The DNA methylation of FOXO3 and TP53 as a blood biomarker of late-onset asthma
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Journal of Translational Medicine Open Access
RESEARCH
The DNA methylation of FOXO3 and TP53 as a blood biomarker of late‑onset asthma Lin Yuan1,2,3, Leyuan Wang2, Xizi Du2, Ling Qin1,3, Ming Yang4, Kai Zhou2, Mengping Wu2, Yu Yang2, Zhiyuan Zheng1,3, Yang Xiang2, Xiangping Qu2, Huijun Liu2, Xiaoqun Qin2 and Chi Liu1,2,5*
Abstract Background: Late-onset asthma (LOA) is beginning to account for an increasing proportion of asthma patients, which is often underdiagnosed in the elderly. Studies on the possible relations between aging-related genes and LOA contribute to the diagnosis and treatment of LOA. Forkhead Box O3 (FOXO3) and TP53 are two classic aging-related genes. DNA methylation varies greatly with age which may play an important role in the pathogenesis of LOA. We supposed that the differentially methylated sites of FOXO3 and TP53 associated with clinical phenotypes of LOA may be useful biomarkers for the early screening of LOA. Methods: The mRNA expression and DNA methylation of FOXO3 and TP53 in peripheral blood of 43 LOA patients (15 mild LOA, 15 moderate LOA and 13 severe LOA) and 60 healthy controls (HCs) were determined. The association of methylated sites with age was assessed by Cox regression to control the potential confounders. Then, the correlation between differentially methylated sites (DMSs; p-value 20 years of age) were selected from the Respiratory Department and the Medical Examination Center of Xiangya Hospital in China from October 2018 to January 2019. After Written informed consent was obtained from each patient, questionnaire information (general condition, smoking history and other respiratory diseases), pulmonary function testing, peripheral blood and induced sputum samples were collected. Lung function test included the spirometric values of F EV1, FEV1% predicted, FVC, the ratio of F EV1 to the FVC, PEF, FEF75, FEF50 and F EF25. The study was approved by No. 20180308 of the Xiangya Hospital Ethics Review Committee and participants provided written consents to participate in this study. Sample collection
Peripheral blood and induced sputum samples were collected from the enrolled 60 HCs and 43 LOA patients, respectively. Peripheral blood was collected into 5 ml EDTA anticoagulation tubes and then transferred to a centrifuge tube. After adding 2 volumes of erythrocyte lysate and lysing for 5 min, peripheral blood cells were pelleted by centrifugation.
Yuan et al. J Transl Med
(2020) 18:467
For sputum induction, each volunteer inhaled a 4.5% saline atomized solution three times for 5 min each time and coughed sputum into a separate cup. Then, fourparts 0.1% dithiothreitol was added to one-part sputum and mixed for 15 min before adding four-parts phosphate-buffered saline. Finally, sputum cells were pelleted by centrifugation and used for RNA extraction after filtering [35]. RNA extraction, RT‑PCR and quantitative RT‑PCR
Total mRNA was purified from peripheral blood and induced sputum cells using Trizol (Invitrogen) and quantified by an ultraviolet spectrophotom
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