|
| Status |
Public on Sep 15, 2016 |
| Title |
Transcription of the human 5-Hydroxytryptamine receptor 2B (HTR2B) gene is under the regulatory influence of the transcription factors NFI and RUNX1 in human uveal melanoma |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
|
| Summary |
Introduction: Uveal melanoma (UM) is the most common type of primary intraocular tumor in the adult population. UM will propagate to the liver as the first metastatic site. Once this organ is invaded, survival becomes a matter of months for the patient as no treatment has proved effectiveness. 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 tumors of UM patients that will progress toward liver metastases. Our study aims at characterizing the molecular mechanisms that lead to this aberrant expression of HTR2B in metastatic UM cell lines.Materials and Methods: Expression of HTR2B was monitored by microarrays and validated by Western blot in a variety of UM cell lines. A 2 Kbp segment covering both the promoter and 5 '-flanking region of the HTR2B gene was cloned upstream the CAT reporter gene in the plasmid pCATbasic (to yield the -2000/HTR2B construct). Deletion mutants were generated by double digestion of the -2000/HTR2B construct with restriction enzymes and transfected, along with the parental -2000/HTR2B construct, in UM cell lines. The genomic areas of interest were 5’ end-labeled and used as probes in electrophoretic mobility shift assays (EMSAs). DMS methylation footprinting was also used to precisely position the DNA target sites for transcription factors (TFs) that bind the HTR2B regulatory regions.Results and Discussion: Transfection analyses revealed that the upstream regulatory regions of HTR2B promoter is made up of a combination of alternative positive and negative regulatory elements. Repressive regions have an abnormally elevated number of target sites for the TFs NFI. EMSA analyses provided evidence that both the NFI isoforms NFIC and NFIX could interact with the promoter and the upstream negative regulatory element from the HTR2B gene. In addition, the TF RunX1 was shown by DMS methylation interference footprinting to bind a target site from the distal silencer element.Conclusion: This project will help understand better the molecular mechanisms accounting for the abnormal 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 formation.
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| Overall design |
Primary culture of humal uveal melanoma cell lines (T97, T98, T108, T111, T128, T131, T132, T142, T143, T151, T157) were analyzed by gene profiling on microarrays at low culture passages (between passage 2 to 7). and their transcriptome analyzed by gen profiling.
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| Contributor(s) |
Benhassine M, Zaniolo K, Guérin SL |
| Citation(s) |
30347896 |
|
https://doi.org/10.3390/ijms23031564
|
| Submission date |
Sep 14, 2016 |
| Last update date |
Feb 10, 2022 |
| Contact name |
Karine Zaniolo |
| E-mail(s) |
karine.zaniolo@gmail.com
|
| Organization name |
Centre de recherche du CHU de Québec
|
| Department |
CUO-Recherche
|
| Lab |
Sylvain Guérin
|
| Street address |
1050 Chemin Sainte-Foy
|
| City |
Québec |
| State/province |
Québec |
| ZIP/Postal code |
G1S 4L8 |
| Country |
Canada |
| |
|
| Platforms (1) |
| GPL13607 |
Agilent-028004 SurePrint G3 Human GE 8x60K Microarray (Feature Number version) |
|
| Samples (11)
|
| GSM2310301 |
Uveal_melanoma_derivative_cell_line_T97_rep1 |
| GSM2310302 |
Uveal_melanoma_derivative_cell_line_T98_rep1 |
| GSM2310303 |
Uveal_melanoma_derivative_cell_line_T108_rep1 |
| GSM2310304 |
Uveal_melanoma_derivative_cell_line_T111_rep1 |
| GSM2310305 |
Uveal_melanoma_derivative_cell_line_T128_rep1 |
| GSM2310306 |
Uveal_melanoma_derivative_cell_line_T131_rep1 |
| GSM2310307 |
Uveal_melanoma_derivative_cell_line_T132_rep1 |
| GSM2310308 |
Uveal_melanoma_derivative_cell_line_T142_rep1 |
| GSM2310309 |
Uveal_melanoma_derivative_cell_line_T143_rep1 |
| GSM2310310 |
Uveal_melanoma_derivative_cell_line_T151_rep1 |
| GSM2310311 |
Uveal_melanoma_derivative_cell_line_T157_rep1 |
|
| Relations |
| BioProject |
PRJNA342951 |
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