Effects Of MnSOD Gene Silencing On Self-renewal And Migration Of H446 Lung Cancer Stem Cells

Objective:To investigate the biological characteristics of human small cell lung cancer cell line NCI-H446,MnSOD expression was silenced in the sphere-forming cells(refer to lung cancer stem-like cells,LCSLCs)in NCI-H446 cell line to explored by using transfection of short hairpin RNA(shRNA)to determine whether MnSOD expression mediates the self-renewal and migration of LCSLCs from NCI-H446 cell line by affecting the expression of uPAR,to provide a reference for the study of new therapeutic targets for small cell lung cancer stem cells.Methods:(1)The primary sphere-forming cells from NCI-H446 cell line were cultured in stem cell-conditioned ulture medium with ultra-low attachment surface plates,then to several passages.The highest sphere-forming efficiency spheres were used for all subsequent experiments;(2)The protein level of MnSOD in NCI-H446 parental cells and LCSLCs were analyzed by Western blot;(3)The migration and colony formation capacity of NCI-H446 parental cells and LCSLCs in vitro were respectively detected by wound healing assay and soft agar colony formation assay;(4)The MnSOD gene of LCSLCs from NCI-H446 was silenced by adenovirus expression technique.The green fluorescence protein(GFP)as a fluorescent control while the untreated group as a blank control,the MnSOD silencing efficiency was analyzed by Western blot;(5)Tumor sphere-forming assay,wound healing assay and soft agar colony formation assay were respectively used to detect the capacity of self-renewal,migration and colony formation of LCSLCs from NCI-H446 cell line after MnSOD gene silencing.(6)The protein level of uPAR in sphere-forming cells of NCI-H446 cells after MnSOD gene silencing were analyzed by Western blot.Results:(1)The results showed that the sphere-forming rate of the second passage of sphere-forming cells in NCI-H446 cell line in the third sphere culture was the highest;which refer to LCSLCs.(2)Western blot analysis showed that the protein level of MnSOD of LCSLCs from NCI-H446 cell line was higher than that of NCI-H446 parental cells;(3)Wound healing assay and soft agar colony formation assay showed that the wound healing rate and colony formation rate of LCSLCs from NCI-H446 cell line were higher than the NCI-H446 parental cells;(4)The protein level of uPAR of LCSLCs from NCI-H446 cell line was higher than the NCI-H446 parental cells;(5)The MnSOD protein of LCSLCs from NCI-H446 cell line can be effectively silenced by MnSOD shRNA;(6)MnSOD gene silencing can effectively inhibit the rates of sphere-forming,migration and colony formation of LCSLCs from NCI-H446 cells in vitro;(7)uPAR can be downregulated by MnSOD gene silencing in LCSLCs from NCI-H446 cell line.Conclusions:(1)The higher level of MnSOD was related to the reinforcing tumorigenic ability of LCSLCs from NCI-H446 cell line in vitro.(2)MnSOD gene silencing can effectively inhibits the capacity of self-renewal,migration and colony formation of NCI-H446 lung cancer stem-like cells in vitro,downregulate the expression of uPAR;...