Homeobox genes are regulatory genes often associated with tumors in the adult organisms. We investigated their comparative expression by immunohistochemical and real-time PCR analysis, in normal and inflammatory nasal mucosae and in sinonasal neoplasms in order to use them as possible diagnostic and therapeutic targets.
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Method Article
Identification of OTX1 and OTX2 As Two Possible Molecular Markers for Sinonasal Carcinomas and Olfactory Neuroblastomas
* These authors contributed equally
In This Article
Summary
Abstract
OTX homeobox (HB) genes are expressed during embryonic morphogenesis and during the development of olfactory epithelium in adult organisms. Mutations occurring in these genes are often related to tumorigenesis in human. No data are available today regarding the possible correlation between OTX genes and tumors of the nasal cavity. The aim of this work is to understand if OTX1 and OTX2 can be considered as molecular markers in the development of nasal tumors. We selected nasal and sinonasal adenocarcinomas to investigate the expression of OTX1 and OTX2 genes through immunohistochemical and real-time PCR analyses.Both OTX1 and OTX2 were absent in all the samples of sinonasal Intestinal-Type Adenocarcinomas (ITACs). OTX1 mRNA was identified only in Non-Intestinal Type Adenocarcinomas (NITACs) while OTX2 mRNA was expressed only in Olfactory Neuroblastomas (ONs). We have demonstrated that the differential gene expression for both OTX1 and OTX2 genes might be a useful molecular marker to distinguish the different types of sinonasal tumors.
Introduction
OTX HB genes are the vertebrate homologue of the Drosophila orthodenticle genes (otd) and they encode for transcription factors which are normally expressed during embryonic morphogenesis, but they can also be expressed in the adult organism with different functions. During embryonic development they control the specification of cell identity, cell differentiation, and the positioning of the body axis¹. The OTX family includes OTX1 and OTX2 genes which display different functions. OTX1 is involved in brain and sensory organ development. In the adult organism, it is expressed in sensory organs and is transcribed at low ....
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Protocol
All the studies were performed according the Declaration of Helsinki (1975) with written informed consent and approved by the Ethical Committee of the University of Insubria in Varese.
1. Collection of the Samples
- Collect and divide all the human Formalin-Fixed Paraffin-Embedded (FFPE) samples into different subgroups according the WHO classification of Head and Neck Tumors14.
NOTE: Here we used the following samples: Normal sinonasal mucosa as control (10 samples); Inverted Papilloma (IP, ICD-O code 8121/1); Sinonasal ITAC (ICD-O code 8144/3, 32 samples); NITAC (ICD-O code 8140/3,....
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Results
In the normal mucosa we observed strong and homogenous nuclear reactivity for OTX genes both in the ciliated pseudostratified respiratory-type epithelium and in the submucosal glandular cells (Figure 1A). We found nuclear expression for OTX1 in all NITACs samples (Figure 1B), while little or absent immunoreactivity was highlighted in ITACs (Figure 1C). Intense immunoreactivity was present in all ONs .......
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Discussion
This study shows for the first time that, based on mRNA levels, the HB genes OTX1 and OTX2 are expressed in normal sinonasal mucosa and submucosal glands, inflammatory polyps, sinonasal Schneiderian papillomas, and in the different epithelial and neuroectodermal neoplasms, including squamous carcinomas, non-intestinal type sinonasal adenocarcinomas, salivary gland-type tumors, neuroendocrine neoplasms, and ONs.
Modifications and Troubleshooting:
To avoid RNA degradatio.......
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Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by Centro Grandi Strumenti Università dell'Insubria, Fondazione Comunitaria del Varesotto, Fondazione del Monte di Lombardia, and Fondazione Anna Villa e Felice Rusconi.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| RecoverAll Total Nucleic Acid Isolation | Applied Biosystem | AM1975 | |
| High-Capacity cDNA Reverse Transcription Kit | Applied Biosystem | 4368814 | |
| TaqMan Universal PCR Master Mix, no AmpErase UNG | Applied Biosystem | 4326614 | |
| ABI Prism 7000 | Applied Biosystem | 270001857 | |
| ACTB probe | Applied Biosystem | Out of production. Sequence protected by copyright | |
| OTX1 probe | Applied Biosystem | Out of production. Sequence protected by copyright | |
| OTX2 probe | Applied Biosystem | Out of production. Sequence protected by copyright | |
| Acqueous Hydrogen Peroxide | Merk | 1072090250 | |
| Citrate Buffer | Sigma-Aldrich | 20276292 | |
| Triton | Sigma-Aldrich | 101473728 | |
| Tris | Merk | 108382 | |
| NaCl | Merk | 106404 | |
| Goat Anti-OTX2 Antibody | Vector Laboratories | Out of production. Catalog number not available | |
| Rabbit Anti-Goat Antibody | Vector Laboratories | BA5000 | |
| ABC-Peroxidase Complex | Vector Laboratories | PK6100 | |
| 3,3'-diaminobenzidine tetrahydrocloride (DAB) | Sigma-Aldrich | D5905 | |
| Harris Hematoxylin | Bioptica | 0506004/L | |
| Pertek | Kaltek SRL | 1560 | |
| BioClear | Bioptica | W01030799 |
References
- Boncinelli, E., Simeone, A., Acampora, D., Gulisano, M. Homeobox genes in the developing central nervous system. Ann genet. 36 (1), 30-37 (1993).
- Omodei, D., et al. Expression of the Brain Transcription Factor....
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