摘要
This study investigated the effect of etoposide,an anticancer chemotherapy drug,on B7-H1 expression in retinoblastoma(Rb) cells and the role of miR-513a-5p in the process.Rb cells were divided into control and etoposide groups.In the etoposide group,cells were treated with etoposide at different concentrations(2.5,5,10,20 and 40 μg/mL) for 24 h.Those given no treatment of etopside served as controls.Reverse transcription polymerase chain reaction(RT-PCR),fluorescence quantitative PCR and flow cytometry were performed to measure the mRNA and protein expression of B7-H1 in Rb cells.The mRNA expression of miR-513a-5p in Rb cells before and after etoposide treatment was also detected by fluorescence quantitative PCR.The miR-513a-5p mimics and the miR-513a-5p inhibitor were transfected into Rb cells separately,and fluorescence quantitative PCR and flow cytometry were used to detect the effect of the miR-513a-5p mimics or inhibitor on B7-H1 expression.TargetScan5.2 was employed to predict the miR-513a-5p binding sites in the 3’-untranslated region of B7-H1 mRNA.Luciferase reporter plasmids carrying this site were prepared and transfected into Rb cells and luciferase activity analyzed.The results showed that etoposide stimulated the mRNA and protein expression of B7-H1 in Rb cells,which reached a maximal level after treatment with 5 μg/mL etoposide(P<0.05).However,miR-513a-5p expression was decreased in Rb cells after etoposide treatment.When the miR-513a-5p inhibitor was added,B7-H1 expression was increased with the concentration of the miR-513a-5p inhibitor(P<0.05).Moreover,B7-H1 expression was decreased gradually with the concentration of the miR-513a-5p mimics increased(P<0.01).Additionally,the miR-513a-5p mimics were found to inhibit the luciferase activity.It was concluded that etoposide can promote B7-H1 expression in Rb cells,which may be associated with chemoresistance.The promoting effect of etoposide on B7-H1 expression can be reversed by miR-513a-5p mimics.MiR-513a-5p inhibits the mRNA and protein expression of B7-H1 via binding to the 3’-UTR of B7-H1 mRNA.
This study investigated the effect of etoposide,an anticancer chemotherapy drug,on B7-H1 expression in retinoblastoma(Rb) cells and the role of miR-513a-5p in the process.Rb cells were divided into control and etoposide groups.In the etoposide group,cells were treated with etoposide at different concentrations(2.5,5,10,20 and 40 μg/mL) for 24 h.Those given no treatment of etopside served as controls.Reverse transcription polymerase chain reaction(RT-PCR),fluorescence quantitative PCR and flow cytometry were performed to measure the mRNA and protein expression of B7-H1 in Rb cells.The mRNA expression of miR-513a-5p in Rb cells before and after etoposide treatment was also detected by fluorescence quantitative PCR.The miR-513a-5p mimics and the miR-513a-5p inhibitor were transfected into Rb cells separately,and fluorescence quantitative PCR and flow cytometry were used to detect the effect of the miR-513a-5p mimics or inhibitor on B7-H1 expression.TargetScan5.2 was employed to predict the miR-513a-5p binding sites in the 3'-untranslated region of B7-H1 mRNA.Luciferase reporter plasmids carrying this site were prepared and transfected into Rb cells and luciferase activity analyzed.The results showed that etoposide stimulated the mRNA and protein expression of B7-H1 in Rb cells,which reached a maximal level after treatment with 5 μg/mL etoposide(P<0.05).However,miR-513a-5p expression was decreased in Rb cells after etoposide treatment.When the miR-513a-5p inhibitor was added,B7-H1 expression was increased with the concentration of the miR-513a-5p inhibitor(P<0.05).Moreover,B7-H1 expression was decreased gradually with the concentration of the miR-513a-5p mimics increased(P<0.01).Additionally,the miR-513a-5p mimics were found to inhibit the luciferase activity.It was concluded that etoposide can promote B7-H1 expression in Rb cells,which may be associated with chemoresistance.The promoting effect of etoposide on B7-H1 expression can be reversed by miR-513a-5p mimics.MiR-513a-5p inhibits the mRNA and protein expression of B7-H1 via binding to the 3'-UTR of B7-H1 mRNA.
