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L. Touzel, M. Gaudreault, S. Leclerc, S. L. Guérin; Lentivirus Mediated shRNA Suppression of Nf1 Isoforms in Uveal Melanoma. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4625.
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© ARVO (1962-2015); The Authors (2016-present)
The NFI family of transcription factors (TFs) comprises four isoforms, NFI- A, -B, -C and -X, that can function either as activators or repressors of gene transcription. Recently, we demonstrated that NFI represses the transcription of the gene encoding the human alpha5 subunit from the alpha5beta1 integrin in primary cultured corneal epithelial cells. In the present study, we attempted to demonstrate how suppression of this TF would alter the expression of the alpha5 gene in the highly aggressive T97 uveal melanoma cell line (that has a very high level of NFI but no expression of alpha5) as well as the poorly invasive T115 (that expresses very low level of this TF but has a high level of alpha5).
Both the T97 and T115 cell lines were stably infected with lentivirus expressing shRNAs directed against each of the four NFI isoforms. Endogenous expression of NFI was monitored through both Western blot and electrophoretic mobility shift assays (EMSAs). The influence of suppressing NFI on the expression of the alpha5 gene was also monitored by transfection of recombinant constructs bearing the CAT reporter gene fused to various segments from the human alpha5 gene promoter into both T97 and T1115 cells stably infected with the various NFI shRNAs. Endogenous expression of alpha5 was also monitored by RT-PCT analyses in the stably infected cells.
Data from the shRNA suppression experiments and Western blot analyses indicated that highly invasive T97 cells primarily express both the NFI-A and NFI-C isoforms whereas the low level of NFI that is typical of poorly invasive T115 cells appears to rely on the expression of NFI-A. Transfection of alpha5/CAT recombinant constructs into stably infected T97/shNFI-C yielded a dramatic increase in CAT activity relative to T97 cells infected with the empty lentiviral vector suggesting that NFI-C is the NFI isoform that accounts for the extinction of alpha5 gene expression in T97 cells.
The strong negative regulatory influence of NFI-C combined to an increase in its level of expression caused a major shift in the NFI/AP-1/Sp1 ratio that resulted into a dramatic repression of alpha5 gene transcription in T97 cells. Our results therefore highlighted how critical any subtle change in the expression/affinity of any of these three TFs will ultimately alter alpha5 gene expression in any given cell.
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