Long non‐coding RNAs in nucleus pulposus cell function and intervertebral disc degeneration

Intervertebral disc degeneration (IDD) is the major cause of low back pain which incurs a significant public‐health and economic burden. The aetiology of IDD is complex, with developmental, genetic, biomechanical and biochemical factors contributing to the disease development. Deregulated phenotypes of nucleus pulposus cells, including aberrant differentiation, apoptosis, proliferation and extracellular matrix deposition, are involved in the initiation and progression of IDD. Non‐coding RNAs, including long non‐coding RNAs (lncRNAs), have recently been identified as important regulators of gene expression. Research into their roles in IDD has been very active over the past 5 years. Our review summarizes current research regarding the roles of deregulated lncRNAs (eg, RP11‐296A18.3, TUG1, HCG18) in modulating nucleus pulposus cell functions in IDD. These exciting findings suggest that specific modulation of lncRNAs or their downstream signalling pathways might be an attractive approach for developing novel therapeutics for IDD.     

MicroRNA in intervertebral disc degeneration

Aetiology of intervertebral disc degeneration (IDD) is complex, with genetic, developmental, biochemical and biomechanical factors contributing to the disease process. It is becoming obvious that epigenetic processes influence evolution of IDD as strongly as the genetic background. Deregulated phenotypes of nucleus pulposus cells, including differentiation, migration, proliferation and apoptosis, are involved in all stages of progression of human IDD. Non‐coding RNAs, including microRNAs, have recently been recognized as important regulators of gene expression. Research into roles of microRNAs in IDD has been very active over the past 5 years. Our review summarizes current research enlightenment towards understanding roles of microRNAs in regulating nucleus pulposus cell functions in IDD. These exciting findings support the notion that specific modulation of microRNAs may represent an attractive approach for management of IDD.     

Spermidine promotes nucleus pulposus autophagy as a protective mechanism against apoptosis and ameliorates disc degeneration

Spermidine has therapeutic effects in many diseases including as heart diastolic function, myopathic defects and neurodegenerative disorders via autophagy activation. Autophagy has been found to mitigate cell apoptosis in intervertebral disc degeneration (IDD). Accordingly, we theorize that spermidine may have beneficial effects on IDD via autophagy stimulation. In this study, spermidine's effect on IDD was evaluated in tert‐butyl hydroperoxide (TBHP)‐treated nucleus pulposus cells of SD rats in vitro as well as in a puncture‐induced rat IDD model. We found that autophagy was actuated by spermidine in nucleus pulposus cells. In addition, spermidine treatment weakened the apoptotic effects of TBHP in nucleus pulposus cells. Spermidine increased the expression of anabolic proteins including Collagen‐II and aggrecan and decreased the expression of catabolic proteins including MMP13 and Adamts‐5. Additionally, autophagy blockade using 3‐MA reversed the beneficial impact of spermidine against nucleus pulposus cell apoptosis. Autophagy was thus important for spermidine's therapeutic effect on IDD. Spermidine‐treated rats had an accentuated T2‐weighted signal and a diminished histological degenerative grade than vehicle‐treated rats, showing that spermidine inhibited intervertebral disc degeneration in vivo. Thus, spermidine protects nucleus pulposus cells against apoptosis through autophagy activation and improves disc, which may be beneficial for the treatment of IDD.     

Upregulated miR-154 promotes ECM degradation in intervertebral disc degeneration

Intervertebral disc degeneration (IDD), a common global health issue, is a major cause for low back pain (LBP). Given the complex etiology of IDD, micro RNA (miRNA) recently has been demonstrated to play essential roles in the progression of IDD. Therefore, this study aims to investigate functions of the miR-154, which is well-documented in a series of cell activities, IDD, and other relevant mechanisms. Lumbar nucleus pulposus (NP) samples were collected from patients with IDD and the control group. After solexa sequencing and bioinformatical analysis, the results showed that miR-154 was increased in NP cells of patients with IDD. Inhibition of miR-154 increased type II collagen and aggrecan and decreased mRNA expressions of collagenase-3 (MMP13) and aggrecanase-1 (ADAMTS4), whereas overexpression of miR-154 reversed such effects in NP cells. In addition, the luciferase reporter assay revealed that fibroblast growth factor 14 (FGF14) is a direct target of miR-154 and that the overexpression of FGF14 leads to similar effects as inhibition of miR-154 did. In conclusion, the results suggested that miR-154 participates in the development of IDD and its effects are mediated via targeting FGF14. Thus, miR-154 may be thought as a potential etiological factor for IDD and may provide insights into a therapeutic target to treat IDD.     

MicroRNA-10b Promotes Nucleus Pulposus Cell Proliferation through RhoC-Akt Pathway by Targeting HOXD10 in Intervetebral Disc Degeneration

Aberrant proliferation of nucleus pulposus cell is implicated in the pathogenesis of intervertebral disc degeneration. Recent findings revealed that microRNAs, a class of small noncoding RNAs, could regulate cell proliferation in many pathological conditions. Here, we showed that miR-10b was dramatically upregulated in degenerative nucleus pulposus tissues when compared with nucleus pulposus tissues isolated from patients with idiopathic scoliosis. Moreover, miR-10b levels were associated with disc degeneration grade and downregulation of HOXD10. In cultured nucleus pulposus cells, miR-10b overexpression stimulated cell proliferation with concomitant translational inhibition of HOXD10 whereas restored expression of HOXD10 reversed the mitogenic effect of miR-10b. MiR-10b-mediated downregulation of HOXD10 led to increased RhoC expression and Akt phosphorylation. Either knockdown of RhoC or inhibition of Akt abolished the effect of miR-10b on nucleus pulposus cell proliferation. Taken together, aberrant miR-10b upregulation in intervertebral disc degeneration could contribute to abnormal nucleus pulposus cell proliferation through derepressing the RhoC-Akt pathway by targeting HOXD10. Our study also underscores the potential of miR-10b and the RhoC-Akt pathway as novel therapeutic targets in intervertebral disc degeneration.     

Retracted: HO-1 overexpression alleviates senescence by inducing autophagy via the mitochondrial route in human nucleus pulposus cells

Intervertebral disc degeneration (IDD) is closely associated with aging. Our previous studies have confirmed that heme oxygenase-1 (HO-1) can inhibit nucleus pulposus (NP) cell apoptosis. However, whether or not HO-1 is involved in NP cell senescence and autophagy is unclear. Our results indicated that HO-1 expression was reduced in IDD tissues and replicative senescent NP cells. HO-1 overexpression using a lentiviral vector reduced the NP cell senescence level, protected mitochondrial function, and promoted NP cell autophagy through the mitochondrial pathway. Autophagy inhibitor 3-MA pretreatment reversed the anti-senescent and protective effects on the mitochondrial function of HO-1, which promoted the degradation of the extracellular matrix (ECM) in the intervertebral disc. In vivo, HO-1 overexpression inhibited IDD and enhanced autophagy. In summary, these results suggested that HO-1 overexpression alleviates NP cell senescence by inducing autophagy via the mitochondrial route.     

小鼠正常黑素细胞和小鼠B16黑素瘤细胞的环状RNAs表达谱

黑素瘤是一种多发于皮肤的恶性肿瘤,因其侵袭性强,预后差等特点一直是科研人员关注的热点。环状RNAs(circRNAs)是一种新型内源性非编码RNA,广泛参与动物生长发育、细胞分化和信号转导等生理过程,但circRNAs在黑素瘤细胞内的分子机制尚未被充分解析。本研究以小鼠(C57BL/6J)正常黑素细胞及B16黑素瘤细胞为研究对象,采用二代测序技术分析两种细胞间circRNAs表达特性。测序结果显示,小鼠正常黑素细胞和黑素瘤细胞中共有851个circRNAs,其中195个差异表达circRNAs(DECs)。GO及KEGG数据库注释发现,DECs的来源基因主要参与细胞周期(cell cycle)、紧密结合(tight junction)、Rap1信号通路(Rap1 signaling pathway)、TGF-beta信号通路(TGF-beta signaling pathway)等与细胞增殖、迁移相关的信号通路;探究发现,黑素瘤细胞中显著性高表达的circE2F5(circ-3:14578602|14606309)通过上调E2F5的表达促进黑素瘤细胞增殖。circRNA靶基因预测发现,...     

CircRNA: a rising star in plant biology

Circular RNAs (circRNAs) are covalently closed single-stranded RNA molecules, which are widespread in eukaryotic cells. As regulatory molecules, circRNAs have various functions, such as regulating gene expression, binding miRNAs or proteins, and being translated into proteins, which are important for cell proliferation and cell differentiation, individual growth and development, as well as many other biological processes. However, compared with that in animal models, studies of circRNAs in plants lags behind and, particularly, the regulatory mechanisms of biogenesis and molecular functions of plant circRNAs remain elusive. Recent studies have shown that circRNAs are wide spread in plants with tissue- or development-specific expression patterns and are responsive to a variety of environmental stresses. In this review, we summarize these advances, focusing on the regulatory mechanisms of biogenesis, molecular and biological functions of circRNAs, and the methods for investigating circRNAs. We also discuss the challenges and the prospects of plant circRNA studies.     

lncRNA HOTAIR upregulates autophagy to promote apoptosis and senescence of nucleus pulposus cells

Intervertebral disc degeneration (IDD) is a complex and chronic disease that involves disc cell senescence, death, and extracellular matrix (ECM) degradation. HOTAIR, a long non-coding RNA (lncRNA) is reportedly associated with autophagy, whereas autophagy is shown to promote IDD. However, how it affects nucleus pulposus (NP) cells, the primary component of intervertebral discs is still unclear. We hypothesized that HOTAIR promotes NP cell apoptosis and senescence through upregulating autophagy. Thus, silencing HOTAIR should inhibit autophagy and exert a therapeutic effect on IDD. Our in vitro experiments in human NP cells revealed that HOTAIR expression positively correlated with IDD grade, and overexpression enhanced autophagy. Autophagy inhibition via 3-methyladenine reversed HOTAIR stimulatory effects on apoptosis, senescence, and ECM catabolism, while the AMP-activated protein kinase (AMPK) inhibitor Compound C suppressed HOTAIR-induced autophagy through regulating AMPK/mTOR/ULK1 pathways. Our in vivo experiment then illustrated that silencing HOTAIR ameliorates IDD in rats. Collectively, we demonstrated that HOTAIR stimulates autophagy to promote NP cell apoptosis, senescence, and ECM catabolism. Therefore, silencing HOTAIR has the potential to become a treatment option for IDD.     

Down‐regulation of insulin‐like growth factor binding protein 5 is involved in intervertebral disc degeneration via the ERK signalling pathway

It is obvious that epigenetic processes influence the evolution of intervertebral disc degeneration (IDD). However, its molecular mechanisms are poorly understood. Therefore, we tested the hypothesis that IGFBP5, a potential regulator of IDD, modulates IDD via the ERK signalling pathway. We showed that IGFBP5 mRNA was significantly down‐regulated in degenerative nucleus pulposus (NP) tissues. IGFBP5 was shown to significantly promote NP cell proliferation and inhibit apoptosis in vitro, which was confirmed by MTT, flow cytometry and colony formation assays. Furthermore, IGFBP5 was shown to exert its effects by inhibiting the ERK signalling pathway. The effects induced by IGFBP5 overexpression on NP cells were similar to those induced by treatment with an ERK pathway inhibitor (PD98059). Moreover, qRT‐PCR and Western blot analyses were performed to examine the levels of apoptosis‐related factors, including Bax, caspase‐3 and Bcl2. The silencing of IGFBP5 up‐regulated the levels of Bax and caspase‐3 and down‐regulated the level of Bcl2, thereby contributing to the development of human IDD. Furthermore, these results were confirmed in vivo using an IDD rat model, which showed that the induction of Igfbp5 mRNA expression abrogated the effects of IGFBP5 silencing on intervertebral discs. Overall, our findings elucidate the role of IGFBP5 in the pathogenesis of IDD and provide a potential novel therapeutic target for IDD.     

Melatonin ameliorates intervertebral disc degeneration via the potential mechanisms of mitophagy induction and apoptosis inhibition

Intervertebral disc degeneration (IDD) is a complicated disease in patients. The pathogenesis of IDD encompasses cellular oxidative stress, mitochondrion dysfunction and apoptosis. Melatonin eliminates oxygen free radicals, regulates mitochondrial homoeostasis and function, stimulates mitophagy and protects against cellular apoptosis. Therefore, we hypothesize that melatonin has beneficial effect on IDD by mitophagy stimulation and inhibition of apoptosis. The effects of melatonin on IDD were investigated in vitro and in vivo. For the former, melatonin diminished cellular apoptosis caused by tert‐butyl hydroperoxide in nucleus pulposus (NP) cells. Mitophagy, as well as its upstream regulator Parkin, was activated by melatonin in both a dose and time‐dependent manner. Mitophagy inhibition by cyclosporine A (CsA) partially eliminated the protective effects of melatonin against NP cell apoptosis, suggesting that mitophagy is involved in the protective effect of melatonin on IDD. In addition, melatonin was demonstrated to preserve the extracellular matrix (ECM) content of Collagen II, Aggrecan and Sox‐9, while inhibiting the expression of matrix degeneration enzymes, including MMP‐13 and ADAMTS‐5. In vivo, our results demonstrated that melatonin treatment ameliorated IDD in a puncture‐induced rat model. To conclude, our results suggested that melatonin protected NP cells against apoptosis via mitophagy induction and ameliorated disc degeneration, providing the potential therapy for IDD.     

Therapeutic potential of melatonin in the intervertebral disc degeneration through inhibiting the ferroptosis of nucleus pulpous cells

Ferroptosis, a novel type of cell death mediated by the iron-dependent lipid peroxidation, contributes to the pathogenesis of the intervertebral disc degeneration (IDD). Increasing evidence demonstrated that melatonin (MLT) displayed the therapeutic potential to prevent the development of IDD. Current mechanistic study aims to explore whether the downregulation of ferroptosis contributes to the therapeutic capability of MLT in IDD. Current studies demonstrated that conditioned medium (CM) from the lipopolysaccharide (LPS)-stimulated macrophages caused a series of changes about IDD, including increased intracellular oxidative stress (increased reactive oxygen species and malondialdehyde levels, but decreased glutathione levels), upregulated expression of inflammation-associated factors (IL-1β, COX-2 and iNOS), increased expression of key matrix catabolic molecules (MMP-13, ADAMTS4 and ADAMTS5), reduced the expression of major matrix anabolic molecules (COL2A1 and ACAN), and increased ferroptosis (downregulated GPX4 and SLC7A11 levels, but upregulated ACSL4 and LPCAT3 levels) in nucleus pulposus (NP) cells. MLT could alleviate CM-induced NP cell injury in a dose-dependent manner. Moreover, the data substantiated that intercellular iron overload was involved in CM-induced ferroptosis in NP cells, and MLT treatment alleviated intercellular iron overload and protected NP cells against ferroptosis, and those protective effects of MLT in NP cells further attenuated with erastin and enhanced with ferrostatin-1(Fer-1). This study demonstrated that CM from the LPS-stimulated RAW264.7 macrophages promoted the NP cell injury. MLT alleviated the CM-induced NP cell injury partly through inhibiting ferroptosis. The findings support the role of ferroptosis in the pathogenesis of IDD, and suggest that MLT may serve as a potential therapeutic approach for clinical treatment of IDD.     

Inhibition of TNFR1 Attenuates LPS Induced Apoptosis and Inflammation in Human Nucleus Pulposus Cells by Regulating the NF-KB and MAPK Signalling Pathway

Neurochemical Research - Intervertebral disc degeneration (IDD) is accompanied by nucleus pulposus (NP) cell apoptosis, inflammation, and extracellular matrix degradation. Tumour necrosis factor...     

Nucleus pulposus related lncRNA and mRNA expression profiles in intervertebral disc degeneration

In the present study, we aimed to have a comprehensive understanding of nucleus pulposus related long noncoding RNA (lncRNA) and mRNA expression profiles in intervertebral disc degeneration (IDD). In total, 2418 mRNAs and 528 lncRNAs were found to be differentially expressed in the IDD group compared with the Control group. Combining microarray datasets and sequencing data, 5 overlapping DEMs and 7 overlapping DELs were identified. NF-κB signaling pathway, PI3K-Akt signaling pathway and Wnt/β-catenin signaling pathway were strongly linked with enriched GO terms and KEGG pathways. The ceRNA network suggested that lnc-TMEM44-AS1-hsa-miR-206-HDAC4 may be one crucial axis in IDD. PPI network analysis was constructed with 309 nodes and 129 edges. And the highest connectivity degrees were ALB, APOB and CCL2. This study suggested that specific lncRNAs and ceRNA axes may be crucial in the development of IDD. It provides a new perspective for delaying IDD process and enhancing intervertebral disc repair.     

Profiling and integrated analysis of differentially expressed circRNAs in cervical cancer

Circular RNAs (circRNAs) are a new type of regulatory RNAs, which have been identified to play critical role in various tumors. However, the profiles and roles of circRNAs in cervical cancer (CCa) have not been fully understood and need to be further explored. In the present study, we performed circRNA array and mRNA-sequencing (mRNA-Seq) to profile the differentially expressed circRNAs and mRNAs in CCa tissues. A total of 397 differentially expressed circRNAs and 2138 differentially expressed mRNAs were detected, respectively. Subsequently, a circRNA-miRNA-mRNA regulatory network was constructed and indicated that hsa_circ_0026377 was downregulated in CCa. Overexpression of hsa_circ_0026377 inhibited HeLa and SiHa cells proliferation, migration and invasion. Collectively, this study provided new insights into the circRNA profiles in CCa and suggested that hsa_circ_0026377 might play important roles in CCa development.     

Circular RNAs Co-Precipitate with Extracellular Vesicles: A Possible Mechanism for circRNA Clearance

Backspliced circular RNAs (circRNAs) are prevalent in many eukaryotic systems and are spliced from thousands of different genes. Where examined, circRNAs are often highly stable and the mechanisms by which cells degrade and/or clear circRNAs from the cells are unknown. Here we investigated the possibility that cells can eliminate circRNAs into extracellular space, possibly within released vesicles such as exosomes and microvesicles. From three different cell lines and examining multiple circRNAs, we show that extracellular vesicle (EVs) preparations recovered from cell culture conditioned media contain established circRNAs. Moreover, these circRNAs are enriched over their linear counterparts within EV preparations when compared to the producing cells. This supports the idea that expulsion from cells into extracellular space, as by EVs release, can be a mechanism by which cells clear circRNAs. Moreover, since EVs can be taken up by other cells, excreted circRNAs could contribute to cell to cell communication.