摘要
目的探究circZNF292在调节人晶状体上皮细胞(LECs)氧化应激损伤中的作用及可能的分子机制。方法收集单纯年龄相关性白内障(ARC)患眼的晶状体前囊膜组织(ARC组)和排除眼部疾病眼球捐献者的晶状体前囊膜组织(对照组)各3例,进行RNA提取及转录组学测序。应用qRT-PCR检测两组样本中circZNF292、miR-23b-3p和SIRT1 mRNA表达水平。利用双荧光素酶报告基因检测系统检测荧光素酶活性来验证circZNF292、miR-23b-3p、SIRT1之间的靶向关系。体外培养人永生化LECs细胞系HLE-B3,分为空白对照组、NC siRNA组、circZNF292 siRNA组、NC inhibitor组、miR-23b inhibitor组、NC mimics组、miR-23b mimics组、circZNF292 siRNA+miR-23b inhibitor组。使用Lipo2000试剂转染,48 h后收获细胞。分别用MTT法、流式细胞术和荧光探针示踪法检测细胞活力、凋亡率及丙二醛(MDA)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)、活性氧(ROS)含量。结果ARC组和对照组晶状体前囊膜组织样本之间鉴定得到469个下调circRNA和1231个上调circRNA,对前10位变化最明显的circRNA进行qRT-PCR验证,最终确定circZNF292在ARC组样本中下调最明显;且circZNF292与miR-23b-3p表达、miR-23b-3p与SIRT1 mRNA表达均呈负相关(r=-0.935,-0.951,均为P<0.05)。NC siRNA组和空白对照组细胞活力、细胞凋亡率及ROS、MDA、SOD、GSH-Px含量比较,差异均无统计学意义(均为P>0.05)。应用H_(2)O_(2)刺激后,与NC siRNA组相比,NC siRNA组+H_(2)O_(2) HLE-B3细胞凋亡率及ROS、MDA含量均明显升高(均为P<0.05),细胞活力及SOD、GSH-Px含量均明显降低(均为P<0.05);而circZNF292 siRNA组+H_(2)O_(2)细胞凋亡率及ROS、MDA含量则较NC siRNA组+H_(2)O_(2)均进一步升高(均为P<0.05),细胞活力及SOD、GSH-Px含量均进一步降低(均为P<0.05)。此外,circZNF292 siRNA+miR-23b inhibitor组上述指标较circZNF292 siRNA组则被逆转(均为P<0.05)。结论circZNF292可通过miR-23b-3p/SIRT1轴调节人LECs氧化应激损伤,从而有望为ARC提供新的治疗靶标。
Objective To investigate the role of circZNF292 in regulating oxidative stress-induced damage in human lens epithelial cells(LECs)and its possible molecular mechanism.Methods The anterior lens capsules from 3 simple age-related cataract patients(ARC group)and 3 volunteers without any eye disease(control group)were collected for RNA extraction and transcriptome sequencing.The mRNA levels of circZNF292,miR-23b-3p and sirtuin 1(SIRT1)were evaluated with the quantitative real-time polymerase chain reaction(qRT-PCR).The luciferase activity was detected with the dual-luciferase reporter assay to verify the targeting relationship among circZNF292,miR-23b-3p,and SIRT1.The immortalized HLE-B3 cells cultured in vitro were divided into the blank control group,NC siRNA group,circZNF292 siRNA group,NC inhibitor group,miR-23b inhibitor group,NC mimics group,miR-23b mimics group,and circZNF292 siRNA+miR-23b inhibitor group.The cells were transfected with Lipo2000 reagent and harvested 48 hours later.Cell viability and apoptosis,as well as the levels of malondialdehyde(MDA),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),and reactive oxygen species(ROS)were measured by MTT assay,flow cytometry,and fluorescence probe.Results There were 469 down-regulated circRNAs and 1231 up-regulated circRNAs in the ARC and control groups.Among the first 10 circRNAs with the most obvious changes,qRT-PCR showed that circZNF292 in the ARC group was down-regulated most significantly.The expression levels of circZNF292 and miR-23b-3p,miR-23b-3p and SIRT1 mRNA were negatively correlated,respectively(r=-0.935,-0.951,both P<0.05).There were no significant differences in cell viability,apoptosis,ROS,MDA,SOD,and GSH-Px levels between the NC siRNA group and the blank control group(all P>0.05).But after H_(2)O_(2) treatment,the cell apoptosis,ROS and MDA levels were significantly increased,while the cell viability,SOD and GSH-Px levels were significantly decreased in the NC siRNA group(all P<0.05).Besides,the cell apoptosis,ROS and MDA levels in the circZNF292 siRNA group were higher than those in the NC siRNA group(all P<0.05),while the cell viability,SOD and GSH-Px levels were lower than those in the NC siRNA group(all P<0.05).The above indexes were reversed in the circZNF292 siRNA+miR-23b inhibitor group compared with the circZNF292 siRNA group(all P<0.05).Conclusion circZNF292 can regulate oxidative stress-induced damage in human LECs through the miR-23b-3p/SIRT1 axis,which provides a new therapeutic target for ARC.
作者
邢媛
鲍宁
XING Yuan;BAO Ning(Department of Ophthalmology,the Second Affiliated Hospital of Anhui Medical University,Hefei 230601,Anhui Province,China)
出处
《眼科新进展》
CAS
北大核心
2022年第7期514-519,共6页
Recent Advances in Ophthalmology
基金
2020年安徽省重点研究与开发计划项目(编号:202004j07020029)。
作者简介
邢媛(ORCID:0000-0002-6066-9211),女,1985年8月出生,上海人。研究方向:眼底病。E-mail:renzin_2000@163.com;通信作者:鲍宁(ORCID:0000-0002-8055-2627),男,1978年10月出生,安徽合肥人,博士,副主任医师。研究方向:眼底病、玻璃体视网膜疾病。E-mail:baoningleon@163.com。
