotonin receptor gene HTR2B and its expression in uveal melanoma. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3171.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose : Uveal melanoma (UM) is the most common type of primary intraocular tumor in the adult population. UM propagates to the liver as the first metastatic site. Once this organ is invaded, survival becomes a matter of months for the patient as no effective treatments are available. Among the candidates from the class II gene signature, the serotonin receptor-encoding gene HTR2B appears to be the most discriminating as its expression strongly increases in the UM tumors that will progress toward liver metastases. Our study aims at characterizing the molecular mechanisms that lead to this deregulated expression of HTR2B in metastatic UM cell lines.

Methods : Expression of HTR2B was monitored by microarrays and validated by Western blot in a variety of UM cell lines. The promoter and 5'-flanking region of the HTR2B gene was cloned upstream the CAT reporter gene to yield the -2000/HTR2B construct. DMS methylation footprinting was used to position the DNA target sites for transcription factors (TFs) that bind the HTR2B regulatory regions. Deletion mutants of the -2000/HTR2B construct as well as site-directed mutants of the TFs target sites identified were produced, and their impact on HTR2B promoter function was evaluated by transfection in UM cells. Electrophoretic mobility shift assay (EMSA) was used to monitor binding of the TFs NFI and Runx1 to the HTR2B promoter.

Results : Transfection analyses revealed that the upstream regulatory regions of the HTR2B gene is made up of a combination of alternative positive and negative regulatory elements. EMSA analyses provided evidence that both the NFI isoforms NFIC and NFIX could interact with the promoter and upstream negative regulatory element from the HTR2B gene. In addition, the TF RunX1 was shown, by DMS methylation interference footprinting and EMSA, to bind a target site from the distal silencer element. Site-directed mutagenesis and RNAi experiments provided evidence that RunX1 functions as a repressor of HTR2B gene promoter activity in UM cells.

Conclusions : This project will help understand better the molecular mechanisms accounting for the deregulated expression of the HTR2B gene in uveal melanoma. In the long term, this study will allow us to identify new potential targets that could help screening patients at risk of progressing to liver metastasis.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.


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