Inhibition of lncRNA H19/miR-370-3p pathway mitigates neuronal apoptosis in an in vitro model of spinal cord injury (SCI).

Xin Li,Li Huang,Kaiwen Zou,Rui Tao,Kaihua Tang,Yang Li,Chunyan Gong,Lindong Liu,Yan Qian

doi:10.1515/tnsci-2021-0013

Abstract

BackgroundSpinal 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.MethodsThe 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.ResultsSCI 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.ConclusionInhibition 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.

Highlights

With the development of economy worldwide, the incidence rate of spinal cord injury (SCI) shows an annual increasing trend
Transfection of miR-370-3p inhibitor could upwardly exchange IκBα and p65 phosphorylation ratio again. These results suggested that silencing Long ncRNAs (lncRNAs) H19 could inhibit NFκB signaling through miR-370-3p mediation
Great efforts have been made to improve the function of SCI patients, secondary injuries by SCI are still the main cause of neurological dysfunction, characterized by neuronal apoptosis and inflammatory injury in the central nervous system [28]

Summary

Introduction

With the development of economy worldwide, the incidence rate of spinal cord injury (SCI) shows an annual increasing trend. There is about 10.4–83 million SCI cases suffering every year globally [1]. SCI is the most complication of spinal injury, which often leads to severe dysfunction of the limb below the injured segment, accompanied by sensory disturbance, spinal shock, motor dysfunction, autonomic nervous dysfunction, abnormal reflexes, and bladder dysfunction [2]. Once suffering from SCI, people maintain worse life standard with lower income. The treatment of SCI is always a big challenge in clinical application for doctors and scientists [3]. According to the degree of severity of SCI, the universal treatment approaches including drug

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Conclusion