circRNA_0051079通过靶向miR⁃26a⁃5p/PTEN轴诱导视网膜神经变性的分子机制

Molecular mechanism of circular ribonucleic acid_0051079 inducing retinal neurodegeneration by targeted micro⁃ribonucleic acid⁃26a⁃5p/PTEN axis

目的探讨circ_0051079对缺血/再灌注(I/R)损伤诱导的视网膜神经变性的影响及机制。方法从C57BL/6J乳鼠眼球中分离视网膜神经节细胞(RGC),随机分为对照组、siRNA组(转染阴性siRNA)、si_circ_0051079组(转染si⁃circ⁃0051079干扰RNA)、模拟物对照组(转染Scr mimic)、miR⁃26a⁃5p组(转染miR⁃26a⁃5p mimic)、miR⁃26a⁃5p+vector组(转染pcDNA 3.1)、miR⁃26a⁃5p+PTEN组(转染pcDNA 3.1⁃PTEN),分别在正常、缺氧(体积分数1%O_(2)暴露)或氧化应激(50μmol·L^(-1) H_(2) O_(2)暴露)条件下培养24 h,进行RT⁃qPCR、CCK⁃8、TUNEL、RNA免疫沉淀(RIP)等检测。于15只C57BL/6小鼠左眼中建立I/R损伤模型,对侧眼保持正常眼压作为对照,I/R损伤后0 d、3 d和7 d各取5只小鼠收集视网膜检测circ_0051079表达。另取20只C57BL/6小鼠分为正常对照组(用针头插入左眼前房但不升高眼压)、I/R组(左眼I/R损伤处理)、I/R+对照shRNA组(左眼注射无序shRNA并建立I/R损伤模型)、I/R+circ_0051079 shRNA组(左眼注射circ_0051079 shR⁃NA并建立I/R损伤模型)。I/R损伤后7 d,取视网膜进行蛋白免疫荧光染色。收集2020年11月至2021年3月急性闭角型青光眼和白内障患者的房水样本各20例,进行RT⁃qPCR测定。结果缺氧或氧化应激条件下培养的RGC中circ_0051079表达量较正常条件下培养的RGC增加(均为P<0.05)。在缺氧或氧化应激条件下培养,si_circ_0051079组RGC细胞活力均较siRNA组增加,RGC凋亡细胞数均减少(均为P<0.05)。荧光素酶报告基因分析和RIP检测结果表明,circ_0051079可以通过调节miR⁃26a⁃3p/PTEN信号转导进而影响RGC功能。动物实验中,I/R损伤可致视网膜组织中circ_0051079表达量升高(P<0.05)。与正常对照组(1.00±0.07)相比,I/R+circ_0051079 shRNA组小鼠视网膜中circ_0051079表达水平(0.37±0.04)下降(P<0.05),同时I/R诱导的视网膜神经变性减轻。临床研究中,与白内障患者房水样本相比,青光眼患者房水样本中circ_0051079和PTEN mRNA表达增加,miR⁃26a⁃3p表达降低(均为P<0.001)。结论circ_0051079可能是缺血性视网膜病变诊断和治疗的潜在靶点。

Objective To investigate the effect and mechanism of circ_0051079 on retinal neurodegeneration induced by ischemia/reperfusion(I/R).Methods Retinal ganglion cells(RGCs)were isolated from the eyeballs of C57BL/6J suckling mice and divided into the control group,siRNA group(transfected with negative siRNA),si_circ_0051079 group(transfected with si_circ_0051079 interfering RNA),mimic control group(transfected with Scr mimic),miR⁃26a⁃5p group(transfected with miR⁃26a⁃5p mimic),miR⁃26a⁃5p+vector group(transfected with pcDNA 3.1),and miR⁃26a⁃5p+PTEN group(transfected with pcDNA 3.1⁃PTEN).RGCs were cultured under the normal,hypoxia(1%O_(2) exposure)and oxida⁃tive stress(50μmol·L^(-1) H_(2) O_(2) exposure)conditions for 24 h,respectively,and then detected by reverse transcription⁃quantitative polymerase chain reaction(RT⁃qPCR),Cell Counting Kit⁃8,terminal deoxynucleotidyl transferase⁃mediated dUTP nick end labeling and RNA immunoprecipitation(RIP).I/R injury model was established in the left eye of 15 C57BL/6 mice,and normal intraocular pressure was maintained in the contralateral eye as a control.Five mice were selected to collect retinas at 0 d,3 d and 7 d after I/R injury modeling to detect the expression of circ_0051079.Another 20 C57BL/6 mice were selected and divided into the control group(left eye was injected with a needle without increasing intraocular pressure),I/R group(I/R injury model was established),I/R+control shRNA group(left eye was injected with disorder shRNA and I/R injury model was established),I/R+circ_0051079 shRNA group(left eye was injected with circ_0051079 shRNA and I/R injury model was established).Retinal tissues were taken 7 d after I/R injury,and detected by protein im⁃munofluorescence staining.Aqueous humor(AH)samples from 20 patients with acute angle⁃closure glaucoma and 20 pa⁃tients with cataract in our hospital from November 2020 to March 2021 were collected and determined by RT⁃qPCR.Re⁃sults The expression level of circ_0051079 in RGCs cultured under hypoxia or oxidative stress was significantly higher than in normal conditions(both P<0.05).Under hypoxia or oxidative stress,the activity of RGCs in the si_circ_0051079 group increased compared with the siRNA group,and the number of apoptotic RGCs decreased(both P<0.05).Luciferase reporter assay and RIP showed that circ_0051079 could affect the functions of RGCs by regulating miR⁃26a⁃3p/PTEN signal transduction.The animal experiments showed that the expression level of circ_0051079 in retinal tissue significantly increased after I/R injury(P<0.05).Compared with the control group(1.00±0.07),the expression of circ_0051079 in retinal tissue significantly decreased in the I/R+circ_0051079 shRNA group(0.37±0.04)(P<0.05),and I/R⁃induced retinal neurodegen⁃eration was relieved.In clinical studies,compared with the AH samples of cataract patients,the expressions of circ_0051079 and PTEN significantly increased,and the expression of miR⁃26a⁃3p significantly decreased in the AH of glaucoma patients(all P<0.001).Conclusion The circ_0051079 may be a promising target for the diagnosis and treatment of ischemic retinopa⁃thy.