CircTM7SF3 通过靶向 miR-206/ASPH 轴在体外动脉粥样硬化细胞模型中促进氧化型低密度脂蛋白诱导的细胞凋亡、炎症和氧化应激。
CircTM7SF3 contributes to oxidized low-density lipoprotein-induced apoptosis, inflammation and oxidative stress through targeting miR-206/ASPH axis in atherosclerosis cell model in vitro.
机构信息
Department of Heart Center, The First Hospital of Lanzhou University, No.1 Donggang West Road, Chengguan District, Lanzhou, 730030, Gansu, China.
出版信息
BMC Cardiovasc Disord. 2021 Feb 2;21(1):51. doi: 10.1186/s12872-020-01800-x.
BACKGROUND
Atherosclerosis (AS) is a chronic inflammatory disorder. The aim of our study was to explore the role of circular RNA (circRNA) transmembrane 7 superfamily member 3 (circTM7SF3) in AS progression.
METHODS
Experiments were conducted using oxidized low-density lipoprotein (ox-LDL)-induced THP-1-derived macrophages and differentiated human monocyte-derived macrophages (hMDMs). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of circTM7SF3, its linear form TM7SF3, microRNA-206 (miR-206) and aspartyl (asparaginyl) β-hydroxylase (ASPH) messenger RNA (mRNA). Cell viability and apoptosis were examined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. Cell inflammation was analyzed by measuring the production of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) using enzyme-linked immunosorbent assay (ELISA) kits. Cell oxidative stress was assessed through analyzing the levels of oxidative stress markers using their corresponding commercial kits. Dual-luciferase reporter assay and RNA-pull down assay were used to confirm the interaction between miR-206 and circTM7SF3 or ASPH. The protein level of ASPH was examined by Western blot assay.
RESULTS
CircTM7SF3 level was markedly increased in the serum samples of AS patients and ox-LDL-induced THP-1-derived macrophages compared with their matching counterparts. ox-LDL induced-damage in THP-1 cells was partly attenuated by the interference of circTM7SF3. MiR-206 was a downstream molecular target of circTM7SF3. Si-circTM7SF3-mediated effects in ox-LDL-induced THP-1-derived macrophages were partly ameliorated by the addition of anti-miR-206. MiR-206 directly interacted with ASPH mRNA. CircTM7SF3 silencing reduced the expression of ASPH partly through up-regulating miR-206 in THP-1-derived macrophages. ASPH overexpression partly counteracted the effects induced by miR-206 overexpression in ox-LDL-induced THP-1-derived macrophages.
CONCLUSION
CircTM7SF3 contributed to ox-LDL-induced injury in AS cell model through up-regulating the expression of ASPH via targeting miR-206.
背景
动脉粥样硬化（AS）是一种慢性炎症性疾病。我们的研究旨在探讨环状 RNA（circRNA）跨膜 7 超家族成员 3（circTM7SF3）在 AS 进展中的作用。
方法
采用氧化型低密度脂蛋白（ox-LDL）诱导的 THP-1 源性巨噬细胞和分化的人单核细胞源性巨噬细胞（hMDMs）进行实验。实时定量聚合酶链反应（qRT-PCR）检测 circTM7SF3、其线性形式 TM7SF3、微小 RNA-206（miR-206）和天冬氨酸（天门冬氨酸）β-羟化酶（ASPH）信使 RNA（mRNA）的表达。通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法和流式细胞术检测细胞活力和凋亡。通过酶联免疫吸附试验（ELISA）试剂盒检测肿瘤坏死因子-α（TNF-α）和白细胞介素 6（IL-6）的产生来分析细胞炎症。通过使用相应的商业试剂盒分析氧化应激标志物的水平来评估细胞氧化应激。双荧光素酶报告基因检测和 RNA 下拉实验用于证实 miR-206 与 circTM7SF3 或 ASPH 之间的相互作用。Western blot 检测 ASPH 蛋白水平。
结果
与匹配对照组相比，AS 患者血清样本和 ox-LDL 诱导的 THP-1 源性巨噬细胞中 circTM7SF3 水平显著升高。circTM7SF3 干扰可部分减轻 ox-LDL 诱导的 THP-1 细胞损伤。miR-206 是 circTM7SF3 的下游分子靶标。加入抗 miR-206 可部分改善 ox-LDL 诱导的 THP-1 源性巨噬细胞中 Si-circTM7SF3 介导的作用。circTM7SF3 沉默通过上调 ox-LDL 诱导的 THP-1 源性巨噬细胞中的 miR-206 部分降低了 ASPH 的表达。ASPH 过表达部分抵消了 ox-LDL 诱导的 THP-1 源性巨噬细胞中 miR-206 过表达引起的作用。
结论
circTM7SF3 通过靶向 miR-206 上调 ASPH 的表达促进 ox-LDL 诱导的 AS 细胞模型损伤。
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