Chronic obstructive pulmonary disease (COPD) is a significant public health problem characterized by persistent airflow limitation. Despite previous research into the pathogenesis of COPD, a comprehensive understanding of the cell-type-specific mechanisms in COPD remains lacking. Recent studies have implicated Rab GTPases in regulating chronic immune response and inflammation via multiple pathways. In this study, the molecular regulating mechanism of RAB32 in COPD was investigated by multiple bioinformatics mining and experimental verification. We collected lung tissue surgical specimens from Zhongshan Hospital, Fudan University, and RT-qPCR and western blotting were used to detect the expression of Rabs in COPD lung tissues. Four COPD microarray datasets from the Gene Expression Omnibus (GEO) were analyzed. COPD-related epithelial cell scRNA-seq data was obtained from the GSE173896 dataset. Weighted gene co-expression network analysis (WGCNA), mfuzz cluster, and Spearman correlation analysis were combined to obtain the regulatory network of RAB32 in COPD. The slingshot algorithm was used to identify the regulatory molecule, and the co-localization of RAB32 and GPRC5A was observed with immunofluorescence. WGCNA identified 771 key module genes significantly associated with the occurrence of COPD, including five Rab genes. RAB32 was up-regulated in lung tissues from subjects with COPD as contrast to those without COPD on both mRNA and protein levels. Integrating the results of WGCNA, Mfuzz clusters, and Spearman analysis, nine potential interacting genes with RAB32 were identified. Among these genes, GPRC5A exhibited a similar molecular expression pattern to RAB32. Co-expression density analysis at the cell level demonstrated that the co-expression density of RAB32 and GPRC5A was higher in type I alveolar epithelial cells (AT1s) than in type II alveolar epithelial cells (AT2s). The immunofluorescence also confirmed the co-localization of RAB32 and GPRC5A, and the Pearson correlation analysis found the relationship between RAB32 and GPRC5A was significantly stronger in the COPD lungs (r = 0.65) compared to the non-COPD lungs (r = 0.33). Our study marked endeavor to delineate the molecular regulatory axis of RAB32 in COPD by employing diverse methods and identifying GPRC5A as a potential interacting molecule with RAB32. These findings offered novel perspectives on the mechanism of COPD.

中文翻译：

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解读慢性阻塞性肺疾病中 RAB32/GPRC5A 轴的分子调控

慢性阻塞性肺疾病（COPD）是一个重大的公共卫生问题，其特征是持续气流受限。尽管先前对 COPD 的发病机制进行了研究，但仍缺乏对 COPD 细胞类型特异性机制的全面了解。最近的研究表明 Rab GTP 酶通过多种途径调节慢性免疫反应和炎症。本研究通过多重生物信息学挖掘和实验验证，探讨RAB32在COPD中的分子调控机制。采集复旦大学附属中山医院肺组织手术标本，采用RT-qPCR和Western blotting检测COPD肺组织中Rabs的表达情况。对来自基因表达综合库 (GEO) 的四个 COPD 微阵列数据集进行了分析。COPD 相关上皮细胞 scRNA-seq 数据从 GSE173896 数据集中获得。结合加权基因共表达网络分析（WGCNA）、mfuzz聚类和Spearman相关分析，获得RAB32在COPD中的调控网络。采用弹弓算法识别调节分子，并通过免疫荧光观察RAB32和GPRC5A的共定位。WGCNA 鉴定出 771 个与 COPD 发生显着相关的关键模块基因，其中包括 5 个 Rab 基因。与非 COPD 受试者相比，患有 COPD 受试者的肺组织中 RAB32 在 mRNA 和蛋白质水平上均上调。整合 WGCNA、Mfuzz 聚类和 Spearman 分析的结果，鉴定出 9 个潜在与 RAB32 相互作用的基因。在这些基因中，GPRC5A 表现出与 RAB32 相似的分子表达模式。细胞水平的共表达密度分析表明，RAB32和GPRC5A在I型肺泡上皮细胞（AT1s）中的共表达密度高于II型肺泡上皮细胞（AT2s）。免疫荧光还证实了 RAB32 和 GPRC5A 的共定位，Pearson 相关分析发现，与非 COPD 肺部 (r = 0.33) 相比，COPD 肺部 (r = 0.65) 中 RAB32 和 GPRC5A 之间的关系明显更强。我们的研究标志着通过采用多种方法来描绘 COPD 中 RAB32 的分子调节轴，并将 GPRC5A 鉴定为与 RAB32 潜在的相互作用分子。这些发现为慢性阻塞性肺病的机制提供了新的视角。