Suppression of LINC00707 alleviates lipopolysaccharide-induced inflammation and apoptosis in PC-12 cells by regulated miR-30a-5p/Neurod 1

ABSTRACT

Long noncoding RNA (lncRNA) has emerged as a pivotal regulator improving neural regeneration in the progression of spinal cord injury (SCI). However, whether lncRNAs can be targeted for therapeutic intervention of SCI remains unclear. In this study, we found that LINC00707 expression was significantly up-regulated in lipopolysaccharide (LPS)-treated PC-12, a model that mimics nerve cell injury in an inflammatory environment after SCI. Suppression of LINC00707 alleviated LPS-induced inflammation and apoptosis in PC-12 cells. Furthermore, we found that LINC00707 adsorbed miR-30a-5p and silenced miR-30a-5p or overexpressed Neurod 1 reversed the effect of LINC00707 on the inflammation and apoptosis of LPS-treated PC-12 cells. These findings revealed that LINC00707 alleviates LPS-induced inflammation and apoptosis in PC-12 cells by targeting miR-30a-5p/Neurod 1, providing a preliminary theoretical basis for the clinical application of LINC00707 in SCI.     

Peripheral Macrophage-derived Exosomes promote repair after Spinal Cord Injury by inducing Local Anti-inflammatory type Microglial Polarization via Increasing Autophagy

Treatment for spinal cord injury (SCI) remains a challenge worldwide, and inflammation is a major cause of secondary injury after SCI. Peripheral macrophages (PMs) have been verified as a key factor that exert anti-inflammatory effects after SCI, but the mechanism is unidentified. As local macrophages, microglia also exert significant effects after SCI, especially polarization. Exosomes show source cell-like biological functions to target cells and have been the subject of much research in recent years. Thus, we hypothesized the PM-derived exosomes (PM-Exos) play an important role in signal transmission with local microglia and can be used therapeutic agents for SCI in a series of in vivo and in vitro studies. For the in vivo experiment, three groups of Sprague-Dawley (SD) rats subjected to spinal cord contusion injury were injected with 200 µg/ml PM-Exos, 20 µg/ml PM-Exos or PBS via the tail vein. Recovery of the rats and of spinal cord function were observed. In vitro, we investigated the potential anti-inflammatory mechanism of PM-Exos and evaluated microglial autophagy, anti-inflammatory type microglia polarization and the upstream signaling pathway. The results showed that spinal cord function and recovery were better in the PM-Exo groups than the control group. In the in vitro study, microglial autophagy levels and the expression of anti-inflammatory type microglia were higher in the experimental groups than the control group. Moreover, the expression of proteins related to the PI3K/AKT/mTOR autophagic signaling pathway was suppressed in the PM-Exo groups. PM-Exos have a beneficial effect in SCI, and activation of microglial autophagy via inhibition of the PI3K/AKT/mTOR signaling pathway, enhancing the polarization of anti-inflammatory type microglia, that may play a major role in the anti-inflammatory process.     

HSP70 alleviates spinal cord injury by activating the NF-kB pathway

Objectives:Spinal cord injury (SCI) is an acute traumatic lesion of neurons in the spinal cord which has a high prevalence in the world, and has no effective surgical treatment. HSP70 is a molecular chaperone protein, serves a protective role in several different models of nervous system injury. The aim of the present study was to investigate the anti-inflammatory role of HSP70 in spinal cord injury and explore its mechanism.Methods:In vivo and in vitro models were constructed to mimic SCI. The Basso Mouse Scale (BMS) was applied to assess SCI degrees of the mouse model. Immunofluorescence (IF) was used for visualizing HSP70 and Iba1 in the spinal cord. Western blot assay was employed to quantify HSP70 and p65, and ELISA was for IL-1β and TNF-α.Results:The results showed that HSP70 expression decreased after SCI. HSP70 and Iba1 showed a decrease of co-localization in SCI mice. Further studies revealed that p65 was upregulated during the process of SCI. Overexpression of HSP70 inhibited the expression of p65 both in vitro and in vivo, and promoted the recovery of SCI mice.Conclusions:HSP70 was involved in the pathological process of spinal cord injury, HSP70 alleviated the spinal cord injury via inhibiting NF-κB signaling pathway.     

miR-31 promotes neural stem cell proliferation and restores motor function after spinal cord injury

This study aims to examine whether miR-31 promotes endogenous NSC proliferation and be used for spinal cord injury management. In the present study, the morpholino knockdown of miR-31 induced abnormal neuronal apoptosis in zebrafish, resulting in impaired development of the tail. miR-31 agomir transfection in NSCs increased Nestin expression and decreased ChAT and GFAP expression levels. miR-31 induced the proliferation of mouse NSCs by upregulating the Notch signaling pathway, and more NSCs entered G1; Notch was inhibited by miR-31 inactivation. Injection of a miR-31 agomir into mouse models of spinal cord injury could effectively restore motor functions after spinal cord injury, which was achieved by promoting the proliferation of endogenous NSCs. After the injection of a miR-31 agomir in spinal cord injury mice, the expression of Nestin and GFAP increased, while GFAP expression decreased. In conclusion, the zebrafish experiments prove that a lack of miR-31 will block nervous system development. In spinal cord injury mouse models, miR-31 overexpression might promote spinal cord injury repair.     

Radiosensitizer EXO-miR-197-3p Inhibits Nasopharyngeal Carcinoma Progression and Radioresistance by Regulating the AKT/mTOR Axis and HSPA5-mediated Autophagy

The biological functions of exosomes and microRNAs (miRs) in nasopharyngeal carcinoma (NPC) remain largely unexplored. Here, miR-197-3p was screened and identified, and whose level was reduced in serum and exosomes of patients with NPC. MiR-197-3p might be a good diagnostic and prognostic indicator. Our data showed that miR-197-3p expression was closely related to radioresistance, apoptosis, proliferation, migration, and survival of NPC. Inhibition of miR-197-3p expression in vitro could promote the proliferation and migration of NPC cells, while promotion of miR-197-3p expression in vivo could significantly inhibit the growth and enhance the radiosensitivity of NPC cells. From the perspective of mechanism, miR-197-3p could inhibit AKT/mTOR phosphorylation activation, inhibit an activated pathway of AKT/mTOR, target Heat Shock 70-kDa Protein 5(HSPA5) related to endoplasmic reticulum homeostasis, inhibit HSPA5-mediated autophagy, and reverse the radioresistance of NPC. Interestingly, exosomal miR-197-3p (EXO-miR-197-3p) reduced the proliferation and migration potential of NPC cells in vitro, and tumor growth and radioresistance of NPC cells in vivo. EXO-miR-197-3p inhibited NPC progression and radioresistance by regulating AKT/mTOR phosphorylation activation and HSPA5-mediated autophagy. In conclusion, our results highlight the potential of EXO-miR-197-3p as an effective radiosensitizer and therapeutic agent for refractory NPC.     

Astragaloside IV alleviates Schwann cell injury in diabetic peripheral neuropathy by regulating microRNA-155-mediated autophagy

BackgroundMicroRNA-155(miR-155) is closely associated with diabetic peripheral neuropathy (DPN). Astragaloside IV (AST) is a significant extract of Astragalus membranaceus, which has been found to be effective in the treatment of DPN. However, whether astragaloside IV alleviate DPN via regulating miR-155-mediated autophagy remains unclear.PurposeThis study was designed to evaluate the effects of AST on DPN myelin Schwann cells injury and explore the mechanism of AST in treating DPN for the first time.MethodsGK rats fed with high-fat diet and RSC96 cells cultured in high glucose were used to establish DPN Schwann cells injury in vivo and in vitro model. The effects of AST on DPN were explored through blood glucose detection, nerve function detection, pathological detection and the expression of Neuritin detected by immunohistochemical. To study the effect of AST on the DPN Schwann cells autophagy and the upstream PI3K/Akt/mTOR pathway, the expressions of beclin-1 and LC3 were detected by western blot (WB) in sciatic nerves and by immunofluorescence (IFC) in RSC96 cells. The real-time polymerase chain reaction (RT-PCR) was applied to detect the expressions of miR-155, ATG5, ATG12 both in vivo and in vitro. The binding effect of miR-155 and target gene PI3KCA was verified by luciferase reporter gene assay. The expressions of PI3K, p-Akt/Akt, p-mTOR/mTOR were detected by WB and the expressions of PI3KCA were detected by RT-PCR in vitro. The apoptosis was detected by flow cytometry. Meanwhile, the influence of miR-155 overexpression and knocked down on the above indicators was also detected in RSC96 cells. At last, further mechanism experiments were conducted to verify the mechanism of AST regulating the autophagy and apoptosis of RSC96 cells.ResultsAST reduced blood glucose levels, alleviated peripheral nerve myelin sheath injury, and improved neurological function in DPN rats. In addition, AST enhanced the autophagy activity and alleviated the apoptosis in RSC96 cell. Mechanism study shown that AST promote autophagy via regulating miR-155-mediated PI3K/Akt/mTOR signaling pathways. AST reduced RSC96 cells apoptosis by promoting autophagy.ConclusionAST alleviate the myelin sheath injury of DPN caused by the apoptosis of Schwann cells via enhancing autophagy, which was attributed to inhibiting the activation of the PI3K/Akt/mTOR signaling pathway by upregulating miR-155 expression.     

microRNA-125b and its downstream Smurf1/KLF2/ATF2 axis as important promoters on neurological function recovery in rats with spinal cord injury

The purpose of this study is to investigate the role of microRNA-125b (miR-125b) and its mechanism in spinal cord injury (SCI) by targeting Smurf1. After loss- and gain-function approaches were conducted in SCI rat models and neural stem cells (NSCs) isolated from foetal rats, the Basso-Beattie-Bresnahan (BBB) score was calculated, and related protein expression was determined by Western blot analysis and cell apoptosis by TUNEL staining. NSC viability was detected by CCK-8, migration abilities by Transwell assay and apoptosis by flow cytometry. The relationship between miR-125b, Smurf1 and KLF2 was evaluated by dual-luciferase reporter gene experiments, Co-IP and in vivo ubiquitin modification assays. Inhibition of miR-125b and KLF2 and the up-regulation of Smurf1 and ATF2 were observed in SCI rats. BBB scores were elevated, the expression of Nestin, NeuN, GFAP, NF-200 and Bcl-2 protein was enhanced but that of Bax protein was reduced, and cell apoptosis was inhibited in SCI rats after up-regulating miR-125b or silencing ATF2. Smurf1 was a target gene of miR-125b, which promoted KLF2 degradation through its E3 ubiquitin ligase function, and KLF2 repressed the expression of ATF2 in NSCs. The results in vivo were replicated in vitro. miR-125b overexpression promotes neurological function recovery after SCI.     

Cancer-associated fibroblasts-derived exosomes upregulate microRNA-135b-5p to promote colorectal cancer cell growth and angiogenesis by inhibiting thioredoxin-interacting protein

ObjectiveThe role of exosomes in human cancers has been identified, while the effect of cancer-associated fibroblasts (CAFs)-derived exosomes (CAF-exos) transmitting microRNAs (miRNAs) on colorectal cancer (CRC) remains largely unknown. We aim to explore the impact of CAF-derived exosomal miR-135b-5p on CRC progression by targeting thioredoxin-interacting protein (TXNIP).MethodsCRC tissues were collected to obtain CAF-exos, which were used to co-culture with LoVo and HT29 cells. The effect of miR-135b-5p and TXNIP on the in vivo growth, in vitro proliferation, apoptosis, migration, invasion and angiogenesis of CRC cells. miR-135b-5p and TXNIP expression in exosomes and CRC cells were detected and their targeting relationship was confirmed.ResultsMiR-135b-5p was upregulated whereas TXNIP was downregulated in CRC tissues and cells. The CAF-exos and CAF-exos upregulating miR-135b-5p promoted in vivo growth, in vitro proliferation, migration and invasion, and suppressed apoptosis of CRC cells, and also promoted the HUVEC angiogenesis. TXNIP was confirmed as a target of miR-135b-5p and overexpression of TXNIP could weaken the pro-CRC effect of exosomal miR-135b-5p,ConclusionCAF-exos upregulate miR-135b-5p to promote CRC cell growth and angiogenesis by inhibiting TXNIP.     

Upregulation of miRNA-223-3p ameliorates RIP3-mediated necroptosis and inflammatory responses via targeting RIP3 after spinal cord injury

Spinal cord injury (SCI) has been a major burden on the society because of the high rate of disability. Receptor-interacting protein 3 (RIP3)-mediated necroptosis is a newly discovered pathway of programmed cell death and is involved in multiple pathologies of various human diseases. Micro RNAs (miRNAs) have been shown to be a potential target for therapeutic interventions after SCI. The aim of the present study is to explore the potential role of miR-223-3p and possible mechanism in SCI. We found that miR-223-3p was significantly downregulated in spinal neurons after H₂O ₂-induced damage, while RIP3-mediated necroptosis was elevated. Accordingly, RIP3-mediated necroptosis and the inflammatory factor secretion could be significantly inhibited by Nec-1 treatment. In adittion, overexpression of miR-223-3p in spinal neurons protected against H ₂O ₂-induced necroptosis, and ablation of miR-223-3p exhibited the opposite effect. We found that miR-223-3p bound to the 3′-untranslated region of RIP3 mRNA to negatively regulate the expression of RIP3. Moreover, the activated RIP3 reversed the inhibition of RIP3 and MLKL expression and the levels of TNF-α, IL-1β, and lactate dehydrogenase, which were induced by transfection with miR-223-3p in a H ₂O ₂-induced model. Finally, these results indicate that miR-223-3p negatively regulates the RIP3 necroptotic signaling cascades and inflammatory factor secretion, which significantly relieves injury of spinal neurons. The miR-223-3p/RIP3 pathway offers a novel therapeutic target for the protection of spinal neurons after SCI.     

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.     

Upregulation of miR-199a-5p Protects Spinal Cord Against Ischemia/Reperfusion-Induced Injury via Downregulation of ECE1 in Rat

Ischemia–reperfusion (I/R)-induced spinal cord injury can cause apoptotic damage and subsequently act as a blood–spinal cord barrier damage. MicroRNAs (miRNAs) contributed to the process of I/R injury by regulating their target mRNAs. miR-199a-5p is involved in brain and heart I/R injury; however, its function in the spinal cord is not yet completely clarified. In this study, we investigated the role of miR-199a-5p on spinal cord I/R via the endothelin-converting enzyme 1, especially the apoptosis pathway. In the current study, the rat spinal cord I/R injury model was established, and the Basso Beattie Bresnahan scoring, Evans blue staining, HE staining, and TUNEL assay were used to assess the I/R-induced spinal cord injury. The differentially expressed miRNAs were screened using microarray. miR-199a-5p was selected by unsupervised hierarchical clustering analysis. The dual-luciferase reporter assay was used for detecting the regulatory effects of miR-199a-5p on ECE1. In addition, neuron expression was detected by immunostaining assay, while the expressions of p-ERK, ERK, p-JNK, JNK, caspase-9, Bcl-2, and ECE1 were evaluated by Western blot. The results indicated the successful establishment of the I/R-induced spinal cord injury model; the I/R induced the damage to the lower limb motor. Furthermore, 18 differentially expressed miRNAs were detected in the I/R group compared to the sham group, and miR-199a-5p protected the rat spinal cord injury after I/R. Moreover, miR-199a-5p negatively regulated ECE1, and silencing the ECE1 gene also protected the rat spinal cord injury after I/R. miR-199a-5p or silencing of ECE1 also regulated the expressions of caspase-9, Bcl-2, p-JNK, p-ERK, and ECE1 in rat spinal cord injury after I/R. Therefore, we demonstrated that miR-199a-5p might protect the spinal cord against I/R-induced injury by negatively regulating the ECE1, which could aid in developing new therapeutic strategies for I/R-induced spinal cord injury.     

The PI3K/AKT signalling pathway in inflammation,cell death and glial scar formation after traumatic spinal cord injury: Mechanisms and therapeutic opportunities

ObjectsTraumatic spinal cord injury (TSCI) causes neurological dysfunction below the injured segment of the spinal cord, which significantly impacts the quality of life in affected patients. The phosphoinositide 3kinase/serine‐threonine kinase (PI3K/AKT) signaling pathway offers a potential therapeutic target for the inhibition of secondary TSCI. This review summarizes updates concerning the role of the PI3K/AKT pathway in TSCI.Materials and MethodsBy searching articles related to the TSCI field and the PI3K/AKT signaling pathway, we summarized the mechanisms of secondary TSCI and the PI3K/AKT signaling pathway; we also discuss current and potential future treatment methods for TSCI based on the PI3K/AKT signaling pathway.ResultsEarly apoptosis and autophagy after TSCI protect the body against injury; a prolonged inflammatory response leads to the accumulation of pro‐inflammatory factors and excessive apoptosis, as well as excessive autophagy in the surrounding normal nerve cells, thus aggravating TSCI in the subacute stage of secondary injury. Initial glial scar formation in the subacute phase is a protective mechanism for TSCI, which limits the spread of damage and inflammation. However, mature scar tissue in the chronic phase hinders axon regeneration and prevents the recovery of nerve function. Activation of PI3K/AKT signaling pathway can inhibit the inflammatory response and apoptosis in the subacute phase after secondary TSCI; inhibiting this pathway in the chronic phase can reduce the formation of glial scar.ConclusionThe PI3K/AKT signaling pathway has an important role in the recovery of spinal cord function after secondary injury. Inducing the activation of PI3K/AKT signaling pathway in the subacute phase of secondary injury and inhibiting this pathway in the chronic phase may be one of the potential strategies for the treatment of TSCI.

During secondary injury after spinal cord injury (SCI), recovery is impaired by inflammation, cell death and glial scar formation. Therefore, these pathological processes may be potential treatment targets for SCI. The phosphoinositide 3‐kinase/serine‐threonine kinase (PI3K/AKT) signalling pathway has a key role in the secondary injury after SCI. PI3K/AKT signalling pathway can inhibit the inflammation and cell death after SCI, and can also promote the formation of glial scar.     

MiR-19b-3p accelerates bone loss after spinal cord injury by suppressing osteogenesis via regulating PTEN/Akt/mTOR signalling

Rapid and extensive bone loss, one of the skeletal complications after spinal cord injury (SCI) occurrence, drastically sacrifices the life quality of SCI patients. It has been demonstrated that microRNA (miRNA) dysfunction plays an important role in the initiation and development of bone loss post-SCI. Nevertheless, the effect of miR-19b-3p on bone loss after SCI is unknown and the accurate mechanism is left to be elucidated. The present work was conducted to explore the role of miR-19b-3p/phosphatase and tensin homolog deleted on chromosome ten (PTEN) axis on osteogenesis after SCI and further investigates the underlying mechanisms. We found that miR-19b-3p level was increased in the femurs of SCI rats with decreased autophagy. The overexpression of miR-19b-3p in bone marrow mesenchymal stem cells (BMSCs) targeted down-regulation of PTEN expression, facilitated protein kinase B (Akt) and mammalian target of rapamycin (mTOR) phosphorylation, and thereby suppressing BMSCs osteogenic differentiation via autophagy. Besides, the inhibiting effects of miR-19b-3p on osteogenic differentiation of BMSCs could be diminished by autophagy inducer rapamycin. Meanwhile, bone loss after SCI in rats was also reversed by antagomir-19b-3p treatment, suggesting miR-19b-3p was an essential target for osteogenic differentiation via regulating autophagy. These results indicated that miR-19b-3p was involved in bone loss after SCI by inhibiting osteogenesis via PTEN/Akt/mTOR signalling pathway.     

Long noncoding RNA PTENP1 affects the recovery of spinal cord injury by regulating the expression of miR-19b and miR-21

Exosomes derived from differentiated P12 cells and MSCs were proved to suppress apoptosis of neuron cells, and phosphatase and tensin homolog pseudogene 1 (PTENP1) was reported to inhibit cell proliferation. In this study, we aimed to investigate the role of PTENP1 in the process of post-spinal cord injury (SCI) recovery, so as to evaluate the therapeutic effects of exosomes derived from MSCs transfected with PTENP1 short hairpin RNA (shRNA), as a type of novel biomarkers in the treatment of SCI. Electron microscopy was used to observe the morphology of different exosomes. Real-time polymerase chain reaction and western blot, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, flow cytometry, Nissl staining, immunohistochemistry assay, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay were conducted to investigate and validate the underlying molecular signaling pathway. PTENP1-shRNA downregulated PTENP1 and PTEN while upregulating miR-21 and miR-19b. PTENP1-shRNA also accelerated cell apoptosis and reduced cell viability. In addition, PTENP1 reduced the miR-21 and miR-19b expression by directly targeting miR-21 and miR-19b. Meanwhile, both miR-21 and miR-19b reduced the expression of PTEN by directly targeting the 3′-untranslated region of PTEN. Furthermore, PTEN level and apoptosis index of neuron cells was the highest in the SCI group, while the treatment with exosomes+PTENP1-shRNA reduced the PTEN expression to a level similar to that in the sham group. Finally, PTENP1 inhibited miR-21 and miR-19b expression but upregulated PTEN expression. The upregulation of miR-21/miR-19b also suppressed the apoptosis of neuron cells by downregulating the PTEN expression. PTENP1 is involved in the recovery of SCI by regulating the expression of miR-19b and miR-21, and exosomes from PTENP1-shRNA-transfected cells may be used as a novel biomarker in SCI treatment.