SPOP protein regulates Hh signal pathway and promotes proliferation and apoptosis of esophageal squamous cells
Objective To investigate the expression of SPOP in esophageal squamous cell carcinoma(ESCC)cells and its effect on its proliferation and apoptosis and underlying mechanisms.Methods The expression of SPOP in normal esoph-ageal epithelial cells and esophageal squamous cell cells was compared by qRT-PCR.CCK8 and colony formation assay were performed to detect the proliferation of ESCC cells.Cell apoptosis and cell cycle were detected by flow cytometry.qRT-PCR and Western blot were used to detect the gene expression of Hh signal pathway in ESCC cells after siRNA transfection.Results Compared with HEEC cells,mRNA expression of SPOP gene was increased in ECA109,EC9706 and TE-1 cells,and t values were 2.77,3.00 and 3.23,respectively,P values were 0.047,0.038 and 0.026.The results of CCK8 experiment showed that compared with the control group,the activity of ECA-109 cells decreased gradually after SPOP was knocked down on the 1st,2nd,3rd,4th and 5th days(F=10.12,P＜0.001).The results of cell clonal for-mation experiment showed that compared with the control group,the number of ECA-109 cell clonal formation decreased after SPOP knocking down(t=6.31,P=O.002).Flow cytometry was used to detect the effect of SPOP on apoptosis of ECA-109 cells.The results showed that compared with the control group,the number of apoptosis increased in the SPOP knockout group(t=6.98,P=0.001).Compared with the control group,the proportion of ECA-109 cells in G1 phase increased in SPOP knockdown group(t=4.02,P=0.012),and there was no statistical significance in the proportion of S phase and G2/M phase cells(P＞0.05).qRT-PCR results showed that compared with the control group,after SPOP gene expression was inhibited,the mRNA expression of Cul3 gene was increased(t=3.91,P=0.014),and the Gli3 gene expression was decreased(t=14.20,P＜0.001).There was no significant difference in the expression of Gli1 and Gli2 genes(P＞0.05).Western blot results showed that the expression levels of Gli3 and Gli1 were decreased after siSPOP transfection(t values were 4.89 and 7.21,P values were 0.005 and 0.001),while the expression levels of Gli2,Cul3 and PSMC3 were increased(t values were 4.19,3.01 and 5.13,respectively,P values were 0.011,0.038 and 0.005).Con-clusion The high expression of SPOP in ESCC may promote the proliferation of ESCC cells,inhibit cell apoptosis and induced G1/S phase transition by regulating the Hh signal pathway.
esophageal carcinomaSPOPproliferationapoptosisHh signal pathwayGli protein
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