Expression of P2X7 receptor in RGC-5 cell line and selection of an effective siRNA sequence down-regulating P2X7 receptor / 中国医师杂志

ABSTRACT

Objective This study aims to investigate the gene expression of P2X7 receptor in retinal ganglion cell line (RGC-5),and to design and select the small interfering RNA (siRNA) which can specifically and effectively downregulate P2X7 receptor expression.Methods The mRNA and protein expressions of P2X7 receptor in RGC-5 cells were examined by real-time polymerase chain reaction (qRT-PCR),Western blot and Immunofluorescence,and compared with those of mouse retina and other tissues.Three P2X7 receptor-specific double stranded siRNAs (P2X7-siRNA-1-3) were designed and delivered into RGC-5 cells by riboFECTTM CP.60 hours after transfection,the expression level of P2X7 receptor was analyzed by qRT-PCR,Western blot and Immunofluorescence.Results The expression of P2X7 receptor was detected in both RGC-5 cell line and mouse retina,and the receptor was mainly expressed in the nucleus and cytoplasm of RGC-5 cells.P2X7-siRNA-3 can effectively down-regulate the mRNA and protein expression of P2X7 at the same time.Conclusions RGC-5 cell line can serve as a good model for studying the biological function of P2X7 receptor in retinal ganglion cells in vitro.We have managed to design and synthesize the siRNA pair that specifically and effectively lowering P2X7 receptor expression.The current study has laid the foundation for future investigations on treating glaucomatous retinopathy by downregulation of P2X7 receptor.