Li Hai-Bo, You Qi-Sheng, Xu Li-Xin, Sun Li-Xin, Abdul Majid Aman Shah, Xia Xiao-Bo, Ji Dan
Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha, China.
Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing, China.
Cell Physiol Biochem. 2017;43(5):2117-2132. doi: 10.1159/000484231. Epub 2017 Oct 24.
BACKGROUND/AIMS: The aim of the present study is to investigate the effect of long non-coding RNA-MALAT1 (LncRNA-MALAT1) on retinal ganglion cell (RGC) apoptosis mediated by the PI3K/Akt signaling pathway in rats with glaucoma.
RGCs were isolated and cultured, and monoclonal antibodies (anti-rat Thy-1, Brn3a and RBPMS) were examined by immunocytochemistry. An overexpression vector MALAT1-RNA activation (RNAa), gene knockout vector MALAT1-RNA interference (RNAi), and control vector MALAT1-negative control (NC) were constructed. A chronic high intraocular pressure (IOP) rat model of glaucoma was established by episcleral vein cauterization. The RGCs were divided into the RGC control, RGC pressure, RGC pressure + MALAT1-NC, RGC pressure + MALAT1-RNAi and RGC pressure + MALAT1-RNAa groups. Sixty Sprague-Dawley (SD) rats were randomly divided into the normal, high IOP, high IOP + MALAT1-NC, high IOP + MALAT1-RNAa and high IOP + MALAT1-RNAi groups. qRT-PCR and western blotting were used to detect the expression levels of LncRNA-MALAT1 and PI3K/Akt. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and flow cytometry were used to detect RGC apoptosis.
Immunocytochemistry revealed that the cultured RGCs reached 90% purity. Compared with the RGC pressure + MALAT1-NC group, the RGC pressure + MALAT1-RNAa group exhibited elevated expression levels of MALAT1, lower total protein levels of PI3K and Akt and decreased RGC apoptosis, while these expression levels were reversed in the RGC pressure + MALAT1-RNAi group. RGC numbers and PI3K/Akt expression levels in the high IOP model groups were lower than those in the normal group. In the high IOP + MALAT1-RNAa group, the mRNA and protein expression levels of PI3K/Akt were reduced but higher than those in the other three high IOP model groups. Additionally, RGC numbers in the high IOP + MALAT1-RNAa group were lower than those in the normal group but higher than those in the other three high IOP model groups.
Our study provides evidence that LncRNA-MALAT1 could inhibit RGC apoptosis in glaucoma through activation of the PI3K/Akt signaling pathway.
背景/目的：本研究旨在探讨长链非编码RNA-MALAT1（LncRNA-MALAT1）对青光眼大鼠中由PI3K/Akt信号通路介导的视网膜神经节细胞（RGC）凋亡的影响。
分离并培养RGC，通过免疫细胞化学检测单克隆抗体（抗大鼠Thy-1、Brn3a和RBPMS）。构建过表达载体MALAT1-RNA激活（RNAa）、基因敲除载体MALAT1-RNA干扰（RNAi）和对照载体MALAT1阴性对照（NC）。通过巩膜静脉烧灼建立慢性高眼压（IOP）青光眼大鼠模型。将RGC分为RGC对照、RGC压力、RGC压力+MALAT1-NC、RGC压力+MALAT1-RNAi和RGC压力+MALAT1-RNAa组。将60只Sprague-Dawley（SD）大鼠随机分为正常、高眼压、高眼压+MALAT1-NC、高眼压+MALAT1-RNAa和高眼压+MALAT1-RNAi组。采用qRT-PCR和蛋白质印迹法检测LncRNA-MALAT1和PI3K/Akt的表达水平。采用末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）和流式细胞术检测RGC凋亡。
免疫细胞化学显示培养的RGC纯度达到90%。与RGC压力+MALAT1-NC组相比，RGC压力+MALAT1-RNAa组MALAT1表达水平升高，PI3K和Akt的总蛋白水平降低，RGC凋亡减少，而这些表达水平在RGC压力+MALAT1-RNAi组中则相反。高眼压模型组的RGC数量和PI3K/Akt表达水平低于正常组。在高眼压+MALAT1-RNAa组中，PI3K/Akt的mRNA和蛋白表达水平降低，但高于其他三个高眼压模型组。此外，高眼压+MALAT1-RNAa组的RGC数量低于正常组，但高于其他三个高眼压模型组。
我们的研究提供了证据，表明LncRNA-MALAT1可通过激活PI3K/Akt信号通路抑制青光眼大鼠的RGC凋亡。
