Regulação de genes da família PPAR por epigenética: contribuição à cuidados com a saúde em câncer de cabeça e pescoço

Abstract

Objectives: To evaluate the regulation of PPAR family genes in head and neck cancer (HNC) through epigenetic mechanisms. Methods: HNC cells from the oral cavity (HN13) and hypopharynx (FaDu) were cultured. The transfection assays with the mimics (miR-9-5p, miR 17-5p and miR-21-5p, positive and negative control) were carried out in 24-well plates for 48 hours, containing around 80,000 cells/well, using 100µL of Opti-MEN medium (Invitrogen), 500µL of DMEM medium with 1µL of LipofectamineTM RNAiMAX Tranfection Reagent (Invitrogen) and 10mM of mimic for each miRNA. After transfection, total RNA was extracted with Tryzol (Applied Biosystems) and protein with RIPA. Gene expression was analyzed using the quantitative polymerase chain reaction (qPCR) method for PPARα and PPARᵧ TaqmanTM (Thermo Fisher Scientific) and GAPDH (Thermo Fisher Scientific) as a reference gene, comparing both to the negative control (RQ=1). As a method for analyzing protein expression, the Western Blotting (WB) technique was used for PPARα and PPARᵧ, following the manufacturer's concentration instructions. Beta-actin antibody was used as a normalizer for the technique. Results: The results showed that there was a decrease in PPARα gene expression in HN13 cells, with a 40% reduction for miR-9-5p (p=0.031), 30% for miR 17-5p (p=0.0.35) and 25% for miR-21-5p (0.0.31). For the FaDu strain, PPARα expression was reduced after miR-9-5p transfection (50%, p=0.031), followed by miR-21-5p (25%, p=0.031) and miR-17-5p (23%, p=0.093), although it did not reach statistical significance for miR-17-5p. Protein expression showed that the microRNAs miR-17-5p and miR-21-5p were reduced compared to the negative control, while miR-9-5p expression was increased. In the HN13 strain there was an increase in PPARᵧ expression compared to the control group after transfection of all the miRNAs, 85% (p=0.0878) with miR-9-5p, 263% (p=0.0454) miR17-5p and 80% (p=0.0335) with miR-21-5p. The same was observed in the FaDu strain, with 80% (p=0.0374) miR-9-5p, 72% (p=0.0580) miR-17-5p and 25% 9p=0.0365) with miR-21-5p. The protein expression of PPARα by WB showed a reduction after transfection with miR-17-5p in the HN13 strain, around 52 kDa. Samples transfected with miR-9-5p and miR-21-5p showed no reduction. Conclusion: The results show that miR-9-5p and miR-21-5p possibly regulate PPARα gene expression in the HN13 and FaDu lineages. However, miR-17-5p regulates PPARα only in the HN13 lineage. However, PPARᵧ expression was not reduced after transfection of all miRNAs. This highlights the importance of these genes in the metabolic and inflammatory processes related to CCP. Through the results, it is possible to understand the fundamental importance of biomarkers for diagnosis, treatment and monitoring of diseases, such as head and neck cancer, which involves multidisciplinary teams, driving crucial advances in health. Knowledge of this regulatory mechanism could contribute to the health care of patients with HNC