Background Spinal cord injury (SCI) is the most serious complication of spinal injury, often leading to severe dysfunction of the limbs below the injured segment. Conventional therapy approaches are becoming less and less effective, and gene therapy is a new research direction by now. Methods The Sprague-Dawley rats were haphazardly assigned to two groups, namely sham group and SCI model group, and lncRNA H19 and miR-370-3p levels were investigated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Correlation between lncRNA H19 and miR-370-3p was ascertained by luciferase report assay and RT-qPCR. After transfection with si-H19, miR-370-3p inhibitor, negative controls (NC), or both, primary spinal neurons were subjected to the simulation of lipopolysaccharide (LPS) for inducing in vitro model of SCI. Cell viability, apoptotic rate, caspase-3 activity, Bax and Bcl-2 protein, ROS generation, TNF-α, IL-1β, and IL-6 protein, as well as IκBα and p65 phosphorylation ratio were evaluated adopting 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), apoptosis, caspase-3 activity, ROS generation, and western blot assays, thereby searching for the specific action mechanism on LPS-induced spinal never injury. Results SCI resulted in lncRNA H19 higher expression and miR-370-3p lower expression. LPS simulation raised a series of cellular biological changes, such as decreased viability, promoted apoptosis, generated ROS, and released inflammatory factors. lncRNA H19 inhibition reversed above LPS-induced changes. Besides, as the downstream target of lncRNA H19, miR-370-3p was oppositely regulated by lncRNA H19. The above biological changes induced by lncRNA H19 inhibition were reversed by miR-370-3p upregulation. Moreover, lncRNA H19 inhibition could block NF-κB pathway through miR-370-3p upregulation. Conclusion Inhibition of lncRNA H19/miR-370-3p mitigated spinal neuron apoptosis in an in vitro model of SCI. This provided the possibility for clinical use of gene therapy.

中文翻译：

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抑制 lncRNA H19/miR-370-3p 通路可减轻脊髓损伤 (SCI) 体外模型中的神经元凋亡

背景脊髓损伤（SCI）是脊柱损伤最严重的并发症，常常导致受伤节段以下肢体的严重功能障碍。传统治疗方法的效果越来越差，基因治疗成为目前新的研究方向。方法 将Sprague-Dawley大鼠随机分为2组，即假手术组和SCI模型组，采用逆转录定量聚合酶链反应（RT-qPCR）检测lncRNA H19和miR-370-3p水平。通过荧光素酶报告分析和 RT-qPCR 确定了 lncRNA H19 和 miR-370-3p 之间的相关性。转染 si-H19、miR-370-3p 抑制剂、阴性对照 (NC) 或两者后，对原代脊髓神经元进行脂多糖 (LPS) 模拟，以诱导 SCI 体外模型。采用3-(4评估细胞活力、凋亡率、caspase-3活性、Bax和Bcl-2蛋白、ROS生成、TNF-α、IL-1β和IL-6蛋白以及IκBα和p65磷酸化比率,5-二甲基噻唑-2-基)-2,5-二苯基溴化四唑(MTT)、细胞凋亡、caspase-3活性、ROS生成和蛋白质印迹测定，从而寻找LPS诱导的脊髓损伤的具体作用机制。结果 SCI 导致 lncRNA H19 表达较高，miR-370-3p 表达较低。LPS模拟引起一系列细胞生物学变化，如活力降低、促进细胞凋亡、产生ROS、释放炎症因子等。lncRNA H19 抑制逆转了 LPS 诱导的上述变化。此外，作为lncRNA H19的下游靶标，miR-370-3p受到lncRNA H19的反向调控。lncRNA H19 抑制诱导的上述生物学变化可通过 miR-370-3p 上调而逆转。此外，lncRNA H19 抑制可以通过 miR-370-3p 上调阻断 NF-κB 通路。结论 在体外 SCI 模型中，抑制 lncRNA H19/miR-370-3p 可减轻脊髓神经元凋亡。这为基因治疗的临床应用提供了可能。