[Effects of decreased OPTN expression on the survival of the rat retinal ganglion cell line RGC5].
OBJECTIVE
To investigate the effects of decreased OPTN expression on the morphological alteration of sub-cellular structure and the survival of the rat retinal ganglion cell line RGC5.


METHODS
Experimental study. OPTN specific siRNA was designed, synthesized and transfected into the astrocyte cell by Lipofectemine 2000. The mRNA and the protein of OPTN were assessed by real time-polymerase chain reaction (real time-PCR) and Western Blot. Eukaryotic expressing plasimid of OPTN specific siRNA (si-OPTN) was constructed with the most effective RNA interference sequence and transfected into RGC5 by Lipofectemine 2000. The RGC5 was divided into 4 groups: blank control group, pEGFP positive group, si-OPTN group, and liposome negative group. Specific fluorescent labeling dyes were used to mark the corresponding cell organelle. The distribution of optineurin and the morphology alteration of sub-cellular structure induced by the abnormal expression of optineurin in RGC5 were observed with confocal fluorescence microscopy. The effect of si-OPTN on cell survival was monitored by morphologic observation and PI-Hoechst33342 fluorescence staining of cells. One-way ANOVA was used between groups.


RESULTS
After a 24 h transfection, the expression of OPTN mRNA was significantly inhibited by transfection of si-OPTN-001, si-OPTN-002 and si-OPTN-003 which was (61.71 ± 0.84)%, (48.13 ± 0.92)% and (46.22 ± 0.73)% respectively comparing with negative control. Forty-eight hours after transfection, the protein expression of OPTN decreased to (64.44 ± 2.01)%, (57.78 ± 1.97)% and (37.78 ± 1.84)% comparing with negative control. When cells were transfected after 24 h and 48 h, the transfecting efficiency of plasmids to RGC5 was (17.43 ± 0.94)% and (20.13 ± 1.24)% respectively. The distribution of si-OPTN was similar to EGFP. The green fluorescence distributed evenly in the cytoplasm and the nucleus. Compared with negative control, the morphology of filaments, mitochondria, lysosomes, and Golgi body had no significant alteration in si-OPTN group. By using fluorescent double staining of Hoechst33342 and PI and comparing with blank control group (0.74 ± 0.34)% and negative control group (0.96 ± 0.41)%, increased cell apoptosis was observed in positive control group and si-OPTN group obviously after a 24 h transfection (F = 5.457, P < 0.05), but the apoptosis rate decreased in si-OPTN group comparing with that of positive control group (F = 6.541, P < 0.05). As the transfecting time extended, the apoptosis rate of positive control group increased when tranfecteing cultures reached 48 h. The apoptosis rate in si-OPTN group increased slightly with no significance (F = 3.212, P > 0.05).


CONCLUSIONS
Si-OPTN-003 is the most effective siRNA in inhibiting the OPTN expression. The expression of si-OPTN distributed evenly in the whole cell with no obvious injuries in the transfected cell. The down regulation of optineurin induced by si-OPTN could be decreased RGC5 cell death.
Department of Ophthalmology, Shenzhen Nanshan District People's Hospital, Shenzhen 518052, China.
Zhirong
Z
Xu
Email:chenfei72@126.com.
Fei
F
Chen
Hao
H
Yan
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology; D015536:Down-Regulation; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D051381:Rats; D012165:Retinal Ganglion Cells / Q000502:physiology* / Q000648:ultrastructure; D013997:Time Factors; D035582:Transcription Factor TFIIIA / Q000378:metabolism*; D014162:Transfection / Q000379:methods
D005136:Eye Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D035582:Transcription Factor TFIIIA