NRP2 downregulation in FB-2 cells inhibits cell proliferation, migration, and invasion.

<p>(A) NRP2 downregulation in PAX8 clones was analyzed by Western blot with a specific anti-NRP2 antibody. (B) Growth curves of FB-2, FB2-FLAG, FB2-P8cl25 and FB2-P8cl31 cells are shown. Triplicate of 8 x 10⁴ cells were seeded into 60-mm plate. Cell numbers were counted on days 1, 2, 3 and 4 after seeding. (C) Wound-healing migration assay for FB-2, FB2-FLAG, FB2-P8cl25 and FB2-P8cl31 cells were performed. The healing of the wounds by migrating cells was imaged at time 0, 8 and 16 h. (D) Matrigel invasion assay of FB-2, FB2-FLAG, FB2-P8cl25 and FB2-P8cl31 cells. The cells were seeded on 8 μm pore size Transwell filters and allowed to migrate toward 10% fetal bovine serum. After 16 h, the upper surface of the filter was wiped clean and cells on the lower surface were stained and photographed. This figure is representative of three independent experiments.</p

Neuropilin-2 Is a Newly Identified Target of PAX8 in Thyroid Cells

<div><p>PAX8 is a transcription factor essential for thyroid gland development, as well as for the maintenance of the thyroid differentiated state in the adult. In particular, PAX8 has been comprehensively shown to regulate genes that are considered markers of thyroid differentiation. However, a better knowledge of genes transcriptionally regulated by PAX8 is desirable to clarify its role in endocrine syndromes and cancer susceptibility. In order to further investigate PAX8 downstream targets, we recently performed a genome-wide expression analysis following PAX8 knockdown in FRTL-5 thyroid cells and Neuropilin-2 was identified as a potential transcriptional target of PAX8. In this study, we determined the role of the transcription factor PAX8 in the regulation of Neuropilin-2 expression. Indeed, in thyroid cells PAX8 directly binds the Neuropilin-2 promoter leading to its transcriptional repression. Interestingly, we observed an inverse correlation between the expression of PAX8 and Neuropilin-2 in thyroid carcinoma tissues and cell lines compared to non-tumor counterparts, suggesting a critical role of PAX8 in regulating Neuropilin-2 expression in vivo. Notably, ectopic overexpression of PAX8 in FB-2 thyroid cancer cells promotes Neuropilin-2 downregulation producing a significant reduction in cell proliferation, migration ability, and invasion activity and reverting the cell phenotype from mesenchymal to a more epithelial one. These findings uncover the novel interplay between PAX8 and Neuropilin-2, which is likely to be important in the pathogenesis of thyroid diseases.</p></div

PAX8 directly modulates NRP2 gene expression.

<p>(A) FRTL-5 cells were transfected with a siRNA that specifically targets rat PAX8. PAX8 and Neuropilin-2 (NRP2) expression levels were measured on total RNA by qRT-PCR 24h, 48h and 72h after siRNA transfection. The values are means ± SD of three independent experiments in duplicate, normalized by the expression of β-actin and expressed as fold change with respect to the untransfected FRTL-5 cells, whose value was set at 1.0. Statistical analysis uses t test (p ≤ 0.05). (B) Chromatin extracted from cross-linked FRTL-5 cells was immunoprecipitated using in parallel an unrelated antibody (anti-tubulin) or an antibody against PAX8. The immunoprecipitates were analyzed by PCR with oligonucleotides corresponding to the rat NRP2 promoter region. Parallel PCR were performed with total input DNA obtained from unprecipitated aliquot of similarly treated chromatin sample and from no template control (NTC). Schematic representation of the upstream region of the rat NRP2 gene. PAX8-binding site is represented as a striped box.</p

NRP2 expression in experimental models of thyroid carcinogenesis.

<p>(A) NRP2 and PAX8 expression was measured by qRT- PCR in five human thyroid cancer cell lines: WRO from follicular thyroid cancer, FB-2 and BCPAP from papillary thyroid carcinoma, Cal62 and FRO from anaplastic thyroid carcinoma. RNA from six non-pathological thyroids was used as control. ABL1 was used as reference gene; results are reported as 2^-ΔCt. Statistical analysis uses t-test (p ≤ 0.05). (B) qRT-PCR analysis was performed on total RNA prepared from 6 tissue specimens of PTC. A pool of six normal thyroid tissues (N.T.) was used as control. Statistical analysis uses t-test (p ≤ 0.05). (C) qRT-PCR analysis was performed on total RNA prepared from thyroid carcinomas developed in Tg-TRK, Tg-PTC3, Tg-N-ras and Tg-SV40 transgenic mice expressing TRK, RET/PTC3, N-ras and SV40 T-antigen under the transcriptional control of the Tg promoter. Relative quantities of NRP2 mRNA were normalized to cyclophilinA as reference gene. Each amplification was performed in duplicate and the data are expressed as fold change with respect to the normal mouse thyroid tissues used as a control, whose value was set at 1.0. Statistical analysis uses t-test (p ≤ 0.05).</p

Altered expression of mitochondrial and extracellular matrix genes in the heart of human fetuses with chromosome 21 trisomy-3

<p><b>Copyright information:</b></p><p>Taken from "Altered expression of mitochondrial and extracellular matrix genes in the heart of human fetuses with chromosome 21 trisomy"</p><p>http://www.biomedcentral.com/1471-2164/8/268</p><p>BMC Genomics 2007;8():268-268.</p><p>Published online 7 Aug 2007</p><p>PMCID:PMC1964766.</p><p></p>H4, CDH5, CDH6 and DH1, DH3, DH4, DH5, DH6) and individual normal hearts (H1, H2, H3, H4, H5) show an inverse correlation (r = -066) between the two genes

Altered expression of mitochondrial and extracellular matrix genes in the heart of human fetuses with chromosome 21 trisomy-1

<p><b>Copyright information:</b></p><p>Taken from "Altered expression of mitochondrial and extracellular matrix genes in the heart of human fetuses with chromosome 21 trisomy"</p><p>http://www.biomedcentral.com/1471-2164/8/268</p><p>BMC Genomics 2007;8():268-268.</p><p>Published online 7 Aug 2007</p><p>PMCID:PMC1964766.</p><p></p>rom the t-test is represented on the y-axis. Genes upregulated in the trisomic samples are on the right of the horizontal axis 0 value; genes downregulated are on the left. Red dots indicate 473 genes that are significantly up- or down-regulated in the trisomic samples compared to the control samples (p < 0.05). Yellow dots indicate genes with no significant variation

Altered expression of mitochondrial and extracellular matrix genes in the heart of human fetuses with chromosome 21 trisomy-2

<p><b>Copyright information:</b></p><p>Taken from "Altered expression of mitochondrial and extracellular matrix genes in the heart of human fetuses with chromosome 21 trisomy"</p><p>http://www.biomedcentral.com/1471-2164/8/268</p><p>BMC Genomics 2007;8():268-268.</p><p>Published online 7 Aug 2007</p><p>PMCID:PMC1964766.</p><p></p>ted pathways are: Oxidative Phosphorylation (cluster 1), containing 16 genes downregulated in trisomic samples, and Focal Adhesion (cluster 2), containing at least 7 genes upregulated in trisomic samples. Cluster 3 is a network of Cell Adhesion genes, mostly upregulated in trisomic samples. Downregulated genes in cluster 1 are all mitochondrial genes; upregulated genes in clusters 2 and 3 are mostly ECM genes. Green indicates downregulated genes (darker green = more downregulated); red indicates upregulated genes (darker red = more upregulated)