LncRNA PVT1/miR-124轴靶向Jagged-1/Notch通路抑制晶状体纤维化的分子机制研究  

Molecular mechanism of LncRNA PVT1/miR-124 axis inhibiting lens fibrosis by targeting Jagged-1/Notch pathway

作　　者：汪涛[1] 张振华 陈文兵 刘建军 张彩霞 吕嘉华 WANG Tao;ZHANG Zhen-hua;CHEN Wen-bing;LIU Jian-jun;ZHANG Cai-xia;LYU Jia-hua(Department of Ophthalmology,Suzhou Science and Technology City Hospital Affiliated to Nanjing Medical University,Suzhou Jiangsu 215153,China;Department of Ophthalmology,Suzhou Ophthalmology and Otolaryngology Hospital,Suzhou Jiangsu 215006,China;Department of Ophthalmology,First Affiliated Hospital of Suzhou University,Suzhou Jiangsu 215006,China;Department of Ophthalmology,Eye&Ent Hospital of Fudan University,Shanghai 200031,China)

机构地区：[1]南京医科大学附属苏州科技城医院眼科,江苏苏州215153 [2]苏州眼耳鼻喉科医院眼科,江苏苏州215006 [3]苏州大学附属第一医院眼科,江苏苏州215006 [4]上海复旦大学附属眼耳鼻喉科医院眼科,上海200031

出　　处：《局解手术学杂志》2022年第9期774-781,共8页Journal of Regional Anatomy and Operative Surgery

基　　金：江苏省卫生计生委年度面上科研课题项目(H201518)。

摘　　要：目的 探究环状LncRNA PVT1调节人晶状体上皮细胞(LECs)纤维化的作用及可能的分子机制。方法 收集新鲜晶状体前囊膜标本,包括前囊下白内障(ASC)手术眼和健康死后眼各9例。体外培养SRA01/04细胞,并分为空白组、TGFβ2组、si-control组、si-PVT1组、si-PVT1+miR-NC组、si-PVT1+miR-124-inhibitor组。除空白组外,其余各组在转染相应序列后,用5 ng·m LTGFβ2作用48 h。提取组织或细胞RNA并进行微阵列分析。应用实时荧光定量PCR法检测组织样本或细胞中LncRNA PVT1、miR-124表达。采用双荧光素酶报告系统和RNA结合蛋白免疫共沉淀(RIP)法分析LncRNA PVT1、miR-124、Jagged-1之间的靶向关系。Western blot法检测各组细胞Jagged-1、Notch-1、Notch-2、Notch-3、Snail、Slug、纤维蛋白(FN)、Ⅳ型胶原蛋白(ColⅣ)、肌动蛋白α (α-SMA)等蛋白表达。结果 LncRNA PVT1在ASC眼的前囊膜组织或TGFβ组SRA01/04细胞中表达上调,同时miR-124表达水平降低(P<0.05),且LncRNA PVT1与miR-124表达水平均呈负相关性(P<0.05)。与空白组相比,TGFβ2组和si-control组FN、α-SMA、ColⅣ、Snail、Slug蛋白表达上调;与TGFβ2组和si-control组比较,si-PVT1组和si-PVT1+miR-NC组上述蛋白表达下调,但si-PVT1+miR-124-inhibitor组可逆转si-PVT1对上述蛋白表达的下调作用(P<0.05)。双荧光素酶报告系统和RIP法分析结果显示,LncRNA PVT1通过“海绵吸附”miR-124,负性调节miR-124的表达。与空白组相比,TGFβ2组Jagged-1、Notch-1、Notch-2、Notch-3蛋白表达显著上调;与TGFβ2组和si-control组比较,si-PVT1组Jagged-1、Notch-1、Notch-2、Notch-3蛋白表达下调,同时si-PVT1+miR-124-inhibitor组可逆转si-PVT1对上述蛋白表达的下调作用(P<0.05)。结论 TGFβ2通过一种LncRNA PVT1依赖机制诱导LECs发生上皮—间充质转化,其机制可能是LncRNA PVT1通过“海绵吸附”miR-124负性调节其表达,进而诱导下游Jagged-1/Notch信号通路激活。Objective To investigate the role and possible molecular mechanism of circ LncRNA PVT1 in regulating human lens epithelial cells (LECs) fibrosis.Methods The fresh lens anterior capsule specimens were collected,including eyes from 9 patients underwent anterior subcapsular cataract surgery and 9 healthy post-mortem eyes.SRA01/04 cells were cultured in vitro and divided into the blank group,the TGFβ2 group,the si-control group,the si-PVT1 group,the si-PVT1+miR-NC group,and the si-PVT1+miR-124-inhibitor group.Except the blank group,the other groups were treated with 5 ng·m LTGFβ2 for 48 hours after transfection with the corresponding sequences.RNA was extracted from tissues or cells and analyzed by microarray analysis.The real-time fluorescence quantitative RT-PCR was used to detect the expressions of LncRNA PVT1 and miR-124 in tissues or cells,the targeting relationships between LncRNA PVT1,miR-124and Jagged-1 were analyzed by double-luciferase reporter system and RNA binding protein immunoprecipitation (RIP) method,the expressions of Jagged-1,Notch-1,Notch-2,Notch-3,Snail,Slug,fibronectin (FN),collagen typeⅣ(ColⅣ),and alpha-smooth muscle actin(α-SMA) of cells in each groups were detected by Western blot.Results LncRNA PVT1 was up-regulated in the anterior capsule of ASC eyes or in the SRA01/04 cells of TGFβ2 group,and the expression level of miR-124 decreased (P<0.05),and the expression levels of LncRNA PVT1 and miR-124 were negatively correlated (P<0.05).The expression of FN,α-SMA,ColⅣ,Snail and Slug proteins were upregulated in the TGFβ2 group and the si-control group compared with those in the blank group,but the expressions of these proteins in the si-PVT1 group and the si-PVT1+miR-NC group were lower than those in the TGFβ2 group and the si-control group,however,the si-PVT1+miR-124-inhibitor group could reverse the down-regulation effect of si-PVT1 on the expressions of the above proteins (P<0.05).The double-luciferase reporter system and RIP method showed that LncRNA PVT1 negatively regulated mi

关 键 词：LncRNA PVT1 miR-124 Jagged-1/Notch通路 人晶状体上皮细胞 晶状体纤维化 

分 类 号：R776[医药卫生—眼科]