HOXA1 Contributes to Bronchial Epithelial Cell Cycle Progression by Regulating p21/CDKN1A

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 homeobox (HOX) A1 expression is reduced in airway stem cells isolated from chronic obstructive pulmonary disease. HOXA1 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 HOXA1 may contribute to cell proliferation by regulating cell cycle progression via p21/CDKN1A in airway epithelial cells.