Non-coding RNAs (ncRNAs) have shown to act as crucial mediators in atherosclerosis (AS) development. The purpose of our study was to explore the role of Astragaloside IV (ASV) and circular RNA_0000231 (circ_0000231) in AS using AS cell model. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry were used to analyze cell viability and apoptosis. Migration ability was assessed by transwell migration assay and wound healing assay. The inflammatory response was evaluated via enzyme-linked immunosorbent assay (ELISA). Oxidative status was assessed via matching commercial kits. Western blot assay was conducted to detect the expression of monocyte chemoattractant protein 1 (MCP1), intercellular adhesion molecule 1 (ICAM1), and chloride intracellular channel 4 (CLIC4). The levels of circ_0000231, its linear form Rho GTPase activating protein 12 (ARHGAP12), microRNA-135a-5p (miR-135a-5p), and CLIC4 messenger RNA (mRNA) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Circ_0000231–miRNA interactions were established using Starbase and Circbank softwares, while the targets of miR-135a-5p were explored by Starbase software. Dual-luciferase reporter assay and RNA-pull down assay were used to verify these target interactions. ASV suppressed the apoptosis, inflammation, and oxidative stress while recovered the viability and migration ability of HUVECs which were mediated by oxidized low-density lipoprotein (ox-LDL). Circ_0000231 overexpression antagonized the protective role of ASV in ox-LDL-induced HUVECs. MiR-135a-5p was verified as a direct target of circ_0000231, and circ_0000231 contributed to ox-LDL-induced cell injury of HUVECs through down-regulating miR-135a-5p. MiR-135a-5p directly interacted with the 3′ untranslated region (3′-UTR) of CLIC4 mRNA in HUVECs, and miR-135a-5p protected HUVECs against ox-LDL-induced injury through down-regulating CLIC4. ASV protected HUVECs against ox-LDL-induced injury through targeting circ_0000231/miR-135a-5p/CLIC4 axis. Targeting circ_0000231/miR-135a-5p/CLIC4 axis might provide a novel insight to develop effective strategy for AS treatment.
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Enzyme-linked immunosorbent assay
Monocyte chemoattractant protein 1
Intercellular adhesion molecule 1
Chloride intracellular channel 4
Oxidized low-density lipoprotein
Reactive oxygen species
Libby P, Ridker PM, Maseri A (2002) Inflammation and atherosclerosis. Circulation 105:1135–1143. https://doi.org/10.1161/hc0902.104353
Lusis AJ (2000) Atherosclerosis. Nature 407:233–241. https://doi.org/10.1038/35025203
Leppänen P, Koota S, Kholová I, Koponen J, Fieber C, Eriksson U, Alitalo K, Ylä-Herttuala S (2005) Gene transfers of vascular endothelial growth factor-A, vascular endothelial growth factor-B, vascular endothelial growth factor-C, and vascular endothelial growth factor-D have no effects on atherosclerosis in hypercholesterolemic low-density lipoprotein-receptor/apolipoprotein B48-deficient mice. Circulation 112:1347–1352. https://doi.org/10.1161/circulationaha.105.534107
Sun C, Wu MH, Yuan SY (2011) Nonmuscle myosin light-chain kinase deficiency attenuates atherosclerosis in apolipoprotein E-deficient mice via reduced endothelial barrier dysfunction and monocyte migration. Circulation 124:48–57. https://doi.org/10.1161/circulationaha.110.988915
Peng N, Meng N, Wang S, Zhao F, Zhao J, Su L, Zhang S, Zhang Y, Zhao B, Miao J (2014) An activator of mTOR inhibits oxLDL-induced autophagy and apoptosis in vascular endothelial cells and restricts atherosclerosis in apolipoprotein E−/− mice. Sci Rep 4:5519. https://doi.org/10.1038/srep05519
Steinberg D, Witztum JL (2010) Oxidized low-density lipoprotein and atherosclerosis. Arterioscler Thromb Vasc Biol 30:2311–2316. https://doi.org/10.1161/atvbaha.108.179697
Qi JC, Liu PG, Wang C, Zheng AD, Wan Z (2017) Tacrolimus protects vascular endothelial cells from injuries caused by Ox-LDL by regulating endoplasmic reticulum stress. Eur Rev Med Pharmacol Sci 21:3966–3973
Liu J, Yao S, Wang S, Jiao P, Song G, Yu Y, Zhu P, Qin S (2014) D-4F, an apolipoprotein A-I mimetic peptide, protects human umbilical vein endothelial cells from oxidized low-density lipoprotein-induced injury by preventing the downregulation of pigment epithelium-derived factor expression. J Cardiovasc Pharmacol 63:553–561. https://doi.org/10.1097/FJC.0000000000000080
Qian W, Cai X, Qian Q, Zhuang Q, Yang W, Zhang X, Zhao L (2019) Astragaloside IV protects endothelial progenitor cells from the damage of ox-LDL via the LOX-1/NLRP3 inflammasome pathway. Drug Des Devel Ther 13:2579–2589. https://doi.org/10.2147/dddt.s207774
Altesha MA, Ni T, Khan A, Liu K, Zheng X (2019) Circular RNA in cardiovascular disease. J Cell Physiol 234:5588–5600. https://doi.org/10.1002/jcp.27384
Zhuang JB, Li T, Hu XM, Ning M, Gao WQ, Lang YH, Zheng WF, Wei J (2020) Circ_CHFR expedites cell growth, migration and inflammation in ox-LDL-treated human vascular smooth muscle cells via the miR-214-3p/Wnt3/β-catenin pathway. Eur Rev Med Pharmacol Sci 24:3282–3292. https://doi.org/10.26355/eurrev_202003_20696
Li CY, Ma L, Yu B (2017) Circular RNA hsa_circ_0003575 regulates oxLDL induced vascular endothelial cells proliferation and angiogenesis. Biomed Pharmacother 95:1514–1519. https://doi.org/10.1016/j.biopha.2017.09.064
Pan J, Xu X, Wang G (2020) lncRNA ZFAS1 Is Involved in the Proliferation, Invasion and Metastasis of Prostate Cancer Cells Through Competitively Binding to miR-135a-5p. Cancer Manag Res 12:1135–1149. https://doi.org/10.2147/cmar.s237439
Guo LM, Ding GF, Xu W, Ge H, Jiang Y, Chen XJ, Lu Y (2018) MiR-135a-5p represses proliferation of HNSCC by targeting HOXA10. Cancer Biol Ther 19:973–983. https://doi.org/10.1080/15384047.2018.1450112
Shan H, Guo D, Zhang S, Qi H, Liu S, Du Y, He Y, Wang B, Xu M, Yu X (2020) SNHG6 modulates oxidized low-density lipoprotein-induced endothelial cells injury through miR-135a-5p/ROCK in atherosclerosis. Cell Biosci 10:4. https://doi.org/10.1186/s13578-019-0371-2
Malekova L, Tomaskova J, Novakova M, Stefanik P, Kopacek J, Lakatos B, Pastorekova S, Krizanova O, Breier A, Ondrias K (2007) Inhibitory effect of DIDS, NPPB, and phloretin on intracellular chloride channels. Pflugers Arch 455:349–357. https://doi.org/10.1007/s00424-007-0300-9
Guo D, Xie W, Xiong P, Li H, Wang S, Chen G, Gao Y, Zhou J, Zhang Y, Bu G, Xue M, Zhang J (2018) Cyclin-dependent kinase 5-mediated phosphorylation of chloride intracellular channel 4 promotes oxidative stress-induced neuronal death. Cell Death Dis 9:951. https://doi.org/10.1038/s41419-018-0983-1
He G, Ma Y, Chou SY, Li H, Yang C, Chuang JZ, Sung CH, Ding A (2011) Role of CLIC4 in the host innate responses to bacterial lipopolysaccharide. Eur J Immunol 41:1221–1230. https://doi.org/10.1002/eji.201041266
Fernández-Salas E, Sagar M, Cheng C, Yuspa SH, Weinberg WC (1999) p53 and tumor necrosis factor alpha regulate the expression of a mitochondrial chloride channel protein. J Biol Chem 274:36488–36497. https://doi.org/10.1074/jbc.274.51.36488
Zhang X, Wang Z, Li W, Huang R, Zheng D, Bi G (2020) MicroRNA-217-5p ameliorates endothelial cell apoptosis induced by ox-LDL by targeting CLIC4. Nutr Metab Cardiovasc Dis 30:523–533. https://doi.org/10.1016/j.numecd.2019.09.027
You L, Fang Z, Shen G, Wang Q, He Y, Ye S, Wang L, Hu M, Lin Y, Liu M, Jiang A (2019) Astragaloside IV prevents high glucose-induced cell apoptosis and inflammatory reactions through inhibition of the JNK pathway in human umbilical vein endothelial cells. Mol Med Rep 19:1603–1612. https://doi.org/10.3892/mmr.2019.9812
Ma Y, Zhao Y, Zhang R, Liang X, Yin Z, Geng Y, Shu G, Song X, Zou Y, Li L, Yin L, Yue G, Li Y, Ye G, He C (2017) Astragaloside IV inhibits PMA-induced EPCR shedding through MAPKs and PKC pathway. Immunopharmacol Immunotoxicol 39:148–156. https://doi.org/10.1080/08923973.2017.1306868
Xu C, Tang F, Lu M, Yang J, Han R, Mei M, Hu J, Wang H (2016) Pretreatment with Astragaloside IV protects human umbilical vein endothelial cells from hydrogen peroxide induced oxidative stress and cell dysfunction via inhibiting eNOS uncoupling and NADPH oxidase - ROS - NF-κB pathway. Can J Physiol Pharmacol 94:1132–1140. https://doi.org/10.1139/cjpp-2015-0572
Pei X, Ye S, Jin G, Yu Y (2018) Overexpression of circRNA-001175 promotes proliferation and angiogenesis and inhibits apoptosis of the human umbilical vein endothelial cells (HUVECs) induced by high glucose. Int J Clin Exp Pathol 11:359–366
Zhong X, Li P, Li J, He R, Cheng G, Li Y (2018) Downregulation of microRNA-34a inhibits oxidized low-density lipoprotein-induced apoptosis and oxidative stress in human umbilical vein endothelial cells. Int J Mol Med 42:1134–1144. https://doi.org/10.3892/ijmm.2018.3663
Wu CY, Zhou ZF, Wang B, Ke ZP, Ge ZC, Zhang XJ (2018) MicroRNA-328 ameliorates oxidized low-density lipoprotein-induced endothelial cells injury through targeting HMGB1 in atherosclerosis. J Cell Biochem. https://doi.org/10.1002/jcb.27469
Qian W, Qian Q, Cai X, Han R, Yang W, Zhang X, Zhao H, Zhu R (2019) Astragaloside IV inhibits oxidized low-density lipoprotein-induced endothelial damage via upregulation of miR-140-3p. Int J Mol Med 44:847–856. https://doi.org/10.3892/ijmm.2019.4257
Zheng Y, Zheng B, Meng X, Yan Y, He J, Liu Y (2019) LncRNA DANCR promotes the proliferation, migration, and invasion of tongue squamous cell carcinoma cells through miR-135a-5p/KLF8 axis. Cancer Cell Int 19:302. https://doi.org/10.1186/s12935-019-1016-6
Wu Q, Shi M, Meng W, Wang Y, Hui P, Ma J (2019) Long noncoding RNA FOXD3-AS1 promotes colon adenocarcinoma progression and functions as a competing endogenous RNA to regulate SIRT1 by sponging miR-135a-5p. J Cell Physiol 234:21889–21902. https://doi.org/10.1002/jcp.28752
Feng L, Lin T, Che H, Wang X (2020) Long noncoding RNA DANCR knockdown inhibits proliferation, migration and invasion of glioma by regulating miR-135a-5p/BMI1. Cancer Cell Int 20:53. https://doi.org/10.1186/s12935-020-1123-4
Fabian MR, Sonenberg N, Filipowicz W (2010) Regulation of mRNA translation and stability by microRNAs. Annu Rev Biochem 79:351–379. https://doi.org/10.1146/annurev-biochem-060308-103103
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Shao, X., Liu, Z., Liu, S. et al. Astragaloside IV alleviates atherosclerosis through targeting circ_0000231/miR-135a-5p/CLIC4 axis in AS cell model in vitro. Mol Cell Biochem (2021). https://doi.org/10.1007/s11010-020-04035-8
- Astragaloside IV (ASV)