Cigarette Smoke Enhances The Stemness Of Renal Cancer Cells Through ΔNp63α-mediated Activation Of Sonic Hedgehog Signaling

Background:Renal cell carcinoma is a common malignant tumor of urinary system,and its specific mortality ranks the first in urinary system tumors.However,the effect of radiotherapy,chemotherapy and targeted therapy is poor,and the prognosis is extremely poor,which has become one of the important threats to national health.In our country,there are not a few smokers,and the number of cancer patients caused by smoking is also increasing year by year,so smoking cancer is widely recognized in the world,and it is also one of the most important carcinogenic and carcinogenic factors of kidney cancer.Cancer stem cells are cells that occupy a small proportion of tumors and have the ability to self-renew and generate heterogeneous tumor cells.Sonic Hedgehog(HH)signaling pathway is a key signaling pathway that regulates various types of cancer stem cells.Studies have shown that cigarette smoke can induce the increase of Sonic Hedgehog signaling pathway-related proteins in renal cancer stem cells.p63 is a member of the p53 family.In recent years,p63 has been found to be associated with many diseases,including cancer,and plays a crucial role in many aspects of cell signaling.p63 encodes two protein isoforms,TP63 and ΔNp63.The α isoform of ΔNp63 plays a key role in the regulation of stem cells,which is a major regulator to maintain the characteristics of cancer stem cells.Whether ΔNp63α promotes tumor progression in renal tumors remains to be further investigated.Objective: The aim of this study is to investigate the expression of ΔNp63α in renal cancer cells induced by cigarette smoke and its role in Sonic Hedgehog signaling pathway,and to clarify the molecular basis for targeted intervention.Methods: Renal cell carcinoma specimens from smoking and non-smoking patients were obtained from the Department of Urology and Pathology of the Second Affiliated Hospital of Anhui Medical University.Western blot and immunohistochemistry were used to detect the expression levels of ΔNp63α and renal cancer cell stemness markers in renal cancer tissues.CCK-8 assay was used to detect the stemness of renal cancer cells in different concentrations of cigarette smoke suspension(CSE).Western blot and RT-PCR were used to detect the expression levels of stemness markers of renal cancer cells in serum-free medium(SFM)and CSE treatment.The morphological changes of renal cancer cells were observed under microscope.The number of CD44 labeled positive cells was detected by flow cytometry.Plasmid transfection and small interfering RNA were used to construct the ΔNp63α overexpression and knockdown models.Western blot was used to detect the expression levels of stemness markers and Sonic Hedgehog signaling pathway related proteins in renal cancer cells treated with CSE,and the changes of cell morphology were observed under a microscope.Renal cancer cells were treated with Sonic Hedgehog pathway inhibitor Vismodegib,and the expression levels of cell stemness markers and Sonic Hedgehog signaling pathway related proteins in renal cancer cells treated with CSE were detected by Western blot.The morphological changes of renal cancer cells were observed under a microscope.Co-immunoprecipitation(Co-IP)was used to detect the relationship between ΔNp63αand Sonic Hedgehog pathway.Finally,the changes of stemness of renal cancer cells treated with SFM and CSE were detected by tumor formation experiment in nude mice.Results: The expression levels of ΔNp63α and cell stemness markers of renal cancer were significantly increased in smokers compared with non-smokers.After SFM treatment,the morphology of renal cancer cells changed from the original spindle adherent cells to spherical suspension cells,the level of stemness markers of renal cancer cells increased,and the number of CD44 labeled positive cells increased.The results of CCK-8 showed that the CSE concentration was 0,0.05% and 0.1%.After CSE treatment,the pellet diameter and number of renal cancer cells increased,the level of stemness markers of renal cancer cells increased,and the number of CD44 marked positive cells increased.After the overexpression and knockdown model of ΔNp63αwas constructed,the expression level of stemness markers and the protein associated with Sonic Hedgehog signaling pathway was increased and decreased correspondingly,and the pellet-forming ability was also increased and decreased.ΔNp63α interacts with Sonic Hedgehog signaling pathway.In vivo experiments,both SFM and CSE treatments showed an increase in renal carcinoma cell stemness.Conclusions: Cigarette smoke can enhances the stemness of renal cancer cells throughΔNp63α-mediated activation of Sonic Hedgehog signaling,which may provide new ideas for the treatment and prognosis of smoking related renal cancer.