McCluskey Elizabeth, Velli Sathesh Kanna, Kaminski Rafal, Markward Tyler, Leming Hannah, Yu Daohai, Sajjan Umadevi
Center for Inflammation and Lung Research, Lewis-Katz Medical School, Temple University, Philadelphia, PA 19140, USA.
Center for Neurovirology and Gene Editing, Lewis-Katz Medical School, Temple University, Philadelphia, PA 19140, USA.
Int J Mol Sci. 2025 Mar 5;26(5):2332. doi: 10.3390/ijms26052332.
Airway basal cells proliferate and regenerate airway epithelium after injury. The first step during airway epithelial repair is airway basal cell proliferation to close the wound. Previously, we demonstrated that expression is reduced in airway stem cells isolated from chronic obstructive pulmonary disease. is a developmental gene and plays a role in hematopoietic stem cell proliferation and differentiation, but its contribution to airway epithelial cell migration and proliferation is not known. In this study, we generated a HOXA1 knockout bronchial epithelial cell line using CRISPR/CAS9 technology followed by clonal expansion to investigate the role of HOXA1 in airway epithelial cell proliferation and migration. Compared to WT, HOXA1 knockout bronchial epithelial cells generated smaller spheroids than WT type cells, indicating a defect in cell proliferation. In the scratch assay, HOXA1 knockout cells showed substantial delay in migrating to the wounded area. By single-cell RNA sequencing and the clustering of cells based on HOXA1 expression, we identified a downregulation of genes involved in cell cycle progression. A cell cycle analysis by flow cytometry indicated partial cell cycle arrest at the G0/G1 phase in HOXA1 knockout cells. This was associated with a reduced expression of Cyclin E1 and an increased expression of the cyclin-dependent kinase inhibitor p21/CDKN1A. These results indicate that may contribute to cell proliferation by regulating cell cycle progression via p21/CDKN1A in airway epithelial cells.
气道基底细胞在损伤后会增殖并再生气道上皮。气道上皮修复的第一步是气道基底细胞增殖以闭合伤口。此前，我们证明从慢性阻塞性肺疾病患者分离出的气道干细胞中[基因名称]的表达降低。[基因名称]是一个发育基因，在造血干细胞增殖和分化中发挥作用，但其对气道上皮细胞迁移和增殖的作用尚不清楚。在本研究中，我们使用CRISPR/CAS9技术构建了一种HOXA1基因敲除的支气管上皮细胞系，随后进行克隆扩增，以研究HOXA1在气道上皮细胞增殖和迁移中的作用。与野生型相比，HOXA1基因敲除的支气管上皮细胞形成的球体比野生型细胞小，表明细胞增殖存在缺陷。在划痕试验中，HOXA1基因敲除的细胞迁移到损伤区域的时间明显延迟。通过单细胞RNA测序以及基于HOXA1表达对细胞进行聚类分析，我们发现参与细胞周期进程的基因表达下调。流式细胞术进行的细胞周期分析表明，HOXA1基因敲除的细胞在G0/G1期出现部分细胞周期停滞。这与细胞周期蛋白E1表达降低以及细胞周期蛋白依赖性激酶抑制剂p21/CDKN1A表达增加有关。这些结果表明，[基因名称]可能通过在气道上皮细胞中经由p21/CDKN1A调节细胞周期进程来促进细胞增殖。
