Airway Basal Cells Mediate Hypoxia Induced EMT By Increasing Ribosome Biogenesis

Objective:Increased mucus secretion of the airways and accumulation of alveolar fluid are common features of many lung diseases,which not only affect the clearance of respiratory pathogens,but also increase the distance of oxygen supplement between air and the epithelium of the airway,leading to hypoxia of airway epithelium.Epithelial-mesenchymal transition（EMT）refers to the biological process of epithelial cells losing epithelial cell properties and acquiring mesenchymal cell characteristics.EMT-mediated airway remodeling is one of the important reasons that why respiratory diseases such as chronic obstructive pulmonary disease are difficult to cure.However,it is unknown whether EMT is triggered by hypoxia in airway epithelium.In our study,we aim to establish a cell model that can simulate the hypoxia in respiratory epithelium under pathological conditions,and to clarify whether hypoxia can lead to the occurrence of EMT on this model.Furtherly,we try to find the cell type in MTECs that mediate the EMT process,and explore the possible molecular mechanisms of EMT process in specific cell type by analyzing single-cell sequencing datasets.The molecular biology experiments will be applied to determine whether the relevant signaling pathways are involved in the EMT process.The results of this topic will provide new insights and ideas for the clinical prevention and treatment of EMT-related lung diseases caused by hypoxia.Methods:1.To determine whether hypoxia can induce EMT:1)To determine whether the air-liquid interface（ALI）cultured MTECs were consistent with the morphological and biophysical characteristics of mouse tracheal tissue,mouse tracheal epithelial cells（MTECs）were isolated by enzymatic digestion,and the morphological and biophysical characteristics of MTECs were observed by HE staining,transmission electron microscopy and confocal microscopy.2)The markers of basal cells and ciliated cells in MTECs were detected by western blot assay.3)To mimic the pathological situation of excess mucus in airway,excess culture medium was added to the apical side of MTECs to change the culture mode from ALI to liquid-liquid interface（liquid-liquid interface,LLI）.Western blot assay was applied to measure the expression of hypoxia-inducible factors after several hours and days LLI culture.4)The HIF1αexpression level was measured after the H441 cells cultured with 8 ml medium,while the control group was cultured with 2 ml medium.300μM of Co Cl2 was used as a positive control to treat H441 cells and detect HIF1αexpression level.5)HE staining was applied to observe the morphological changes in MTECs after hypoxia.6)The expression levels of epithelial cell marker（epithelial-cadherin）,mesenchymal cell marker（vimentin,α-smooth muscle actin）and basal cell marker（keratin 5）after hypoxia were detected by Western blot and quantitative real-time polymerase chain reaction（qRT-PCR）.2.To explore the cell types and related mechanisms that mediate hypoxia induced EMT:1)To determine whether mesenchymal cells were originated from basal cells under hypoxia,we measured the co-expression of keratin 5 and vimentin in MTECs.2)Fluorescence microscopy was used to investigate the changes of the mesenchymal and basal cell number after hypoxia,as well as the co-expression of these two cell markers.3)Basal cells in MTECs were labeled with keratin 5 antibody and sorted on flow cytometry.4)qRT-PCR was used to detect the change of EMT marker expression level in basal and other cells.5)Seurat was used to analyze the differential genes of basal cells and other cells in single-cell sequencing datasets,and we performed KEGG analysis of differential genes to explore signaling pathway in basal cells that may be involved in the EMT process.3.To explore the role of ribosome biogenesis during the hypoxia induced EMT process:1)The expression levels of ribosome RNA and ribosome protein RNA in basal cells were determined at the transcriptional level.2)After inhibiting the increase of ribosome biogenesis by applying CX5461,the expression levels of epithelial-cadherin,vimentin,α-smooth muscle actin under hypoxia condition were measured by Western blot and qRT-PCR.3)The effect of CX5461 on the morphology of MTECs was observed by HE staining.4)The number of mesenchymal cells was observed by immunofluorescence microscopy after hypoxia.5)Western blot assay was applied to detect the phosphorylation levels of m TOR and AKT.Result:1.Hypoxia induces EMT:1)Identical with the airway epithelium,MTECs cultured in ALI showed pseudostratified columnar structure.Tight junction and cilium were observed at the apical side of MTECs through transmission electron microscopy,and the height of surface liquid was about 8μm measured by confocal microscope,basically in line with the in vitro condition of the airway epithelium.2)The expression of basal and ciliated cell markers were increased after ALI culture.3)The expression level of HIF1αincreased significantly after 6 hours of LLI culture,and the expression level of HIF2αincreased significantly after 2 days LLI culture.Based on the expression of HIF,we could confirm that LLI mode culture caused MTECs hypoxia.4)The expression level of HIF1αin H441 cells increased significantly after 24hours cultured with excess medium,and 300μM Co Cl₂ significantly increased the expression level of HIF1αwithin several hours.5)After hypoxia in MTECs,the pseudostratified columnar structure disappeared,and the morphology of cell changed from cubic to spindle type.6)After 4 days hypoxia treatment of MTECs,the protein and RNA expression levels of epithelial-cadherin,vimentin,α-smooth muscle actin and keratin 5 changed significantly,indicating that hypoxia induced EMT in MTECs.2.Ribosome biogenesis in basal cell may mediate the hypoxia induced EMT:1)The numbers of basal cells and mesenchymal cells increased significantly after hypoxia,and the mesenchymal cells increased under hypoxia conditions positively expressed basal cell marker（keratin 5）,suggesting that basal cells may mediate the EMT process under hypoxia conditions.2)By using immunofluorescent microscopy,we observed that Keratin 5 and Vimentin positive cells were significantly increased after hypoxia,and these two cell markers were co-expressed.This result indicated that the mesenchymal cells might originate from basal cells under hypoxia.3)Change in the expression levels of EMT markers under hypoxia were mainly mediated by basal cells.4)Enrichment analysis showed that the differential genes between basal cells and other cells were mainly enriched in the ribosome signaling pathway.3.Increased ribosome biogenesis during the hypoxia induced EMT process:1)The expression levels of ribosome RNA and ribosome protein RNA under hypoxia conditions increased significantly.2)After the application of ribosome biogenesis inhibitor,the expression change of EMT markers was no longer significant.3)HE staining results showed that CX5461 could significantly inhibit the morphological change after hypoxia.4)CX5461 significantly inhibited the increase of mesenchymal cells under hypoxia.5)CX5461 reduced the phosphorylation levels of m TOR and AKT under hypoxia.Conclusions:Basal cells in airway epithelium may mediate the process of hypoxia induced EMT,partly through ribosome-m TORC2-AKT signaling pathway.