title: Effect of miR-513a-5p on etoposide-stimulating B7-H1 expression in retinoblastoma cells.
abstract: 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.
authors.affiliation: Eye Center, Renmin Hospital of Wuhan University, Wuhan, 430060, China.
authors.forename: Li
authors.initials: L
authors.lastname: Wu
authors.affiliation: Eye Center, Renmin Hospital of Wuhan University, Wuhan, 430060, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Department of Ophthalmology, Ezhou Central Hospital, Ezhou, 436000, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Eye Center, Renmin Hospital of Wuhan University, Wuhan, 430060, China. xyqdr07@yahoo.com.cn.
authors.forename: Yiqiao
authors.initials: Y
authors.lastname: Xing
mesh_terms: D060890:B7-H1 Antigen / Q000235:genetics*; D045744:Cell Line, Tumor; D005047:Etoposide / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D012175:Retinoblastoma / Q000235:genetics*
chemical_list: D060890:B7-H1 Antigen; C423236:CD274 protein, human; C546275:MIRN513A1 microRNA, human; D035683:MicroRNAs; D005047:Etoposide