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-现代医学

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

单位：湖北文理学院附属医院/襄阳市中心医院儿科, 湖北襄阳 441021

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;R575

出版年·卷·期（页码）：2021·49·第九期（1012-1018）

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-128-3p+H/R group, anti-miR-NC+si-lncRNA FOXD3-AS1+H/R group, and anti-miR-128-3p+si-lncRNA FOXD3-AS1+H/R group. Real-time quantitative PCR (RT-qPCR) was used to detect the expression levels of lncRNA FOXD3-AS1 and miR-128-3p. 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 blotwas 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-128-3p 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-128-3p was predicted to contain the complementary binding sites of lncRNA FOXD3-AS1. And functional experiments revealed that miR-128-3p expression is highly expressed in H/R-treated FHC cells, and overexpression of miR-128-3p significantly increased cell viability, but reduced cell apoptosis and oxidative stress in H/R-treated FHC cells. Furthermore, downregulation of miR-128-3p significantly reversed the suppression effects of si-lncRNA FOXD3-AS1 on the oxidative stress damage in H/R-treated FHC cells. Conclusion: LncRNA FOXD3-AS1 could regulate the oxidative stress damage of H/R intestinal mucosal epithelial cells by up-regulating miR-128-3p.

参考文献：

[1] 姜晨, 杨洪涛, 耿芳, 等.扶肾颗粒改善肠上皮细胞屏障损伤的作用机制研究[J]. 中草药, 2020, 51(21):5496-5500.
[2] 雒红霞, 孟亚萍, 王浩江, 等.黄花蒿花粉破坏鼻黏膜上皮细胞紧密连接的实验研究[J]. 中华耳鼻咽喉头颈外科杂志, 2020, 55(5):465-471.
[3] 刘艳红, 陈文霞, 屈重行, 等.LncRNA SOX21-AS1靶向调控miR-202-5p影响结直肠癌细胞生物学行为的实验研究[J]. 胃肠病学, 2019, 24(12):721-728.
[4] 邴艳萍, 宋璇, 姜楠, 等.lncRNA NEAT1调控miR-206对缺氧复氧大鼠心肌细胞氧化应激损伤和凋亡的影响[J]. 中国动脉硬化杂志, 2020, 28(12):1034-1041.
[5] 李莉.LncRNA FTX调控miR-410-3p/Fmr1分子轴减弱缺氧复氧诱导的H9c2细胞损伤[D]. 郑州:郑州大学, 2020.
[6] 夏如冰, 王红英, 戴丹, 等.miR-128-3p靶向Lin28B增加肝癌细胞对奥沙利铂的敏感性[J]. 世界华人消化杂志, 2018, 26(30):1748-1757.
[7] 赵阳, 万银绪, 车吉忠, 等.miR-128-3p过表达靶向MAPK1抑制膀胱癌5637细胞株的侵袭, 迁移和上皮间质转化[J]. 中国临床解剖学杂志, 2019, 37(5):58-65.
[8] 赵建强, 邢金平, 来春林, 等.微RNA-98在姜黄素保护缺氧-复氧心肌细胞损伤中的作用及其机制[J]. 中华生物医学工程杂志, 2019, 25(2):136-141.
[9] 曹露.鱿鱼墨多糖对环磷酰胺所致小鼠肠道黏膜上皮细胞损伤的保护作用研究[D]. 青岛:中国海洋大学, 2013.
[10] 赵京飞.梗阻性黄疸患者肠黏膜上皮细胞增殖与凋亡的研究[D]. 烟台:滨州医学院, 2015.
[11] TONG G, WANG Y, XU C, et al. Long non-coding RNA FOXD3-AS1 aggravates ischemia/reperfusion injury of cardiomyocytes through promoting autophagy[J]. Am J Transl Res, 2019, 11(9):5634-5644.
[12] QU C, LIU X, GUO Y, et al. MiR-128-3p inhibits vascular smooth muscle cell proliferation and migration by repressing FOXO4/MMP9 signaling pathway[J]. Mol Med, 2020, 26(1):116.
[13] WANG D, CHEN F, FANG B, et al. MiR-128-3p alleviates spinal cord ischemia/reperfusion injury associated neuroinflammation and cellular apoptosis via SP1 suppression in rat[J]. Front Neurosci, 2020, 14:609613.
[14] LU Y, HAN Y, HE J, et al. LncRNA FOXD3-AS1 knockdown protects against cerebral ischemia/reperfusion injury via miR-765/BCL2L13 axis[J]. Biomed Pharmacother, 2020, 132:110778.
[15] ZHENG J Y, PENG B T, ZHANG Y W, et al. FOXD3-AS1 knockdown suppresses hypoxia-induced cardiomyocyte injury by increasing cell survival and inhibiting apoptosis via upregulating cardioprotective molecule miR-150-5p in vitro[J]. Front Pharmacol, 2020, 11:1284.
[16] ZHANG D, LEE H, HASPEL J A, et al. Long noncoding RNA FOXD3-AS1 regulates oxidative stress-induced apoptosis via sponging microRNA-150[J]. FASEB J, 2017, 31(10):4472-4481.

服务与反馈：

【文章下载】【发表评论】【查看评论】【加入收藏】

提示：您还未登录，请登录！点此登录