RNAi targeting Nogo Receptor enhanced survival and proliferation of murine retinal ganglion cells during N-methyl-D-aspartateinduced optic nerve crush
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Kun Zeng1,*, Bo Zhong2,*, Xiao-Li Shen1, Min Fang1, Bao-Tao Lin1 and Da-Hui Ma1
1Key Laboratory of Ophthalmology, Shenzhen Eye Hospital, Ophthalmology College of Shenzhen University, Shenzhen 518000, P.R. China
2Department of Stomatology, Shenzhen Second People’s Hospital, Shenzhen 518000, P.R. China
*Considered to be the first author
Kun Zeng, email: [email protected]
Keywords: Nogo receptor, RNA interference, proliferation, apoptosis, mouse retinal ganglion cells
Received: December 15, 2016 Accepted: March 24, 2017 Published: April 21, 2017
We investigated the effects of lentivirus-mediated RNAi targeting of Nogo Receptor (NgR) on the proliferation and survival of murine retinal ganglion cells (mRGCs) in vitro and in vivo. Cultured mRGCs and C57BL/6 male mice were divided into 4 experimental groups: blank, model [100 μM N-methyl-D-aspartate (NMDA)], nscRNA (100 μM NMDA+ nscRNA vectors) and siNgR (100 μM NMDA+ siNgR vectors). CCK-8 and flow cytometry analyses revealed that silencing NgR enhanced proliferation, cell cycling and survival of NMDA-treated mRGCs. H&E staining showed that NgR silencing enhanced mRGC cell density and reduced angiogenesis in NMDA-treated retinal tissues. TUNEL assays showed that mRGC apoptosis was significantly diminished by NgR silencing in NMDA-treated retinal tissues. Western blotting and qRT-PCR analysis in NMDA-treated mRGCs and murine retinal tissues revealed that NgR silencing resulted in downregulation of RhoA signaling (RhoA and ROCK2). Western blotting showed that levels of activated Bax and cleaved caspase 3 were decreased, while Bcl-2 and pro-caspase 3 were increased in NMDA-treated mRGCs and murine retinal tissues, which corroborated the decreased apoptosis. These findings indicate that NgR gene silencing increases proliferation and survival of mRGCs in NMDA-treated murine retinas, which suggests a potential for therapeutic application to preventing optic nerve damage.
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