Yang Deyi, Li Pengchong, Dang Yan, Zhu Shengtao, Shi Haiyun, Wu Ting, Zhang Zinan, Chen Chuyan, Zong Ye
Department of Gastroenterology, State Key Laboratory of Digestive Health, National Clinical Research Center for Digestive Disease, Beijing Digestive Disease Center, Beijing Key Laboratory for Precancerous Lesion of Digestive Disease, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China.
Department of Cardiovascular Medicine, Xiangya Hospital, Central South University, Changsha, Hunan, China.
Gene. 2024 Dec 30;931:148872. doi: 10.1016/j.gene.2024.148872. Epub 2024 Aug 17.
Crohn's disease (CD) is marked by disruption of intestinal epithelial barrier, with unclear underlying molecular mechanisms. This study aimed to investigate key genes regulating the intestinal barrier in CD patients.
Differential gene expression analysis and gene set enrichment analysis were conducted to identify potential key genes involved in CD within the GEO database. Single-cell RNA sequencing from ileum samples in GSE134809 of 59,831 inflamed and uninflamed cells from 11 CD patients and microarray data from ileal tissues in GSE69762 (3 controls and 4 CD patients) and GSE75214 (11 controls and 51 CD patients) with GSE179285 (49 uninflamed and 33 inflamed from CD patients) as the validation set. Protein-protein interaction and logistic regression analyses identified key downregulated genes in CD. A key gene was then investigated through immunohistochemistry of ileal tissues from 5 CD patients and in the Caco-2 cell line with RNA interference and treatment with IFN-γ and TNF-α to stimulate inflammation.
Single-cell RNA-seq identified 33 genes and microarray identified 167 genes with significant downregulation in inflamed CD samples. PCK1 was identified and validated as one of the most promising candidate genes. Reduced PCK1 expression was evident in inflamed ileal tissues. In vitro, knockdown of PCK1 resulted in decreased cell viability, increased apoptosis, and reduced nectin-2 production, while combination of IFN-γ and TNF-α significantly reduced PCK1.
PCK1 is downregulated in inflamed ileal tissues of CD patients and may be a key factor in maintaining epithelial integrity during inflammation in Crohn's disease.
克罗恩病（CD）的特征是肠道上皮屏障破坏，其潜在分子机制尚不清楚。本研究旨在调查调节CD患者肠道屏障的关键基因。
在基因表达综合数据库（GEO）中进行差异基因表达分析和基因集富集分析，以识别参与CD的潜在关键基因。对来自11例CD患者的59831个炎症和非炎症细胞的回肠样本进行单细胞RNA测序，并将基因表达综合数据库GSE69762（3例对照和4例CD患者）、GSE75214（11例对照和51例CD患者）中的回肠组织微阵列数据以及GSE179285（49例非炎症和33例CD患者炎症样本）作为验证集。通过蛋白质-蛋白质相互作用和逻辑回归分析确定CD中关键的下调基因。然后通过对5例CD患者的回肠组织进行免疫组织化学分析，并在Caco-2细胞系中利用RNA干扰以及用干扰素-γ（IFN-γ）和肿瘤坏死因子-α（TNF-α）刺激炎症来研究一个关键基因。
单细胞RNA测序鉴定出33个基因，微阵列鉴定出167个在炎症性CD样本中显著下调的基因。磷酸烯醇式丙酮酸羧激酶1（PCK1）被鉴定并验证为最有前景的候选基因之一。在炎症性回肠组织中PCK1表达明显降低。在体外，敲低PCK1导致细胞活力下降、凋亡增加以及nectin-2产生减少，而IFN-γ和TNF-α联合使用显著降低PCK1。
PCK1在CD患者的炎症性回肠组织中表达下调，可能是克罗恩病炎症期间维持上皮完整性的关键因素。
