title: Effects of optineurin siRNA on apoptotic genes and apoptosis in RGC-5 cells.
abstract: OBJECTIVE
  Optineurin is a pathogenic gene associated with primary open angle glaucoma (POAG), in which the retinal ganglion cells (RGCs) are targeted. However, the functions of optineurin, particularly in RGCs, are currently not clear. We introduced optineurin siRNA into cultured retinal ganglion cell 5 (RGC-5) and PC12 cells to determine the cellular and molecular mechanisms underlying the role of optineurin in POAG.
  
  
  METHODS
  We constructed four optineurin siRNA-expressing plasmids, and transiently transfected them into PC12 cells. Quantitative real-time PCR, western blot, and fluorescent microscopy were used to determine optineurin expression and select the most effective optineurin siRNA to construct RGC-5 and PC12 stable transfected cells. Dimethylthiazolyl diphenyl tetrazolium bromide (MTT) assay and flow cytometry were applied to investigate the role of optineurin siRNA in cell growth and apoptosis. Gene microarray and quantitative real-time PCR were used to screen and validate differentially expressed genes in optineurin siRNA transfected PC12 and RGC-5 cells.
  
  
  RESULTS
  siRNA effectively downregulated optineurin expression in RGC-5 and PC12 stable transfected cells. Optineurin siRNA significantly inhibited cell growth and increased apoptosis in RGC-5 and PC12 cells. Microarray analysis identified 112 differentially expressed genes in optineurin siRNA transfected RGC-5 cells. Quantitative real-time PCR and western blot confirmed that the expression of brain-derived neurotrophic factor (Bdnf), neurotrophin-3(Ntf3), synaptosomal-associated protein 25(Snap25), and neurofilament, light polypeptide(Nefl) was significantly downregulated in RGC-5 and PC12 cells transfected with optineurin siRNA.
  
  
  CONCLUSIONS
  Our study suggested that optineurin downregulation by siRNA in RGCs was an in vitro model for studying the mechanisms of optineurin effects on POAG. Neuroprotective factor and axonal transport genes may be involved in the development of POAG and could be novel targets for treating POAG due to optineurin mutation.
authors.affiliation: China Medical University, Beier Road No. 92, He Ping, Shen Yang, Liaoning province, China.
authors.forename: Hongyang
authors.initials: H
authors.lastname: Li
authors.forename: Xiuqin
authors.initials: X
authors.lastname: Ao
authors.forename: Juan
authors.initials: J
authors.lastname: Jia
authors.forename: Qingzhong
authors.initials: Q
authors.lastname: Wang
authors.forename: Zhongzhi
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D019208:Brain-Derived Neurotrophic Factor / Q000235:genetics / Q000378:metabolism; D002470:Cell Survival / Q000235:genetics; D015536:Down-Regulation; D015870:Gene Expression; D020869:Gene Expression Profiling; D005902:Glaucoma, Open-Angle / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis; D016716:PC12 Cells; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction; D012165:Retinal Ganglion Cells / Q000166:cytology / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics*; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism; D035582:Transcription Factor TFIIIA / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: D019208:Brain-Derived Neurotrophic Factor; D034741:RNA, Small Interfering; D050825:Synaptosomal-Associated Protein 25; D035582:Transcription Factor TFIIIA