环状RNA_014260/微小RNA-384/血小板反应蛋白1通过促进神经元凋亡和内质网应激加重大鼠脊髓损伤。
circ_014260/miR-384/THBS1 aggravates spinal cord injury in rats by promoting neuronal apoptosis and endoplasmic reticulum stress.
作者信息
Yao Yu, Zhang Xin, Xu Jun, Gao Feng, Wu Yanni, Cui Xintao, Wei Li, Jiang Jie, Wang Xintao
机构信息
Department of Orthopedics, The Second Affiliated Hospital of Harbin Medical University Harbin 150001, Heilongjiang Province, China.
Department of Nephrology, The Second Affiliated Hospital of Harbin Medical University Harbin 150001, Heilongjiang Province, China.
出版信息
Am J Transl Res. 2022 Jan 15;14(1):518-533. eCollection 2022.
OBJECTIVE
To explore the mechanism of circ_014260 regulating neuronal apoptosis, oxidative stress, and endoplasmic reticulum stress in rats with spinal cord injury (SCI) miR-384/THBS1 axis.
METHODS
T9-L10 spinal cord segments of Sprague Dawley rats were subjected to compression or contusion injuries after T10 laminectomy to establish rat models of SCI, which were then divided into SCI group, si-circ group and oe-circ group according to the transfection. There was another sham operation group which received no treatment. There were 10 rats in each group. The Basso-Beattie-Bresnahan scale and HE staining were used to evaluate the changes in neuronal motor function in rats with SCI. TUNEL staining was used to determine the neuronal apoptosis. Flow cytometry was used to measure the changes in HO-induced apoptosis of primary neurons. The activities of myeloperoxidase, malondialdehyde, superoxide dismutase and catalase were measured to evaluate the level of oxidative stress. Western blot was used to measure the expressions of CHOP and CRP78 (which are related to endoplasmic reticulum stress). Expression of circ_014260, miR-384 and THBS1 in tissues and cells was measured by qRT-PCR. RNase R restriction enzyme digestion and chromatin fractionation were used to identify the nature of circ_014260. Dual-luciferase reporter assay and RNA immunoprecipitation were used to verify the targeted binding relationship between circ_014260 and miR-384, as well as between miR-384 and THBS1.
RESULTS
Compared with the sham operation group or the untreated rat primary neurons (control group), increased expression of circ_014260 and THBS1 as well as decreased expression of miR-384 were observed in the spinal cord tissue from rats with SCI and in HO-treated primary neurons (all P<0.05). The results of both and experiments showed that knocking down circ_014260 could reduce neuronal apoptosis and inhibit oxidative stress and endoplasmic reticulum stress in rats with SCI (all P<0.05). Circ_014260 targetedly inhibited miR-384 to up-regulate the expression of THBS1. Both miR-384 inhibitor and THBS1 overexpression vector partially reversed the alleviated neuronal damage by knocking down circ_014260 (both P<0.05).
CONCLUSION
Circ_014260 promotes neuronal damage in rats with SCI by inhibiting miR-384 to up-regulate the expression of THBS1. Thus, circ_014260 could possibly be a new molecular target of SCI.
目的
探讨环状RNA_014260（circ_014260）通过微小RNA-384（miR-384）/血小板反应蛋白1（THBS1）轴调控脊髓损伤（SCI）大鼠神经元凋亡、氧化应激和内质网应激的机制。
方法
对10只Sprague Dawley大鼠行T10椎板切除术后，对T9-L10脊髓节段进行压迫或挫伤损伤，以建立SCI大鼠模型，然后根据转染情况将其分为SCI组、环状RNA干扰组（si-circ组）和环状RNA过表达组（oe-circ组）。另设假手术组，不进行任何处理。每组10只大鼠。采用Basso-Beattie-Bresnahan评分和苏木精-伊红（HE）染色评估SCI大鼠神经元运动功能变化。采用末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）染色法检测神经元凋亡情况。采用流式细胞术检测过氧化氢（HO）诱导的原代神经元凋亡变化。检测髓过氧化物酶、丙二醛、超氧化物歧化酶和过氧化氢酶活性，以评估氧化应激水平。采用蛋白质免疫印迹法检测与内质网应激相关的C/EBP同源蛋白（CHOP）和葡萄糖调节蛋白78（GRP78）的表达。采用实时荧光定量聚合酶链反应（qRT-PCR）检测组织和细胞中circ_014260、miR-384和THBS1的表达。采用核糖核酸酶R（RNase R）限制性酶切和染色质分离法鉴定circ_014260的性质。采用双荧光素酶报告基因检测和RNA免疫沉淀法验证circ_014260与miR-384以及miR-384与THBS1之间的靶向结合关系。
结果
与假手术组或未处理的大鼠原代神经元（对照组）相比，SCI大鼠脊髓组织和HO处理的原代神经元中circ_014260和THBS1表达增加，miR-384表达降低（均P<0.05）。RNA干扰和过表达实验结果均显示，敲低circ_014260可减少SCI大鼠神经元凋亡，抑制氧化应激和内质网应激（均P<0.05）。circ_014260靶向抑制miR-384，上调THBS1表达。miR-384抑制剂和THBS1过表达载体均部分逆转了敲低circ_014260减轻的神经元损伤（均P<0.05）。
结论
circ_014260通过抑制miR-384上调THBS1表达，促进SCI大鼠神经元损伤。因此，circ_014260可能是SCI的一个新分子靶点。
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