摘要
目的探讨高糖环境下lncRNA MEG3对人视网膜血管内皮细胞(hRECs)生长的作用及其机制。方法采用qRT-PCR检测高糖和正常环境下hRECs中lncRNA MEG3的表达;构建lncRNA MEG3过表达质粒及沉默质粒,利用CCK-8法、Transwell实验以及划痕实验检测lncRNA MEG3对hRECs增殖、愈合及迁移能力的影响。利用双荧光素酶报告基因实验验证lncRNA MEG3和miR-223-3p的靶向结合,并外加miR-223-3p观察对lncRNA MEG3过表达的作用。Western blot检测lncRNA MEG3对hRECs中FBXW7蛋白表达的影响。过表达或沉默PABPC1后,利用qRT-PCR检测hRECs中lncRNA MEG3表达水平。结果与正常培养环境相比,高糖环境下hRECs中lncRNA MEG3表达下降(F=73.613,P=0.001)。CCK-8法检测发现,lncRNA MEG3过表达可显著降低hRECs增殖,而miR-223-3p的添加可逆转这一现象;划痕实验结果显示,lncRNA MEG3过表达(高糖)组hRECs愈合速度显著低于过表达对照(高糖)组(F=121.193,P<0.001);lncRNA MEG3沉默(高糖)组hRECs愈合速度略高于lncRNA MEG3沉默对照(高糖)组,但两者差异无统计学意义(F=0.689,P=0.453);miR-223-3p可显著降低lncRNA MEG3过表达对hRECs愈合能力的抑制(F=110.016,P<0.001)。Transwell实验结果显示,lncRNA MEG3过表达(高糖)组hRECs迁移能力显著低于过表达对照(高糖)组(F=22.407,P=0.009);lncRNA MEG3沉默(高糖)组hRECs迁移能力显著高于lncRNA MEG3沉默对照(高糖)组(F=17.093,P=0.014);miR-223-3p可显著降低lncRNA MEG3过表达对hRECs迁移能力的抑制(F=33.338,P=0.004)。在双荧光素酶报告基因实验中,共转染miR-223-3p后,突变型lncRNA MEG3(位点1和位点2)的荧光素酶活性均显著下降(野生型F=66.691,P=0.001;突变位点1F=58.657,P=0.002;突变位点2F=29.104,P=0.006)。Western blot检测结果显示,过表达lncRNA MEG3后,hRECs中FBXW7蛋白的相对表达量明显升高(F=185.443,P<0.001),而miR-223-3p的添加逆转了这一趋势(F=162.257,P<0.001)。沉默了PABPC1后,hRECs中lncRNA MEG3相对表达水平显著下降(F=145.293,P<0.001),而PABPC1基因的过表达则导致hRECs中lncRNA MEG3相对表达水平上调(F=70.638,P=0.001)。结论在高糖环境下,lncRNA MEG3通过与miR-223-3p的直接相互作用调控下游的相关靶点,抑制hRECs增殖与迁移;而其上游的调控元件可能为PABPC1。
Objective To explore the mechanism of lncRNA MEG3 on the growth of human retinal endothelial cells(hRECs)under high glucose.Methods The level of lncRNA MEG3 was detected using qRT-PCR in high glucose group and normal group.Overexpression and silent plasmids of lncRNA MEG3 were conducted.The proliferation,migration,and wound healing of hRECs in different groups were measured by cell counting Kit-8(CCK-8)assay,Transwell assay and scarification test.Luciferase reporter genes were used to detect the target interaction between lncRNA MEG3 and miR-223-3p,and the effect of additional miR-223-3p on the overexpression of lncRNA MEG3 was also investigated.Western blot analysis was used to reveal the effect of lncRNA MEG3 on the protein expression level of FBXW7.The level of lncRNA MEG3 was detected by qRT-PCR after polyadenylate-binding protein cytoplasmic 1(PABPC1)was overexpressed or silenced.Results Compared with cells cultured in normal environment,the expression level of lncRNA MEG3 in hRECs was decreased in high glucose environment(F=73.613,P=0.001).In CCK-8 assay,overexpression of lncRNA MEG3 significantly repressed the proliferation of hRECs in high glucose environment,while co-transfected miR-223-3p rescued this phenomenon.In wound healing assay,the wound healing process of hRECs in lncRNA MEG3 overexpressed group(high glucose)was significantly lower than that in overexpression control group(high glucose)(F=121.193,P<0.001),while that of the lncRNA-MEG3 silencing group(high glucose)was slight higher than silencing control group(high glucose)(F=0.689,P=0.453);miR-223-3p could significantly reduce the inhibition of the wound healing of hRECs induced by overexpression of lncRNA MEG3(F=110.016,P<0.001).In transwell assays,the migration of hRECs of lncRNA MEG3 overexpression group(high glucose)was significantly lower than overexpression control group(high glucose)(F=22.407,P=0.009);the migration of hRECs of lncRNA MEG3 silencing group(high glucose)was significantly higher than silencing control group(high glucose)(F=17.093,P=0.014);miR-223-3p could significantly reduce the inhibition of the migration of hRECs induced by the overexpression of lncRNA MEG3(F=33.338,P=0.004).Inluciferase reporter gene assays,after co-transfected with miR-223-3p,the luciferase activities of wild type and single-site(site 1 and site 2)mutated lncRNA MEG3 were significantly decreased(wild typeF=66.691,P=0.001;site 1F=58.657,P=0.002;site 2F=29.104,P=0.006).In Western blot assay,overexpressed lncRNA MEG3 promoted the protein level of FBXW7(F=185.443,P<0.001),while co-transfected mir-223-3p reversed it(F=162.257,P<0.001).The knocked-down of PABPC1 reduced the relative expression level of lncRNA MEG3(F=145.293,P<0.001),while the overexpression of PABPC1 promoted the relative expression level of lncRNA MEG3(F=70.638,P=0.001).Conclusion In hyperglycemia,lncRNA MEG3 can regulate the downstream targets by direct interaction with mir-223-3p,thus repressing the proliferation and migration of hRECs;and the upstream fact of lncRNA MEG3 may be PABPC1.
作者
邵珺
王杨宁致
詹鹏飞
SHAO Jun;WANGYANG Ningzhi;ZHAN Pengfei(Department of Ophthalmology,Wuxi People’s Hospital Affiliated to Nanjing Medical University,Wuxi 214023,Jiangsu Province,China)
出处
《眼科新进展》
CAS
北大核心
2021年第7期621-627,共7页
Recent Advances in Ophthalmology
基金
国家自然科学基金项目(编号81970819)
无锡市双百拔尖人才(编号2020012)
中国博士后67批面上项目(编号2020M671541)。
