Division of Pediatric Pulmonology, Wuxi Children's Hospital Affiliated to Nanjing Medical University, Wuxi, Jiangsu 214023, P.R. China.
Department of Pediatrics, The Fourth Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212000, P.R. China.
Int J Mol Med. 2019 Mar;43(3):1263-1270. doi: 10.3892/ijmm.2019.4054. Epub 2019 Jan 9.
Histone acetylation is important in the modification of gene transcription in asthma and is regulated by histone acetyltransferases (HATs). P300 (P300 HAT) is an enzyme that is able to acetylate a wide variety of proteins. The modification of core histones can further regulate gene transcription, cell proliferation and other cell processes. Airway mucus hypersecretion is one of the most serious pathophysiological symptoms of chronic airway inflammatory diseases, and the human mucin 5AC (MUC5AC) gene has been reported to be a major component of respiratory secretions related to asthma and chronic obstructive pulmonary disease. In the present study, the 5' sequence of the human MUC5AC gene with a 1,348‑bp DNA sequence was amplified from human A549 cells genomic DNA by polymerase chain reaction (PCR), and the product of the PCR was sequenced. By promoter deletion analysis, five promoter segments with different lengths were amplified by PCR. The products were identified by DNA sequencing and the six promoter segments were inserted into pGL3‑enhancer vectors. The core promoter area was identified with a series of 5' deletion promoter plasmids using luciferase reporter assays. MUC5AC promoter activity, and the mRNA and protein expression levels of MUC5AC were observed in P300 wild‑type, P300 mutant, P300 small interfering RNA and P300 control groups. The results showed that the core promoter area of MUC5AC was located within the ‑935/+48 region and that P300 reduced the expression of MUC5AC in A549 cells.
组蛋白乙酰化在哮喘中基因转录的修饰中很重要,并且受组蛋白乙酰基转移酶(HATs)调控。P300(P300 HAT)是一种能够乙酰化多种蛋白质的酶。核心组蛋白的修饰可以进一步调节基因转录、细胞增殖和其他细胞过程。气道黏液高分泌是慢性气道炎症性疾病最严重的病理生理症状之一,并且已经报道人类粘蛋白 5AC(MUC5AC)基因是与哮喘和慢性阻塞性肺疾病相关的呼吸道分泌物的主要成分。在本研究中,通过聚合酶链反应(PCR)从人 A549 细胞基因组 DNA 中扩增人 MUC5AC 基因的 5'序列,长度为 1348bp 的 DNA 序列,并且对 PCR 产物进行测序。通过启动子缺失分析,通过 PCR 扩增了五个不同长度的启动子片段。通过 DNA 测序鉴定产物,并将六个启动子片段插入 pGL3-增强子载体。通过一系列 5'缺失启动子质粒使用荧光素酶报告基因检测鉴定核心启动子区。观察 P300 野生型、P300 突变型、P300 小干扰 RNA 和 P300 对照组中 MUC5AC 启动子活性以及 MUC5AC 的 mRNA 和蛋白表达水平。结果表明,MUC5AC 的核心启动子区位于-935/+48 区域内,并且 P300 降低了 A549 细胞中 MUC5AC 的表达。