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 etoposi...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.展开更多
Background:Circular RNAs(circR NAs)are covalently closed single-stranded RNAs with multiple biological functions.CircRNA.0007127 is derived from the carbon catabolite repression 4-negative on TATA-less(CCR4-NOT)comple...Background:Circular RNAs(circR NAs)are covalently closed single-stranded RNAs with multiple biological functions.CircRNA.0007127 is derived from the carbon catabolite repression 4-negative on TATA-less(CCR4-NOT)complex subunit2(CNOT2),which was found to regulate tumor cell apoptosis through caspase pathway.Methods:Potential circR NA.0007127 target microRNAs(miRNAs)were analyzed by miRanda,TargetScan,and RNAhybrid software,and the miRNAs with binding sites for apoptosis-related genes were screened.The roles of circR NA.0007127 and its downstream target,microR NA(miR)-513a-5p,were validated by quantitative real-time polymerase chain reaction(qPCR),flow cytometry,mitochondrial membrane potential,immunofluorescence,western blot,and caspase-8(CASP8)protein activity in vitro in HO-induced K-562 cells.The circRNA.0007127-miR-513a-5p and CASP8-miR-513a-5p interactions were verified by luciferase reporter assays.Results:Silencing circRNA.0007127 decreased cell apoptosis by inhibiting CASP8 pathway activation in K-562 cells.Compared with the control group,the expression of CASP8 was reduced by 50%and the 43-kD fragment of CASP8 protein was significantly reduced(P≤0.05).The luciferase reporting assay showed that circRNA.0007127 combined with miR-513a-5p or CASP8,with extremely significant differences(P≤0.001).The overexpression of miR-513a-5p inhibited the gene expression level of CASP8in a human myeloid leukemia cell model(75%change)and the level of a 43-kD fragment of CASP8 protein(P small-interfering RNA(siRNA)and the miR-≤0.01).The rescue experiment showed that cotransfection with circRNA.0007127513a-5p inhibitor increased CASP8 gene expression and the apoptosis rate,suggesting that the miR-513a-5p inhibitor is a circRNA.0007127siRNA antagonist.Conclusions:CircRNA.0007127 regulates K-562 cell apoptosis through the miR-513a-5p/CASP8 axis,which can serve as a novel powerful molecular target for K-562 cells.展开更多
文摘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.
基金supported by the Guangzhou Science and Technology Plan Project(No.201904010027)the Key Clinical Technology Program of Guangzhou(No.2019ZD18),China。
文摘Background:Circular RNAs(circR NAs)are covalently closed single-stranded RNAs with multiple biological functions.CircRNA.0007127 is derived from the carbon catabolite repression 4-negative on TATA-less(CCR4-NOT)complex subunit2(CNOT2),which was found to regulate tumor cell apoptosis through caspase pathway.Methods:Potential circR NA.0007127 target microRNAs(miRNAs)were analyzed by miRanda,TargetScan,and RNAhybrid software,and the miRNAs with binding sites for apoptosis-related genes were screened.The roles of circR NA.0007127 and its downstream target,microR NA(miR)-513a-5p,were validated by quantitative real-time polymerase chain reaction(qPCR),flow cytometry,mitochondrial membrane potential,immunofluorescence,western blot,and caspase-8(CASP8)protein activity in vitro in HO-induced K-562 cells.The circRNA.0007127-miR-513a-5p and CASP8-miR-513a-5p interactions were verified by luciferase reporter assays.Results:Silencing circRNA.0007127 decreased cell apoptosis by inhibiting CASP8 pathway activation in K-562 cells.Compared with the control group,the expression of CASP8 was reduced by 50%and the 43-kD fragment of CASP8 protein was significantly reduced(P≤0.05).The luciferase reporting assay showed that circRNA.0007127 combined with miR-513a-5p or CASP8,with extremely significant differences(P≤0.001).The overexpression of miR-513a-5p inhibited the gene expression level of CASP8in a human myeloid leukemia cell model(75%change)and the level of a 43-kD fragment of CASP8 protein(P small-interfering RNA(siRNA)and the miR-≤0.01).The rescue experiment showed that cotransfection with circRNA.0007127513a-5p inhibitor increased CASP8 gene expression and the apoptosis rate,suggesting that the miR-513a-5p inhibitor is a circRNA.0007127siRNA antagonist.Conclusions:CircRNA.0007127 regulates K-562 cell apoptosis through the miR-513a-5p/CASP8 axis,which can serve as a novel powerful molecular target for K-562 cells.
