Li Xin, Qian Yan, Tang Kaihua, Li Yang, Tao Rui, Gong Chunyan, Huang Li, Zou Kaiwen, Liu Lindong
Department of Rehabilitation Medicine, Qujing No. 1 Hospital, Yuanlin No. 1 Road, Qilin District, Qujing 655000, Yunnan, China.
Transl Neurosci. 2021 Mar 1;12(1):103-113. doi: 10.1515/tnsci-2021-0013. eCollection 2021 Jan 1.
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.
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 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.
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.
Inhibition of lncRNA H19/miR-370-3p mitigated spinal neuron apoptosis in an model of SCI. This provided the possibility for clinical use of gene therapy.
脊髓损伤（SCI）是脊柱损伤最严重的并发症，常导致损伤节段以下肢体严重功能障碍。传统治疗方法的效果越来越差，基因治疗是目前新的研究方向。
将Sprague-Dawley大鼠随机分为两组，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。通过荧光素酶报告基因检测和RT-qPCR确定lncRNA H19与miR-370-3p之间的相关性。用si-H19、miR-370-3p抑制剂、阴性对照（NC）或两者转染后，对原代脊髓神经元进行脂多糖（LPS）模拟诱导SCI模型。采用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）、凋亡、caspase-3活性、活性氧生成及蛋白质免疫印迹法检测细胞活力、凋亡率、caspase-3活性、Bax和Bcl-2蛋白、活性氧生成、肿瘤坏死因子-α、白细胞介素-1β和白细胞介素-6蛋白，以及IκBα和p65磷酸化率，从而寻找对LPS诱导的脊髓神经损伤的具体作用机制。
SCI导致lncRNA H19表达升高，miR-370-3p表达降低。LPS模拟引起一系列细胞生物学变化，如活力降低、凋亡增加、活性氧生成及炎症因子释放。lncRNA H19抑制可逆转上述LPS诱导的变化。此外，作为lncRNA H19的下游靶点，miR-370-3p受lncRNA H19的反向调控。lncRNA H19抑制诱导的上述生物学变化可被miR-370-3p上调逆转。此外，lncRNA H19抑制可通过上调miR-370-3p阻断NF-κB通路。
抑制lncRNA H19/miR-370-3p可减轻SCI模型中脊髓神经元的凋亡。这为基因治疗的临床应用提供了可能。
