lncRNA FOXD3-AS1靶向miR-128-3p调控缺氧复氧肠黏膜上皮细胞氧化应激损伤的分子机制研究  

lncRNA FOXD3-AS1 targeting miR-128-3p to regulate oxidative stress injury of hypoxia-reoxygenation intestinal mucosal epithelial cells

作　　者：许诣 陈明 秦建品 沈文婷 XU Yi;CHEN Ming;QIN Jianpin;SHEN Wenting(Department of Pediatrics,Affiliated Hospital of Hubei University of Arts and Science,Xiangyang Central Hospital,Xiangyang 441021,China)

机构地区：[1]湖北文理学院附属医院/襄阳市中心医院儿科,湖北襄阳441021

出　　处：《现代医学》2021年第9期1012-1018,共7页Modern Medical Journal

基　　金：湖北省自然科学基金资助项目(WJ2017Q039)。

摘　　要：目的:探讨lncRNA FOXD3-AS1靶向miR-128-3p调控缺氧复氧肠黏膜上皮细胞氧化应激损伤的分子机制研究。方法:将人正常结直肠黏膜上皮细胞系FHC分为NC组、缺氧/复氧(H/R)组、si-NC+H/R组、si-lncRNA FOXD3-AS1+H/R组、pcDNA组、pcDNA-lncRNA FOXD3-AS1组、miR-NC+H/R组、miR-128-3p+H/R组、anti-miR-NC+si-lncRNA FOXD3-AS1+H/R组、anti-miR-128-3p+si-lncRNA FOXD3-AS1+H/R组。实时荧光定量聚合酶链反应(RT-qPCR)检测细胞lncRNA FOXD3-AS1和miR-128-3p表达水平。细胞计数试剂盒(CCK-8)检测细胞活力。流式细胞术检测细胞凋亡。试剂盒分别检测细胞中活性氧(ROS)活性、丙二醛(MDA)含量和3-硝基酪氨酸(3-NT)水平。蛋白质印迹法(Western blot)检测细胞半胱氨酸天冬氨酸蛋白酶-3(Cleaved caspase-3)、Bax蛋白表达。双荧光素酶报告实验检测lncRNA FOXD3-AS1和miR-128-3p靶向关系。结果:与NC组比较,H/R组FHC细胞中lncRNA FOXD3-AS1表达水平显著提高,miR-128-3p表达水平显著降低(P<0.05),而且H/R处理使细胞活力显著降低,细胞凋亡率显著升高,氧化应激水平显著升高(P<0.05);与si-NC+H/R组比较,si-lncRNA FOXD3-AS1+H/R组FHC细胞中lncRNA FOXD3-AS1表达水平显著降低,细胞活力显著升高,细胞凋亡率显著降低,氧化应激水平显著降低(P<0.05)。与miR-NC+H/R组比较,miR-128-3p+H/R组FHC细胞中miR-128-3p表达水平显著降低,细胞活力显著升高,细胞凋亡率显著降低,氧化应激水平显著降低(P<0.05)。与anti-miR-NC+si-lncRNA FOXD3-AS1+H/R组比较,anti-miR-128-3p+si-lncRNA FOXD3-AS1+H/R组FHC细胞中miR-128-3p表达水平显著降低,细胞活力显著降低,细胞凋亡率显著升高,氧化应激水平显著升高(P<0.05)。结论:抑制lncRNA FOXD3-AS1表达水平可通过上调miR-128-3p抑制H/R肠黏膜上皮细胞氧化应激损伤。Objective:To investigate the function and regulatory mechanism of long non-coding RNA(lncRNA)forkhead box D3 antisense RNA 1(FOXD3-AS1)in the oxidative stress damage of hypoxia-reoxygenation intestinal mucosal epithelial cells.Methods:Human normal colorectal mucosal epithelial cells FHC were divided into NC group,H/R group,small interfere RNA(si)-NC+H/R group,si-lncRNA FOXD3-AS1+H/R group,pcDNA group,pcDNA-lncRNA FOXD3-AS1 group,miR-NC+H/R group,miR-128-3 p+H/R group,anti-miR-NC+silncRNA FOXD3-AS1+H/R group,and anti-miR-128-3 p+si-lncRNA FOXD3-AS1+H/R group.Real-time quantitative PCR(RT-q PCR)was used to detect the expression levels of lncRNA FOXD3-AS1 and miR-128-3 p.Cell viability and apoptosis were detected by cell counting kit(CCK-8)assay and flow cytometry assay,respectively.The activity of reactive oxygen species(ROS),the content of malondialdehyde(MDA)and the level of 3-nitrotyrosine(3-NT)in cells were detected by corresponding kit.Western blot was conducted to detect the protein levels of Cleaved caspase-3 and Bax.Results:Compared with the NC group,the expression level of lncRNA FOXD3-AS1 was significantly increased while miR-128-3 p was significantly decreased in H/R group.Besides,H/R treatment significantly reduced cell viability,but promoted cell apoptosis and increased oxidative stress.In contrast with the si-NC+H/R group,lncRNA FOXD3-AS1 knockdown significantly decreased the expression level of lncRNA FOXD3-AS1,elevated cell viability,inhibited cell apoptosis and reduced oxidative stress in H/R-treated FHC cells.miR-128-3 p was predicted to contain the complementary binding sites of lncRNA FOXD3-AS1.And functional experiments revealed that miR-128-3 p expression is highly expressed in H/R-treated FHC cells,and overexpression of miR-128-3 p significantly increased cell viability,but reduced cell apoptosis and oxidative stress in H/R-treated FHC cells.Furthermore,downregulation of miR-128-3 p significantly reversed the suppression effects of si-lncRNA FOXD3-AS1 on the oxidative stress damage in H/R-tre

关 键 词：lncRNA SOX21-AS1 miR-590-3p 支气管上皮细胞 氧化应激 损伤 

分 类 号：R574.62[医药卫生—消化系统] R575[医药卫生—内科学]