Xiaohong Kong

Targeting RPTPσ with lentiviral shRNA promotes neurites outgrowth of cortical neurons and improves functional recovery in a rat spinal cord contusion model.

After spinal cord injury (SCI), the rapidly upregulated chondroitin sulfate proteoglycans (CSPGs), the prominent chemical constituents and main repulsive factors of the glial scar, play an important role in the extremely limited ability to regenerate in adult mammals. Although many methods to overcome the inhibition have been tested, no successful method with clinical feasibility has been devised to date. It was recently discovered that receptor protein tyrosine phosphatase sigma (RPTPσ) is a functional receptor for CSPGs-mediated inhibition. In view of the potential clinical application of RNA interference (RNAi), here we investigated whether silencing RPTPσ via lentivirus-mediated RNA interference can promote axon regeneration and functional recovery after SCI. Neurites of primary rat cerebral cortical neurons with depleted RPTPσ exhibited a significant enhancement in elongation and crossing ability when they encountered CSPGs in vitro. A contusion model of spinal cord injury in Wistar rats (the New York University (NYU) impactor) was used for in vivo experiments. Local injection of lentivirus encoding RPTPσ shRNA at the lesion site promoted axon regeneration and synapse formation, but did not affect the scar formation. Meanwhile, in vivo functional recovery (motor and sensory) was also enhanced after RPTPσ depletion. Therefore, strategies directed at silencing RPTPσ by RNAi may prove to be a beneficial, efficient and valuable approach for the treatment of SCI.

ERK2 small interfering RNAs prevent epidural fibrosis via the efficient inhibition of collagen expression and inflammation in laminectomy rats.

Laminectomy is a widely accepted treatment for lumbar disorders. Epidural Fibrosis (EF) is a common post-laminectomy or post-discectomy complication, which is thought to cause recurrent pain. RNA interference (RNAi) is a process by which double-stranded RNA triggers the destruction of mRNAs sharing the same sequence. Previously, extra-cellular signal-regulated kinase (ERK) 2 plays crucial roles in suppressing the collagen expression. To investigate the effects of lentiviral ERK2 siRNA on the prevention of post-laminectomy EF formation in a rat model, a controlled double-blinded study was conducted in 75 healthy adult Wistar rats that underwent laminectomy. They were divided randomly into 3 groups according to the treatment method: (1) control group; (2) ERK scrRNA group; (3) ERK siRNA group. All rats were euthanized humanely 4 weeks post-laminectomy. The hydroxyproline content, Rydell score, vimentin cells density, fibroblasts density, inflammatory cells density and inflammatory factors expressions were performed. The hydroxyproline content, Rydell score, vimentin cells density, fibroblasts density, inflammatory cells density and inflammatory factors expressions all suggested better results in ERK siRNA group than other two groups. None of the rats expired and no obvious adverse effects were observed. Local delivery of a lentiviral siRNA targeting ERK2 can prevent epidural scar adhesion in post-laminectomy rat via inhibiting collagen expression and inflammation.

Valproic acid-mediated neuroprotection and neurogenesis after spinal cord injury: from mechanism to clinical potential.

Spinal cord injury (SCI) is difficult to treat because of secondary injury. Valproic acid (VPA) is clinically approved for mood stabilization, but also counteracts secondary damage to functionally rescue SCI in animal models by improving neuroprotection and neurogenesis via inhibition of HDAC and GSK-3. However, a comprehensive review summarizing the therapeutic benefits and mechanisms of VPA for SCI and the issues affecting clinical trials is lacking, limiting future research on VPA and impeding its translation into clinical therapy for SCI. This article presents the current status of VPA treatment for SCI, emphasizing interactions between enhanced neuroprotection and neurogenesis. Crucial issues are discussed to optimize its clinical potential as a safe and effective treatment for SCI.

In vitro characteristics of Valproic acid and all-trans-retinoic acid and their combined use in promoting neuronal differentiation while suppressing astrocytic differentiation in neural stem cells

Multipotent neural stem cells (NSCs) are currently under investigation as a candidate treatment for central nervous system (CNS) injury because of their potential to compensate for neuronal damage and to reconstruct disrupted neuronal connections. To maximize the regenerative effect of the derived neurons and to minimize the side effects of the derived astrocytes, it is necessary to regulate the fate determination of NSCs to produce more neurons and fewer astrocytes. Both valproic acid (VPA) and all-trans-retinoic acid (ATRA), two clinically established drugs, induce neuronal differentiation and facilitate neurite outgrowth at the expense of astrocytic differentiation in NSCs. However, the time-dependent activities and the long-term treatment effects of these drugs have not been explored in NSCs. More importantly, the efficacies of VPA and ATRA in neuronal promotion and astrocytic suppression remain unclear. In this study, we compare the time-dependent characteristics of VPA and ATRA in NSC differentiation and neurite outgrowth in vitro and, for the first time, demonstrate the improved efficacy of their combined application in neuronal induction and astrocytic suppression. These significant effects are closely coupled to the altered expression of a neurogenic transcription factor, a Wnt signaling component, a cell cycle regulator and a neural growth factor, indicating an underlying cross-talk between the mechanisms of action of ATRA and VPA. These findings indicate that a novel strategy combining these two therapeutic drugs may improve the restorative effect of NSC transplantation by altering the expression of their interconnected targets for fate determination.

The roles of microRNAs in spinal cord injury

ABSTRACT Background and purpose: Spinal cord injury (SCI) involves serious damage that can result in abnormal or absent motor and sensory functions and a disruption of autonomic function, and a series of pathological reactions occur after the injury. As a type of small non-coding RNA, microRNAs (miRNAs) have been verified to inhibit gene expression via post-transcriptional regulation. This review mainly focuses on recent advances regarding the roles of miRNAs following SCI. Methods: The Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were adopted. The studies regarding the roles of miRNAs following SCI were identified through PubMed, Embase and Web of Science. We summarise the changes in expression levels of miRNAs and discuss the roles of miRNAs after SCI. Results: A total of 77 empirical studies meeting the inclusion criteria were identified. Existing studies showed that miRNAs were temporally altered and had effects on apoptosis, inflammation, angiogenesis, astrogliosis, oligodendrocyte development, axonal regeneration and remyelination after SCI. The alteration of miRNAs and the regulative action of pathological reactions can also provide opportunities for potential therapeutic interventions. “miRNA replacement therapy” aims to transfer miRNAs into diseased cells via delivery techniques and improve targeting effectiveness in cells, and this novel therapeutic tool provides a promising technique to promote the repair of SCI and reduces functional deficits. Conclusions: This review is helpful for understanding the underlying mechanisms of SCI and the potential clinical value of miRNAs. miRNAs have the potential to be attractive tools and targets for novel diagnostic and therapeutic approaches of SCI.

Bovine ISG15: an antiviral and inducible protein in BIV infected fetal bovine lung cells

Bovine ISG15 (bISG15) is an interferon inducible ubiquitin-like protein that is responsible for the establishment of early pregnancy in ruminant, understanding the properties of bISG15 capable of being inducible in fetal bovine lung (FBL) cells upon infection of bovine immunodeficiency virus (BIV) is of significant importance. In this study, we investigated the expression of bISG15 in poly I:C treated FBL cells. The increased expression of bISG15 was observed, and the inhibition of BIV replication was also detected in FBL cells. Elimination of bISG15 expression by small interfering RNA reversed the bISG15 mediated inhibition of BIV replication. These findings demonstrate that bISG15 plays an important role in inhibition of the BIV replication in FBL cells. Furthermore, real-time PCR and western blot assay revealed that bISG15s expression can also be induced in BIV infected FBL cells. Taken together, bISG15 is an antiviral and inducible protein in BIV infected FBL cells.

Identification of microRNAome in rat bladder reveals miR-1949 as a potential inducer of bladder cancer following spinal cord injury

The costs of spinal cord injury and its complications are high in personal, social and financial terms. Complications include bladder cancer, for which the risk is 16-28 times higher than that of the general population, There is currently little consensus regarding the cause of this discrepancy. As microRNAs are stable biomarkers and potential therapeutic targets of cancer, the present study aimed to explore the underlying mechanisms of this phenomenon by examining changes in the microRNAome. Rats were used to produce models of spinal cord injury. Microarrays and bioinformatics were used to investigate the cancer-associated microRNAs that are upregulated in rat bladders following spinal cord injury. In order to validate the results, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blotting and immunohistochemistry were performed. The expression of miR-1949 was found to be deregulated and abundant in the rat bladder following spinal cord injury. Bioinformatics demonstrated that retinoblastoma 1, which is involved in tumorigenesis, is a target gene of miR-1949. qRT-PCR, western blotting and immunohistochemistry confirmed the results of the microarray analysis. In addition, it was shown that miR-1949 expression was not influenced by aging. Furthermore, the expression of miR-1949 was stable until the third month following spinal cord injury, after which it significantly increased. If this increase was prolonged, the expression of retinoblastoma 1 may decline to a carcinogenic level. The present study suggests a role for miR-1949 in the translational regulation of retinoblastoma 1 and in subsequent bladder tumorigenesis following spinal cord injury.

Gene Expression Analysis at Multiple Time Points Identifies Key Genes for Nerve Regeneration

Introduction: The purpose of this study was to provide a comprehensive understanding of gene expression during Wallerian degeneration and axon regeneration after peripheral nerve injury. Methods: A microarray was used to detect gene expression in the distal nerve 0, 3, 7, and 14 days after sciatic nerve crush. Bioinformatic analysis was used to predict function of the differentially expressed mRNAs. Microarray results and the key pathways were validated by quantitative real‐time polymerase chain reaction (qRT‐PCR). Results: Differentially expressed mRNAs at different time‐points (3, 7, and 14 days) after injury were identified and compared with a control group (0 day). Nine general trends of changes in gene expression were identified. Key signal pathways and 9 biological processes closely associated with nerve regeneration were identified and verified. Conclusions: Differentially expressed genes and biological processes and pathways associated with axonal regeneration may elucidate the molecular‐biological mechanisms underlying peripheral nerve regeneration. Muscle Nerve 55: 373–383, 2017

Time-dependent differential expression of long non-coding RNAs following peripheral nerve injury

Long non-coding RNAs (lncRNAs) are widely accepted as key players in various biological processes. However, the roles of lncRNA in peripheral nerve regeneration remain completely unknown. Thus, in this study, we performed microarray analysis to measure lncRNA expression in the distal segment of the sciatic nerve at 0, 3, 7 and 14 days following injury. We identified 5,354 lncRNAs that were differentially expressed: 3,788 lncRNAs were differentially expressed between days 0 and 3; 3,314 lncRNAs were differentially expressed between days 0 and 7; and 2,400 lncRNAs were differentially expressed between days 0 and 14. The results of RT-qPCR of two dysregulated lncRNAs were consistent with those of microarray analysis. Bioinformatics approaches, including lncRNA classification, gene ontology (GO) analysis and target prediction, were utilized to investigate the functions of these dysregulated lncRNAs in peripheral nerve damage. Importantly, we predicted that several lncRNA-mRNA pairs may participate in biological processes related to peripheral nerve injury. RT-qPCR was performed for the preliminary verification of three lncRNA-mRNA pairs. The overexpression of NONMMUG014387 promoted the proliferation of mouse Schwann cells. Thus, the findings of our study may enhance our knowledge of the role of lncRNAs in nerve injury.

Two retroviruses packaged in one cell line can combined inhibit the replication of HIV-1 in TZM-bl cells

The cellular protein tetherin tethers the HIV-1 viral particles on the cellular membrane to inhibit the replication of HIV-1. However, the HIV-1 accessory protein Vpu counteracts the antiviral function of tetherin. In this study, two retroviral vector plasmids were constructed. One inhibited the vpu gene expression; the other one over-expressed the tetherin. Both retroviral vector plasmids could be packaged in the packaging cell line PT67 to obtain the corresponding retroviruses. The retroviral vector plasmids’ functions of tetherin over-expression or vpu-RNAi were detected at the cell level. Retroviral vector plasmids were transfected to PT67 cells at different ratios from 0T3V to 3T0V, and then mixed retroviruses were harvested. The antiviral functions of mixed retroviruses were detected in HIV-1 infected TZM-bl cells. The results showed that packaged mixed retroviruses could repress the replication of HIV-1 in TZM-bl cells.