Effects of miR-7 regulating KLF4 expression on biological behavior of colon cancer stem cells
Objective To investigate the effect of microRNA 7(miR-7)on the biological behavior of colon cancer stem cells by targetedly regulating Krüppe-like factor 4(KLF4)expression.Methods Colon cancer stem cells were cul-tured in vitro.Colon cancer stem cells of the 15th generation,which were in the logarithmic growth stage and had good growth state,were taken and randomly divided into the control group,mimics NC group and miR-7 mimics group.Cells in the mimics NC group and miR-7 mimics group were transfected with empty plasmid and miR-7 mimics plasmid,respectively,while cells in the control group were not transfected.The transfection efficiency was verified by RT-qPCR.Colorectal can-cer stem cells transfected for 48 h in each group were collected,and cell viability was detected by CCK-8,cell migration ability was detected by cell scratch assay,cell invasion ability was detected by modified Boyden cell assay,and apoptosis rate was detected by flow cytometry.The binding site of miR-7 and KLF4 gene was predicted by TargetScan online web-site,and the regulatory effect of miR-7 on KLF4 was verified by double luciferase reporter gene assay.Colon cancer stem cells transfected for 48 h in each group were collected,and the expression levels of KLF4 mRNA and protein were detected by RT-qPCR and Western blotting,respectively.Results The relative expression of miR-7 was higher in miR-7 mimics group than in the control group and mimics NC group(both P<0.05),but there was no statistically significant difference between the control group and mimics NC group(P>0.05).The cell survival rate,cell mobility and number of transmem-brane cells in the miR-7 mimics group were lower than those in the mimics NC group and control group,and the apoptosis rate was higher than those in the mimics NC group and control group(all P<0.05);there were no significant differences in cell survival rate,cell mobility,number of transmembrane cells or apoptosis rate between the mimics NC group and control group(all P>0.05).As predicted by TargetScan online,miR-7 binding sites existed in the 3'untranslated region of KLF4 gene.Dual luciferase reporter gene experiment confirmed that miR-7 had targeted regulatory effect on KLF4.The relative expression levels of KLF4 mRNA and protein in the miR-7 mimics group were lower than those in the mimics NC group and control group(all P<0.05),but there was no significant difference between the mimics NC group and control group(all P>0.05).Conclusion MiR-7 can inhibit the proliferation,invasion,migration and promote apoptosis of colon cancer stem cells by down-regulating the expression of KLF4.
万方数据
维普
