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Signalling with retinoids in the human lung: validation of new tools for the expression study of retinoid receptors
Stéphane Poulain1, Stéphanie Lacomme, Shyue-Fang Battaglia-Hsu
1Faculté de Médecine, Laboratoire de Pathologie Cellulaire et Moléculaire en Nutrition, INSERM U724, Centre de Ressources Biologiques, CHU de Nancy, 54505 Vandoeuvre-Lès-Nancy, France. Stephane.Poulain@nancy.inserm.fr
Background:
Retinoid Receptors are involved in development and cell homeostasis. Alterations of their expressions have been observed in lung cancer. However, retinoid chemoprevention trials in populations at risk to develop such tumors have failed. Therefore, the pertinence of new clinical trials using second generation retinoid requires prior better understanding of retinoid signalling. This is our aim when validating extensively research tools, focused on Retinoic Acid Receptor beta, whose major role in lung cancer is documented.
Methods:
Biocomputing was used to assess the genomic organization of RAR beta. Its putative RAR-beta1' promoter features were investigated experimentally. Specific measures realized, with qRT-PCR Syber Green assays and a triplex of Taqman probes, were extensively validated to establish Retinoid Receptors mRNAs reference values for in vivo normal human bronchial cells, lung tumors and cell lines. Finally, a pan-RAR-beta antibody was generated and extensively validated by western-blot and immunoprecipitation.
Results:
No promoter-like activity was found for RAR-beta1'. RAR-beta2 mRNAs increase signs the normal differentiation of the human bronchial epithelium while a decrease is observed in most lung cancer cell lines. Accordingly, it is also, along with RXR beta, down-regulated in lung tumors. When using nuclear extracts of BEAS-2B and normal lung cells, only the RAR-beta2 long protein isoform was recognized by our antibody.
Conclusion:
Rigorous samples processing and extensive biocomputing, were the key factors for this study. mRNA reference values and validated tools can now be used to advance researches on retinoid signalling in the lung.
Insights
Retinoic Acid Receptor beta (RAR beta) is crucial in lung cancer. This study validated tools to measure RAR beta mRNA, finding RAR-beta2 is downregulated in lung tumors, suggesting its role in retinoid signaling for lung cancer research.
Area of Science:
- Molecular Biology
- Oncology
- Gene Expression Regulation
Background:
- Retinoid Receptors (RARs) play roles in cell development and homeostasis.
- Altered RAR expression is linked to lung cancer, yet chemoprevention trials have yielded poor results.
- Understanding retinoid signaling is critical for developing new lung cancer therapies.
Purpose of the Study:
- To validate research tools for studying Retinoic Acid Receptor beta (RAR beta) in lung cancer.
- To investigate the role of RAR beta in lung cancer by analyzing its expression and signaling pathways.
Main Methods:
- Genomic organization and promoter activity of RAR beta were assessed using biocomputing.
- Quantitative real-time PCR (qRT-PCR) assays and Taqman probes were validated to quantify RAR mRNAs in various lung samples.
- A validated pan-RAR-beta antibody was generated for protein analysis via western blot and immunoprecipitation.
Main Results:
- No promoter-like activity was detected for RAR-beta1'.
- RAR-beta2 mRNA levels indicate normal bronchial epithelial differentiation but are decreased in lung cancer cell lines and tumors.
- RAR-beta2 and RXR beta are downregulated in lung tumors; only the long RAR-beta2 protein isoform was detected in normal and BEAS-2B lung cells.
Conclusions:
- Validated mRNA reference values and research tools are now available for studying retinoid signaling in lung cancer.
- The findings provide a foundation for advancing research on the role of RAR beta in lung carcinogenesis and potential therapeutic strategies.
