环境因素对激素诱导性股骨头坏死中 PIK3R5 启动子甲基化及其相关通路调控的影响。
Effects of external environment on promoter methylation of PIK3R5 and related pathway regulation in steroid-induced femoral head necrosis.
机构信息
Department of Bone and Joint Surgery, Guangxi Medical University First Affiliated Hospital, Nanning, China.
Department of Rehabilitation, The Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, China.
出版信息
Environ Res. 2023 Dec 1;238(Pt 1):117116. doi: 10.1016/j.envres.2023.117116. Epub 2023 Sep 12.
BACKGROUND
Steroid-induced Avascular Necrosis of the Femoral Head (SANFH) is a condition characterized by the necrosis of the femoral head caused by long-term or high-dose hormone usage. Studies have shown that the PI3K/AKT pathway plays a crucial regulatory role in the development of SANFH. The aim of this study is to determine how external environmental factors induce changes in endogenous hormone levels, how these changes lead to steroid-induced femoral head necrosis, and the interrelationship between the changes in PIK3R5 promoter methylation levels and the regulation of the associated signaling pathways.
METHODS
Femoral head samples underwent molecular sequencing analysis. Candidate genes were screened by differential gene analysis and functional enrichment analysis.Methylation level of candidate gene PIK3R5 was verified by methylation-specific PCR(MS-PCR). SANFH model was constructed in New Zealand white rabbits, and the model results were verified by magnetic resonance imaging (MRI) and haematoxylin-eosin (HE) staining.The expression of PIK3R5, PI3K and AKT in rabbit models and human specimens was verified by real-time fluorescence quantitative PCR(RT-qPCR) and Western Blot(WB), respectively.
RESULTS
Human femoral head sequencing results indicate distinct differences in the methylation level and mRNA expression of PIK3R5 in SANFH. MS-PCR results showed the methylation level of SANFH patients was significantly higher than that of the control group (P < 0.01). The RT-qPCR results showed that PIK3R5 and PI3K expression levels in the SANFH group were lower than those in the control group (P < 0.05), and the WB experiment results were consistent with the RT-qPCR results. The MRI and HE staining results showed that the rabbit model of SANFH was successfully constructed, and the results of RT-qPCR and WB were consistent with the results of human tissues.
CONCLUSION
During the occurrence and development of SANFH, PIK3R5 gene regulates the PI3K/AKT pathway through methylation modification, promotes the oxidative stress response of cells, and accelerates the disease process.
背景
激素性股骨头坏死（SANFH）是一种由长期或大剂量激素使用引起的股骨头坏死的疾病。研究表明，PI3K/AKT 通路在 SANFH 的发生发展中起着至关重要的调节作用。本研究旨在确定外部环境因素如何诱导内源性激素水平的变化，这些变化如何导致激素性股骨头坏死，以及 PIK3R5 启动子甲基化水平变化与相关信号通路调节之间的相互关系。
方法
对股骨头样本进行分子测序分析。通过差异基因分析和功能富集分析筛选候选基因。通过甲基化特异性 PCR（MS-PCR）验证候选基因 PIK3R5 的甲基化水平。构建新西兰大白兔 SANFH 模型，通过磁共振成像（MRI）和苏木精-伊红（HE）染色验证模型结果。通过实时荧光定量 PCR（RT-qPCR）和 Western blot（WB）分别验证兔模型和人标本中 PIK3R5、PI3K 和 AKT 的表达。
结果
人股骨头测序结果表明，PIK3R5 的甲基化水平和 mRNA 表达在 SANFH 中存在明显差异。MS-PCR 结果显示，SANFH 患者的甲基化水平明显高于对照组（P<0.01）。RT-qPCR 结果显示，SANFH 组 PIK3R5 和 PI3K 的表达水平低于对照组（P<0.05），WB 实验结果与 RT-qPCR 结果一致。MRI 和 HE 染色结果表明，SANFH 兔模型构建成功，RT-qPCR 和 WB 结果与人组织结果一致。
结论
在 SANFH 的发生发展过程中，PIK3R5 基因通过甲基化修饰调节 PI3K/AKT 通路，促进细胞氧化应激反应，加速疾病进程。
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