摘要
目的探讨LncRNA NEAT1调节miR-204-5p/10-11易位蛋白1(TET1)对高糖条件下晶状体上皮细胞(LECs)损伤的影响。方法通过RNA pull down、RNA免疫沉淀与双荧光素酶报告基因检测实验进行靶向验证。HLEB3细胞随机分为正常对照组、高糖组、si-NEAT1组、si-NC+NC-miR-204-5p组、si-NEAT1+miR-204-5p inhibitor组,除正常对照组外其余各组均以高糖诱导,慢病毒质粒分组转染后,RT-qPCR与Western blot检测各组HLEB3细胞LncRNA NEAT1、miR-204-5p、TET1表达;CCK-8实验、流式细胞实验分别检测细胞活力与凋亡;Western blot检测凋亡与上皮间质转化(EMT)相关蛋白表达;测定细胞抗氧化酶活性、活性氧(ROS)与炎症相关因子水平。SD大鼠随机分为对照组、糖尿病性白内障(DC)组、NEAT1敲低组、阴性对照组、NEAT1敲低+miR-204-5p抑制剂组,腹腔注射链脲佐菌素诱导DC大鼠模型,慢病毒质粒分组干预后检测大鼠晶状体组织LncRNA NEAT1、miR-204-5p、TET1表达,进行晶状体混浊评分,HE染色检测大鼠晶状体组织形态。结果LncRNA NEAT1可靶向调节miR-204-5p/TET1。与正常对照组相比,高糖组LncRNA NEAT1、TET1蛋白、TET1 mRNA及Caspase-3、Cleaved Caspase-3、Bax、N-cadherin、MMP9和MMP2蛋白相对表达,以及ROS、IL-1β和IL-6水平均升高,miR-204-5p相对表达、细胞活力、Bcl-2和E-cadherin蛋白相对表达,以及SOD、CAT和GSH-Px活性均降低(均为P<0.05);与高糖组相比,si-NEAT1组HLEB3细胞上述指标均被逆转(均为P<0.05);与si-NEAT1组相比,si-NEAT1+miR-204-5p inhibitor组HLEB3细胞TET1蛋白、TET1 mRNA及Caspase-3、Cleaved Caspase-3、Bax、N-cadherin、MMP9和MMP2蛋白相对表达,以及ROS、IL-1β与IL-6水平均升高,miR-204-5p相对表达、细胞活力、Bcl-2和E-cadherin蛋白相对表达,以及SOD、CAT和GSH-Px活性均降低(均为P<0.05)。与对照组相比,DC组大鼠LncRNA NEAT1、TET1蛋白、TET1 mRNA相对表达及晶状体混浊评分均升高,miR-204-5p相对表达降低(均为P<0.05);与DC组相比,NEAT1敲低组上述指标均被逆转(均为P<0.05);与NEAT1敲低组相比,NEAT1敲低+miR-204-5p抑制剂组大鼠TET1蛋白和mRNA相对表达、晶状体混浊评分均升高,miR-204-5p相对表达降低(均为P<0.05)。结论敲低LncRNA NEAT1可通过上调miR-204-5p降低TET1表达,从而减轻高糖条件下LECs损伤,并缓解DC大鼠晶状体混浊与组织损伤症状。
Objective To investigate the effects of LncRNA NEAT1-regulated miR-204-5p/ten-eleven translocation protein 1(TET1)on lens epithelial cell(LEC)injury under high-glucose conditions.Methods Target validation was performed through RNA pull-down,RNA immunoprecipitation,and dual-luciferase reporter assays.HLEB3 cells were randomly divided into normal control,high-glucose,si-NEAT1,si-NC+NC-miR-204-5p,and si-NEAT1+miR-204-5p inhibitor groups.Except for the normal control group,all other groups were induced with high glucose.After lentiviral plasmid transfection,RT-qPCR and Western blot were used to detect the expression of LncRNA NEAT1,miR-204-5p,and TET1 in each group of HLEB3 cells.Cell viability and apoptosis were detected by CCK-8 and flow cytometry assays,respectively.Western blot was used to detect the expression of apoptosis-and epithelial-mesenchymal transition(EMT)-related proteins.The activity of cellular antioxidant enzymes,reactive oxygen species(ROS),and levels of inflammation-related factors were measured.SD rats were randomly divided into control,diabetic cataract(DC),NEAT1 knockdown,negative control,and NEAT1 knockdown+miR-204-5p inhibitor groups.DC rat models were induced by intraperitoneal injection of streptozotocin.After lentiviral plasmid intervention,the expression of LncRNA NEAT1,miR-204-5p,and TET1 in rat lens tissues was detected.Lens opacity was scored,and the morphology of rat lens tissues was examined by HE staining.Results LncRNA NEAT1 can target the regulation of miR-204-5p/TET1.Compared with the normal control group,the relative expression of LncRNA NEAT1,TET1 protein,TET1 mRNA,and the proteins Caspase-3,Cleaved Caspase-3,Bax,N-cadherin,MMP9,and MMP2,as well as the levels of ROS,IL-1β,and IL-6,were increased in the high glucose group,while the relative expression of miR-204-5p,cell viability,and the relative expression of Bcl-2 and E-cadherin proteins,and the activities of SOD,CAT,and GSH-Px were decreased(all P<0.05).Compared with the high glucose group,the above mentioned indicators in HLEB3 cells of the si-NEAT1 group were reversed(all P<0.05).Compared with the si-NEAT1 group,the relative expression of TET1 protein,TET1 mRNA,and the proteins Caspase-3,Cleaved Caspase-3,Bax,N-cadherin,MMP9,and MMP2,as well as the levels of ROS,IL-1β,and IL-6,were increased in the si-NEAT1+miR-204-5p inhibitor group of HLEB3 cells,while the relative expression of miR-204-5p,cell viability,and the relative expression of Bcl-2 and E-cadherin proteins,and the activities of SOD,CAT,and GSH-Px were decreased(all P<0.05).Compared with the control group,the relative expression of LncRNA NEAT1 and TET1 protein and mRNA,and the lens opacity score were increased in the DC group of rats,while the relative expression of miR-204-5p was decreased(all P<0.05).Compared with the DC group,the above mentioned indicators were reversed in the NEAT1 knockdown group(all P<0.05).Compared with the NEAT1 knockdown group,the relative expression of TET1 protein and mRNA and the lens opacity score were increased in the NEAT1 knockdown+miR-204-5p inhibitor group of rats,while the relative expression of miR-204-5p was decreased(all P<0.05).Conclusion Knockdown of LncRNA NEAT1 can alleviate LEC injury under high-glucose conditions and mitigate lens opacity and tissue injury in DC rats by up-regulating miR-204-5p to reduce TET1 expression.
作者
卢毅
陈凡
叶慧玲
钱龙奇
LU Yi;CHEN Fan;YE Huiling;QIAN Longqi(Department of Ophthalmology,Anqing Municipal Hospital,Anqing 246000,Anhui Province,China)
出处
《眼科新进展》
北大核心
2025年第9期703-710,共8页
Recent Advances in Ophthalmology
基金
安庆市2024年度医疗卫生类自筹经费科技计划项目(编号2024Z3010)。
作者简介
卢毅(ORCID:0009-0000-8812-6078),男,1985年1月出生,安徽安庆人,硕士,副主任医师。研究方向:眼科相关。E-mail:rn0140@sina.com。
