摘要
目的探讨微小RNA-23b-3p(miR-23b-3p)对高糖诱导的人晶状体上皮细胞自噬和凋亡的调控作用。方法纳入2019年9月至2020年10月在西安医学院第一附属医院眼科收治的糖尿病性白内障(DC)患者30例为DC组,同期就诊的单纯白内障患者30例为单纯白内障组。2个组均行常规晶状体超声乳化及人工晶状体植入术,术中收集晶状体前囊膜组织。实时荧光定量PCR检测各组晶状体前囊膜组织中miR-23b-3p表达。体外培养人晶状体上皮细胞系HLEB3细胞,将其分为正常对照组、高糖组,分别于正常和高糖培养基中培养。根据生物信息学数据库预测原钙黏附蛋白17(PCDH17)与miR-23b-3p的靶向关系,双荧光素酶报告基因实验验证两者的靶向关系;取高糖培养的HLEB3细胞,分为miR-23b-3p拟似物组、阴性对照(NC)拟似物组、NC-siRNA组、PCDH17-siRNA组、miR-23b-3p拟似物+Vector组、miR-23b-3p拟似物+pcDNA-PCDH17组,分别转染相应试剂后实时荧光定量PCR法检测miR-23b-3p和PCDH17 mRNA表达;Western blot法检测哺乳动物ATG6同源蛋白(Beclin-1)、微管相关蛋白1轻链3(LC3B)、c-Jun氨基末端激酶(JNK)、磷酸化(p-)JNK、c-Jun、p-c-Jun、B淋巴细胞瘤基因-2相关X蛋白(Bax)、B淋巴细胞瘤基因-2蛋白(Bcl-2)蛋白的表达;流式细胞术检测细胞凋亡率。结果DC组晶状体前囊膜组织中miR-23b-3p相对表达量为0.35±0.15,明显低于单纯白内障组的1.00±0.09,差异有统计学意义(t=44.627,P<0.01);正常对照组、高糖组、高糖+NC拟似物组、高糖+miR-23b-3p拟似物组细胞中miR-23b-3p,LC3BⅡ/Ⅰ、Beclin-1、Bcl-2、Bax蛋白相对表达量以及细胞凋亡率总体比较,差异均有统计学意义(F=21.325、28.318、17.634、15.482、22.325、26.537,均P<0.01);其中与正常对照组比较,高糖组细胞凋亡率、LC3BⅡ/Ⅰ、Beclin-1、Bax蛋白表达量显著升高,Bcl-2蛋白表达量下降,与NC拟似物组比较,miR-23b-3p拟似物组细胞凋亡率、LC3BⅡ/Ⅰ、Beclin-1、Bax蛋白表达量显著下降,Bcl-2蛋白表达量显著升高,差异均有统计学意义(均P<0.05);生物信息学和双荧光素酶报告基因实验结果显示,PCDH 17是miR-23b-3p的靶基因,miR-23b-3p拟似物组中PCDH17 mRNA相对表达量较NC拟似物组显著降低(P<0.05)。与NC-siRNA组相比,PCDH17-siRNA组细胞凋亡率、LC3B-Ⅱ/Ⅰ、Beclin-1以及Bax蛋白表达量显著下降,Bcl-2蛋白表达量显著升高,差异均有统计学意义(t=9.116、12.413、5.349、3.273、8.419,均P<0.01)。NC拟似物组、miR-23b-3p拟似物组、miR-23b-3p拟似物+Vector组、miR-23b-3p拟似物+pcDNA-PCDH17组细胞中p-JNK/JNK、p-c-Jun/c-Jun、LC3BⅡ/Ⅰ、Beclin-1、Bcl-2、Bax蛋白相对表达量总体比较,差异均有统计学意义(F=24.724、19.319、23.418、17.562、20.263、15.249,均P<0.05);其中与miR-23b-3p拟似物+Vector组比较,miR-23b-3p拟似物+pcDNA-PCDH17组细胞中p-JNK/JNK、p-c-Jun/c-Jun、LC3BⅡ/Ⅰ、Beclin-1以及Bax蛋白表达量显著升高,Bcl-2蛋白表达量降低,差异均有统计学意义(均P<0.05)。结论miR-23b-3p对高糖环境下HLEB3细胞具有保护作用,其机制主要是通过靶向PCDH17调控JNK信号通路抑制高糖诱导HLEB3细胞的自噬和凋亡。
Objective To investigate the regulatory effects of microRNA-23b-3p(miR-23b-3p)on the autophagy and apoptosis of human lens epithelial cells induced by high glucose.Methods Thirty diabetic cataract(DC)patients as DC group and 30 patients with simple cataract as simple cataract group were enrolled in The First Affiliated Hospital of Xi'an Medical University from September 2019 to October 2020.Conventional phacoemulsification and intraocular lens transplantation were performed in both groups.The anterior capsular tissue was collected during the operation.The expression of miR-23b-3p in the anterior lens capsule was detected by real-time fluorescence quantitative PCR(RT-qPCR).Human lens epithelial cell line HLEB3 cells were cultured in vitro and divided into normal control group and high-glucose group,which were cultured in normal and high-glucose medium,respectively.The targeting relationship between proto-cadherin 17(PCDH17)and miR-23b-3p was predicted according to the bioinformatics database,and was verified by the dual-luciferase reporter gene experiment.High glucose-cultured HLEB3 cells were divided into miR-23b-3p mimics group,negative control(NC)mimics group,NC-siRNA group,PCDH17-siRNA group,miR-23b-3p mimics+Vector group,miR-23b-3p mimics+pcDNA-PCDH17 group,and were transfected with corresponding reagents according to grouping.The expression of miR-23b-3p and PCDH17 mRNA was detected by RT-qPCR.The expressions of a mammalian homolog of yeast Atg6/Vps30(Beclin-1),microtubule-associated protein 1 light chain 3(LC3B),c-Jun N-terminal kinases(JNK),phosphorylated(p-)JNK,c-Jun,p-c-Jun,B-cell lymphoma-2(Bcl-2)and Bcl-2 associated X protein(Bax)proteins were assayed by Western blot.The apoptosis rate was detected by flow cytometry.The study protocol was approved by the Ethics Committee of The First Affiliated Hospital of Xi'an Medical College(No.LSL2019037).Written informed consent was obtained from each patient.Results The relative expression of miR-23b-3p in the anterior lens capsule of DC group was 0.35±0.15,which was significantly lower than 1.00±0.09 of simple cataract group(t=44.627,P<0.01).There were significant differences in the relative expression levels of miR-23b-3p,LC3BⅡ/Ⅰ,Beclin-1,Bcl-2 and Bax proteins among normal control group,high glucose group,high glucose+NC mimics group and high glucose+miR-23b-3p mimics group(F=21.325,28.318,17.634,15.482,22.325,26.537;all at P<0.01).Compared with normal control group,the apoptosis rate,LC3BⅡ/Ⅰ,Beclin-1 and Bax protein expressions in high glucose group were significantly increased,and the Bcl-2 protein expression was significantly decreased(all at P<0.05).Compared with NC mimics group,the apoptosis rate,LC3BⅡ/Ⅰ,Beclin-1,and Bax protein expressions were significantly decreased and the Bcl-2 protein expression was significantly increased in miR-23b-3p mimics group(all at P<0.05).The results of bioinformatics and dual-luciferase reporter gene experiments showed that PCDH 17 was a target gene of miR-23b-3p,and the relative expression of PCDH17 mRNA in miR-23b-3p mimics group was significantly lower than that in NC mimics group(P<0.05).Compared with NC-siRNA group,the apoptosis rate,LC3BⅡ/Ⅰ,Beclin-1 and Bax protein expressions in PCDH17-siRNA group were significantly decreased,and the Bcl-2 protein expression was significantly increased(t=9.116,12.413,5.349,3.273,8.419;all at P<0.01).There were significant differences in the relative expression levels of p-JNK/JNK,p-c-Jun/c-Jun,LC3BⅡ/Ⅰ,Beclin-1,Bcl-2 and Bax proteins in NC mimics group,miR-23b-3p mimics group,miR-23b-3p mimics+Vector group and miR-23b-3p mimics+pcDNA-PCDH17 group(F=24.724,19.319,23.418,17.562,20.263,15.249;all at P<0.05).Compared with the miR-23b-3p mimics+Vector group,the expressions of p-JNK/JNK,p-c-Jun/c-Jun,LC3BⅡ/Ⅰ,Beclin-1 and Bax were significantly increased,and the expression of Bcl-2 protein was decreased in miR-23b-3p mimics+pcDNA-PCDH17 group(all at P<0.05).Conclusions MiR-23b-3p have a protective effect on HLEB3 cells in a high-glucose environment,mainly by targeting PCDH17 to regulate the JNK signaling pathway to inhibit high glucose-induced autophagy and apoptosis.
作者
刘文兰
王莉
杨扬
闫瑾
何媛
Liu Wenlan;Wang Li;Yang Yang;Yan Jin;He Yuan(School of Medical Technology,Xi'an Medical University,Xi'an 710021,China;Department of Ophthamology,The First Affiliated Hospital of Xi'an Medical University,Xi'an 710077,China)
出处
《中华实验眼科杂志》
CAS
CSCD
北大核心
2022年第9期804-812,共9页
Chinese Journal Of Experimental Ophthalmology
基金
陕西省教育厅2019年度科学研究计划项目(19JK0756)
陕西省自然科学基础研究计划项目一般项目(2021JM-500)。
