Effect Of PHF5A On The Maintenance Of Cancer Stem-like Phenotypes In Non-small Cell Lung Cancer And Its Mechanisms

Objective:To identify the biological characteristics of cancer stem-like cells（CSLCs）in non-small cell lung cancer（NSCLC）,and to investigate the role of PHD-finger domain protein 5A（PHF5A）in the stemness maintenance of CSLCs and its potential mechanism of action.This study aims to add more and new experimental data for further characterizing the biological characterizations of NSCLC stem-like cells and clarifying the underlying molecular mechanism of NSCLC stemness maintenance.Methods:Firstly,H1299-Spheres were obtained from human parental cells by serum-free suspension culture.Using parental cells as control,changes in H299-Spheres in both expressions of the target proteins CD133,aldehyde dehydrogenase 1（ALDH1）,and epithelial-mesenchymal transition（EMT）markers tested by immunofluorescence staining,flow cytometry,Western blot and RT-q PCR,and proliferation inhibitory effect of cisplatin determined by CCK-8 assay,were compared.In parental H1299 cells,A549cells and their identified CSLCs,transwell assay was performed to detect the migration and invasive abilities.Subsequently,immunofluorescence staining,RT-q PCR and Western blot were used to detect the expression of PHF5A in H1299 cells and its CSLCs,and then a stable PHF5A-overexpressed H1299 cell model was constructed to investigate the effects of induced PHF5A up-regulation on cell stemness phenotype.Besides,setting H1299-CSLCs as control,the effects of PHF5A on cell stemness were explored by PHF5A knockdown with lentivirus-mediated sh RNA interference.Furthermore,changes in histone deacetylase 8（HDAC8）expression in each group were analyzed,and after inhibition of HDACs activity in H1299-CSLCs with Pan-HDACs inhibitor,the role of HDAC8 in PHF5A maintaining stemness were explored.Finally,the expressions of indicated proteins in human NSCLC tissues before and after recurrence/platinum resistance were also detected.Results:H1299 tumor Spheres could be formed from H1299 cells after 10-12 days of serum-free culture.When compared to H1299 cells,H1299-Spheres exhibited an enhanced expression of stemness markers CD133 and ALDH1,and also an increase in both cell subpopulation of CD133⁺/ALDH1⁺and resistance to cisplatin.CSLCs showed increased migration and invasive abilities（P<0.05）as compared to the monolayers,accompanied by EMT activation.Moreover,H1299-CSLCs showed high expression of PHF5A,and PHF5A overexpression can induce cell stem-like phenotypes.Conversely,targeted knockdown of PHF5A in H1299-CSLCs resulted in decreased cell stemness and HDAC8 expression,inhibition of HDACs activity partially suppressed H1299-CSLCs stemness.Most importantly,the expressions of PHF5A,HDAC8 and CD133 were all altered in before and after recurrent/platinum-resistant NSCLC tissues.Conclusion:Compared to parental NSCLC cells,CSLCs are shown to manifest cancer stem-like phenotypic properties.PHF5A is involved in stemness maintenance of NSCLC-CSLCs,and this process may be related to the activation of HDAC8.