时间性miRNA重塑界定了间歇性缺氧诱导心肌细胞损伤中免疫炎症激活的24小时调控阈值

文献信息

中文摘要

背景： 间歇性缺氧（IH）是阻塞性睡眠呼吸暂停（OSA）的标志性特征，通过氧化应激、炎症和免疫失调等机制导致心脏损伤。然而，miRNA介导的转录反应被激活的时间阈值及其在此过程中的免疫调节作用仍不清楚。
方法： 在H9C2心肌细胞中建立体外IH模型，分别暴露于IH 8小时和24小时。评估细胞损伤、线粒体功能障碍、炎症反应和细胞凋亡。进行小RNA测序和mRNA转录组分析，以鉴定IH应答miRNA及其调控网络。进行整合的miRNA-mRNA分析以揭示潜在的免疫炎症通路，并通过抑制剂介导的功能实验进一步验证miR-146b-5p。
结果： IH诱导严重程度依赖性的心肌细胞损伤，其特征为线粒体膜去极化、促炎细胞因子（TNF-α、IL-6、IL-1β）表达增加以及caspase-3介导的细胞凋亡激活。8小时未检测到差异表达miRNA（DEmiR），而24小时鉴定出18个DEmiR，表明IH诱导miRNA激活存在关键的24小时阈值。整合的miRNA-mRNA分析显示免疫相关通路显著富集，包括：细胞因子-细胞因子受体相互作用、IL-17信号通路、Toll样受体信号通路、PD-L1/PD-1检查点、MAPK、FoxO、Wnt和mTOR通路。Fos、Gsk3b、Adam17、Cd200、Socs6和Smad4等关键枢纽基因被鉴定为炎症和免疫反应的中心调控因子。在DEmiR中，miR-146b-5p、miR-26a-5p和miR-23a-3p被突出显示为IH条件下免疫炎症信号的潜在调节因子。综上所述，我们的研究结果确定miR-146b-5p是关键的IH应答miRNA，并揭示其上调通过激活炎症和凋亡通路促进IH诱导的心肌细胞损伤进展。
结论： 本研究确定了IH诱导心肌细胞损伤中miRNA介导免疫调节的24小时时间阈值。整合的miRNA-mRNA分析揭示IH应答miRNA参与免疫炎症通路，并确定miR-146b-5p为关键候选调控因子。功能验证表明，IH暴露诱导miR-146b-5p上调，其通过促进炎症反应和细胞凋亡导致心肌细胞损伤。重要的是，抑制miR-146b-5p可通过降低TNF-α表达、抑制cleaved caspase-3激活并恢复Bcl-2水平来减轻IH诱导的细胞损伤。这些发现为miRNA在早期免疫失调中的作用提供了机制见解，并提示靶向特定miRNA，尤其是miR-146b-5p，可能为预防IH相关心肌病提供新的治疗策略。

英文摘要

BACKGROUND: Intermittent hypoxia (IH), a hallmark of obstructive sleep apnea (OSA), contributes to cardiac injury through mechanisms including oxidative stress, inflammation, and immune dysregulation. However, the temporal threshold at which miRNA-mediated transcriptional responses are activated, as well as their immunoregulatory roles in this process, remain unclear.
METHODS: An in vitro IH model was established in H9C2 cardiomyocytes exposed to IH for 8 h and 24 h. Cellular injury, mitochondrial dysfunction, inflammatory responses, and apoptosis were assessed. Small RNA sequencing and mRNA transcriptome profiling were performed to identify IH-responsive miRNAs and their regulatory networks. Integrated miRNA-mRNA analysis was conducted to reveal potential immune-inflammatory pathways, and miR-146b-5p was further validated using inhibitor-mediated functional assays.
RESULTS: IH induced severity-dependent cardiomyocyte injury, characterized by mitochondrial membrane depolarization, increased expression of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β), and activation of caspase-3-mediated apoptosis. No DEmiRs were detected at 8 h, whereas 18 DEmiRs were identified at 24 h, indicating a critical 24-hour threshold for IH-induced miRNA activation. Integrated miRNA-mRNA analysis revealed significant enrichment in immune-related pathways, including: Cytokine-cytokine receptor interaction, IL-17 signaling, Toll-like receptor signaling, PD-L1/PD-1 checkpoint, MAPK, FoxO, Wnt, and mTOR pathways. Key hub genes such as Fos, Gsk3b, Adam17, Cd200, Socs6, and Smad4 were identified as central regulators of inflammation and immune responses. Among the DEmiRs, miR-146b-5p, miR-26a-5p, and miR-23a-3p were highlighted as potential modulators of immune-inflammatory signaling under IH conditions. Taken together, our findings identify miR-146b-5p as a critical IH-responsive miRNA and reveal that its upregulation contributes to the progression of IH-induced cardiomyocyte injury through activation of inflammatory and apoptotic pathways.
CONCLUSION: This study identifies a 24-hour temporal threshold for miRNA-mediated immune regulation in IH-induced cardiomyocyte injury. Integrated miRNA-mRNA analysis reveals that IH-responsive miRNAs are involved in immune-inflammatory pathways and identifies miR-146b-5p as a key candidate regulator. Functional validation demonstrates that IH exposure induces miR-146b-5p upregulation, which contributes to cardiomyocyte injury by promoting inflammatory responses and apoptosis. Importantly, inhibition of miR-146b-5p alleviates IH-induced cellular damage by reducing TNF-α expression, suppressing cleaved caspase-3 activation, and restoring Bcl-2 levels. These findings provide mechanistic insights into the role of miRNAs in early immune dysregulation and suggest that targeting specific miRNAs, particularly miR-146b-5p, may offer novel therapeutic strategies for preventing IH-related cardiomyopathy.