The Role Of Sonic Hedgehog Pathway In Cigarette Smoke-Enhanced Stemness In Renal Cancer Stem Cells

Objectives Renal cell carcinoma,originating from renal tubulet epithelium,is the most commom magliganacy in kidney tumors.Substantial evidence indicates that cigarette snoke(CS)is the established risk factor of renal cell carcinoma.Cancer stem cell is featured as self-renewal and differentiation ability,which plays a crucial role in the initiation and advancement of tumor.This study was aimed to investigate the mechanism of CS-enhanced renal cell carcinoma cell stemness.Methods Serum-free medium system and in vitro CS exposure model were applied to renal carcinoma cell lines 786-O and ACHN.These two lines were cultured in serum-supplied medium(SSM)and serum-free medium(SFM),respectively,for 5 days,then renal cancer stem cell characteristics was anayzed by western blot,q RT-PCR and flow cytometry.In addition,CCK-8 was used to detected cell proliferation after 5 days culture.Optical microscope was utilized to observe the tumorsphere formation alterations,.Western blot,q RT-PCR,flow cytometry and immunofluorescence were adopted to explore the effect of cigarette smoke extrace()on 786-O and ACHN.Specific Sonic Hedgehog pathways inhibitor was conducted to explore the role of Sonic Hedgehog pathway in CS-induced stemness enhancement in renal cell carcinoma.Furthermore,immunohistochemistry and western blot were performed to detected the stemness expression and Sonic Hedgehog pathway expression in patients exposed to cigarette smoke or not.Results After cultured in SSM and SFM,respectively,786-O and ACHN cells grew adherently in SSM;under SFM,cells were able to form three-dimensional tumorspheres.And renal cancer stem cell specific markers(CD133,CD44,ALDHA1,Oct4 and Nanog)protein and m RNA expression were upregulated markedly in contrast to cultured in SSM.Additionally,was added in cells for 5 days,and CCK-8 was performed to detect the cell proliferation.After treatment with at 0.05% and 0.1%,effectively promoted the size and numbers of tumorspheres,increased the expression of renal cancer stem cell markers,cell proliferation associated proteins(PCNA and Cyclin D1)and elevated the percentage of CD133-positive cells.Immunofluorescent staining also revealed that enhanced the expression of CD44 protein in 786-O and ACHN sphere-forming cells.Furthermore,inhibition of SHH pathway dampened the promotive effects of CS on renal CSCs.Finally,higher levels of renal cancer stem cell markers and Sonic Hedgehog pathway-related proteins were observed in kidney cancer tissues from smokers than non-smoking cancer tissues.Conclusion These results demonstrated isolation renal cancer stem cell via SFM and the important role of SHH pathway in regulating CS-induced renal CSCs stemness augment.Findings from this study could provide new insight into the molecular mechanisms of CS-elicited stemness of renal CSCs.