环状RNA0001287(circ_0001287)和miR-21在糖尿病视网膜病变发病过程中的作用机制  

Mechanism of cyclic RNA0001287 and miR-21 in the pathogenesis of diabetic retinopathy

作　　者：陈妍鹏[1] 顾婕 袁沣 温登慧 CHEN Yanpeng;GU Jie;YUAN Feng;WEN Denghui(Department of Ophthalmology,the First Affiliated Hospital of Hebei North University,Zhangjiakou 075000,Hebei Province,China)

机构地区：[1]河北北方学院附属第一医院眼科,河北省张家口市075000

出　　处：《眼科新进展》2023年第12期946-951,共6页Recent Advances in Ophthalmology

基　　金：河北省2021年度医学科学研究项目(编号:20211086)。

摘　　要：目的 探讨环状RNA0001287(circ_0001287)和miR-21在糖尿病视网膜病变(DR)发病过程中的作用机制。方法 分离原代人RPE(phRPE)细胞用于circRNA芯片分析。体外培养ARPE-19细胞,并分为空白组、高糖组、阴性组、si-circ组、circ_0001287组、circ_0001287+阴性组、circ_0001287+miR-21组。构建抗circ_0001287的小干扰RNA(siRNA)寡核苷酸和含有靶向miR-21序列的模拟物以及miR-21 mimics质粒。阴性组、si-circ组、circ_0001287组、circ_0001287+阴性组、circ_0001287+miR-21组分别采用Lipofectamine2000试剂盒将空质粒、circ_0001287 siRNA、circ_0001287模拟物、circ_0001287模拟物+miRNA无序序列、circ_0001287模拟物+miR-21 mimics质粒转染到ARPE-19细胞。转染6 h后,用新鲜的正常培养基更换Opti-MEM培养基。空白组和高糖组细胞不作转染处理。空白组细胞用含5.5 mmol·L^(-1)葡萄糖的培养液培养,高糖组细胞逐次用含15.5 mmol·L^(-1)、25.5 mmol·L^(-1)、35.5 mmol·L^(-1)葡萄糖的培养液培养。而其余各组细胞用35.5 mmol·L^(-1)葡萄糖处理48 h。采用RT-PCR检测circ_0001287和miR-21表达情况,CCK-8实验检测细胞增殖活性,双荧光素酶报告基因检测circ_0001287和miR-21的靶向关系,RNA免疫沉淀(RIP)法和生物素偶联探针Pull-down法验证circ_0001287和miR-21的靶向关系,Western blot法检测蛋白表达情况。结果 circRNA芯片筛选发现,hsa_circ_0001287在phRPE细胞中的表达下调。RT-PCR检测结果显示:与空白组相比,高糖组ARPE-19细胞中circ_0001287表达降低(P<0.05),并且具有剂量依赖性,而且随着葡萄糖浓度的增加,ARPE-19细胞中miR-21表达逐渐升高(P<0.05)。将siRNA与circ_0001287模拟物共转染,siRNA也能减少circ_0001287表达,表现为circ_0001287+阴性组circ_0001287相对表达量(0.70±0.03)较阴性组(0.98±0.04)显著降低(P<0.05)。对于转染circ_0001287-WT质粒的细胞,与对照模拟组(0.98±0.03)相比,miR-21模拟组荧光素酶相对酶活性降低(0.59±0.02,PObjective To investigate the action mechanism of cyclic RNA0001287(circ_0001287)and miR-21 in the pathogenesis of diabetic retinopathy(DR).Methods Primary human retinal pigment epithelium(phRPE)cells were isolated for circRNA microarray analysis.Arising retinal pigment epithelium(ARPE)-19 cells were cultured in vitro and divided into the blank group,high-glucose group,negative group,si-circ group,circ_0001287 group,circ_0001287+negative group,and circ_0001287+miR-21 group.Small interfering RNA(siRNA)oligonucleotides against circ_0001287,mimics containing miR-21 sequences and miR-21 mimic plasmids were constructed.In the negative group,si-circ group,circ_0001287 group,circ_0001287+negative group and circ_0001287+miR-21 group,the empty plasmid,circ_0001287 siRNA,circ_0001287 mimics,circ_0001287 mimics+miRNA disordered sequence,and circ_0001287 mimics+miR-21 mimic plasmid were transfected into ARPE-19 cells using Lipofectamine 2000 Transfection Reagent.After transfection for 6 h,the Opti-MEM medium was replaced with a fresh normal medium.Cells in the blank group and the high-glucose group were not transfected.Cells in the blank group were cultured with culture solution containing 5.5 mmol·L^(-1) glucose,and cells in the high-glucose group were cultured with culture solution containing 15.5 mmol·L^(-1),25.5 mmol·L^(-1) and 35.5 mmol·L^(-1) glucose,respectively.Cells in other groups were treated with 35.5 mmol·L^(-1) glucose for 48 h.The expressions of circ_0001287 and miR-21 were detected by reverse transcription polymerase chain reaction(RT-PCR),cell proliferation activity was detected by Cell Counting Kit-8,and the targeting relationship between circ_0001287 and miR-21 was detected by Dual Luciferase Reporter Assay.RNA immunoprecipitation(RIP)assay and biotin-coupled probe pull-down assay were used to verify the targeting relationship between circ_0001287 and miR-21.Western blot was used to detect protein expression.Results After screening by circRNA,the expression of hsa_circ_0001287 in phRPE cells was signif

关 键 词：环状RNA0001287 MIR-21 PTEN 糖尿病视网膜病变 视网膜色素上皮 

分 类 号：R774.1[医药卫生—眼科]