miR-424-5p regulates apoptosis and cell proliferation via targeting Bcl2 in nucleus pulposus cells

ABSTRACT

miRNAs play an important role in the pathogenesis of intervertebral disc degeneration (IDD). The role and the underlying mechanism of miR-424-5p in human nucleus pulposus (NP) are still unknown. We aimed to explore the role of miR-424-5p in IDD.

Real-time PCR was used to detect the expression of miR-424-5p and Bcl2 in IDD tissues and idiopathic scoliosis tissues. Human NP cells were used in our study. MTT and Hoechst apoptosis assays were used to detect the proliferation and apoptosis of NP cells, respectively. Western blotting assays were used to detect the expression levels of Bcl-2, cleaved caspase-3, cleaved caspase-9, caspase-3 and caspase-9 in degenerative NP cells. A luciferase reporter assay was applied to confirm the relationship between miR-424-5p and Bcl2.

Our results showed that the expression of miR-424-5p was increased and Bcl2 was decreased in degenerative NP cells. miR-425-5p expression was negatively correlated with Bcl2 expression in IDD tissues. Suppression of miR-424-5p using an inhibitor increased Bcl2 expression at both the mRNA and protein levels, and it promoted cell viability and inhibited apoptosis. Furthermore, the levels of cleaved caspase-3 and cleaved caspase-9 were downregulated in miR-424-5p-silenced NP cells. Interestingly, we found that silencing miR-424-5p increased p62 expression at both the mRNA and protein levels. Finally, a luciferase reporter assay verified the binding of the miR-424-5p and the 3’UTR of Bcl2.

These results suggested that silencing miR-424-5p suppressed NP cell apoptosis by upregulating Bcl2. Therefore, miR-424-5p might be a novel target for IDD therapies.     

MicroRNA-15b silencing inhibits IL-1β-induced extracellular matrix degradation by targeting SMAD3 in human nucleus pulposus cells

Objectives

To determine the role of microRNA-15b (miR-15b) in interleukin-1 beta (IL-1β)-induced extracellular matrix (ECM) degradation in the nucleus pulposus (NP).

Results

MiR-15b was up-regulated in degenerative NP tissues and in IL-1β-stimulated NP cells, as compared to the levels in normal controls (normal tissue specimens from patients with idiopathic scoliosis). Bioinformatics and luciferase activity analyses showed that mothers against decapentaplegic homolog 3 (SMAD3), a key mediator of the transforming growth factor-β signaling pathway, was directly targeted by miR-15b. Functional analysis demonstrated that miR-15b overexpression aggravated IL-1β-induced ECM degradation in NP cells, while miR-15b inhibition had the opposite effects. Prevention of IL-1β-induced NP ECM degeneration by the miR-15b inhibitor was attenuated by small-interfering-RNA-mediated knockdown of SMAD3. In addition, activation of MAP kinase and nuclear factor-κB up-regulated miR-15b expression and down-regulated SMAD3 expression in IL-1β-stimulated NP cells.

Conclusions

MiR-15b contributes to ECM degradation in intervertebral disc degeneration (IDD) via targeting of SMAD3, thus providing a novel therapeutic target for IDD treatment.

     

miR-154 suppresses colorectal cancer cell growth and motility by targeting TLR2

MicroRNAs play critical roles in the development and progression of colorectal cancer (CRC). miR-154 acts as a tumor suppressor in several tumors; however, its role in CRC is poorly understood. Herein, we found that miR-154 was decreased in CRC tissues and cell lines. Ectopic expression of miR-154 remarkably suppressed cell proliferation and colony formation, migration and invasion in CRC cells. The toll-like receptor 2 (TLR2) was found to be a direct target of miR-154 in CRC cells. Inhibition of TLR2 performed similar effects with miR-154 overexpression on CRC cells, and overexpression of TLR2 could significantly reverse the tumor suppressive effects of miR-154 on CRC cells. This study suggests an essential role for miR-154 in CRC.     

miR-195 Inhibits EMT by Targeting FGF2 in Prostate Cancer Cells

Prostate cancer (PCa) is one of the leading causes of deaths in America. The major cause of mortality can be attributed to metastasis. Cancer metastasis involves sequential and interrelated events. miRNAs and epithelial-mesenchymal transition (EMT) are implicated in this process. miR-195 is downregulated in many human cancers. However, the roles of miR-195 in PCa metastasis and EMT remain unclear. In this study, data from Memorial Sloan Kettering Cancer Center (MSKCC) prostate cancer database were re-analysed to detect miR-195 expression and its roles in PCa. miR-195 was then overexpressed in castration-resistant PCa cell lines, DU-145 and PC-3. The role of miR-195 in migration and invasion in vitro was also investigated, and common markers in EMT were evaluated through Western blot analysis. A luciferase reporter assay was conducted to confirm the target gene of miR-195; were validated in PCa cells. In MSKCC data re-analyses, miR-195 was poorly expressed in metastatic PCa; miR-195 could be used to diagnose metastatic PCa by measuring the corresponding expression. Area under the receiver operating characteristic curve (AUC-ROC) was 0.705 (P = 0.017). Low miR-195 expression was characterised with a shorter relapse-free survival (RFS) time. miR-195 overexpression suppressed cell migration, invasion and EMT. Fibroblast growth factor 2 (FGF2) was confirmed as a direct target of miR-195. FGF2 knockdown also suppressed migration, invasion and EMT; by contrast, increased FGF2 partially reversed the suppressive effect of miR-195. And data from ONCOMINE prostate cancer database showed that PCa patients with high FGF2 expression showed shorter RFS time (P = 0.046). Overall, this study demonstrated that miR-195 suppressed PCa cell metastasis by downregulating FGF2. miR-195 restoration may be considered as a new therapeutic method to treat metastatic PCa.     

Propofol suppresses proliferation,invasion, and migration of human melanoma cells via regulating microRNA-137 and fibroblast growth factor 9

Propofol is an intravenous anesthetic widely used in clinical surgeries, such as tumor resection. Propofol affects the growth of many cancers, though its effect on melanoma is unknown. Our study aimed to explore how propofol affects melanoma cells. Melanoma cells A2058 and WM793B were cultured with propofol for 24 hr. Propofol significantly suppressed proliferation, migration, and invasion of A2058 and WM793B cells. Lower miR-137 level was observed in A2058 and WM793B cells, compared with normal human epidermal melanocyte HEMa-LP cells. Propofol-induced miR-137 upregulation and decreased proliferation, invasive ability, and migrated ability of A2058 and WM793B cells. Transfection with the miR-137 inhibitor reversed these effects. Additionally, miR-137 was verified to target and negatively regulate fibroblast growth factor 9 (FGF9) expression. Propofol efficiently downregulated FGF9 protein expression by upregulating miR-137. Furthermore, FGF9 overexpression abrogated propofol's repressive effects on the malignant potential of A2058 and WM793B cells. These findings indicate that propofol suppressed melanoma cell proliferation, invasion, and migration by regulating miR-137 and FGF9.     

LncRNA TUG1 regulates FGF1 to enhance endothelial differentiation of adipose-derived stem cells by sponging miR-143

Adipose-derived stem cells (ADSCs) have emerged as a cell source for regeneration medicine. ADSCs possess the capacity to differentiate into endothelial cells and serve an essential role in vascular development and function. LncRNA taurine upregulated gene 1 (TUG1) has recently been linked with angiogenesis in hepatoblastoma. However, the roles of TUG1 in endothelial differentiation of ADSCs remain unidentified. Human adipose-derived stem cells (hADSCs) were obtained and characterized by flow cytometry, Oil red O and Alizarin Red staining. HADSCs were maintained in the endothelial differentiation medium and the expressions of TUG1, miR-143, and FGF1 were examined by qRT-PCR. To assess endothelial differentiation, the expressions of CD31, von Willebrand factor (vWF), VE-cadherin were examined by Western blot analysis, qRT-PCR, and immunofluorescence. Tube formation in Matrigel was examined. The interactions between TUG1 and miR-143, miR-143 and FGF1 were validated by luciferase assays. During the endothelial differentiation process, TUG1 and FGF1 were upregulated, whereas miR-143 was downregulated. TUG1 overexpression downregulated miR-143, upregulated FGF1, CD31, vWF, and VE-cadherin, and enhanced capillary tube formation. Luciferase assays showed that TUG1 interacted with miR-143, and FGF1 was a direct target of miR-143. Furthermore, the enhancement of endothelial differentiation induced by TUG1 overexpression was abolished by miR-143 overexpression. Our findings implicated that lncRNA TUG1 promoted endothelial differentiation of ADSCs by regulating the miR-143/FGF1 axis.     

LncRNA LINC00324 is upregulated in intervertebral disk degeneration and upregulates FasL in nucleus pulposus cells

Background

It has been reported that long intergenic non-protein-coding RNA 324 (LINC00324) promotes liver cancer by upregulating Fas ligand (FasL), which is a major player in intervertebral disk degeneration (IDD), indicating the involvement of LINC00324 in IDD. This study was carried out to investigate the interaction between LINC00324 and FasL in IDD.

Methods

Plasma samples were collected from both IDD (n?=?60) and healthy controls (n?=?60). The expression of LINC00324 and FasL in plasma was determined by RT-qPCR. The interactions between LINC00324 and FasL in nucleus pulposus (NP) cells were analyzed by overexpression experiments.

Results

LINC00324 and FasL were upregulated in IDD patients, and they were positively correlated. After treatment, the expression levels of FasL and LINC00324 were significantly decreased. In NP cells, overexpression of LINC00324 increased the expression of FasL at both mRNA and protein levels, while overexpression of FasL did not affect the expression of LINC00324.

Conclusion

LINC00324 may upregulate FasL in IDD to promote disease progression.

     

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.     

SKI knockdown suppresses apoptosis and extracellular matrix degradation of nucleus pulposus cells via inhibition of the Wnt/β-catenin pathway and ameliorates disc degeneration

This study aimed to determine the effects of SKI on interleukin (IL)-1β-induced apoptosis of nucleus pulposus (NP) cells, intervertebral disc degeneration (IDD), and the Wnt signaling pathway. NP tissue specimens of different Pfirrmann grades (II–V) were collected from patients with different grades of IDD. Real-time polymerase chain reaction and western blotting were used to compare SKI mRNA and protein expression in NP tissues from patients. Using the IL-1β-induced IDD model, NP cells were infected with lentivirus-coated si-SKI to downregulate the expression of SKI and treated with LiCl to evaluate the involvement of the Wnt/β-catenin signaling pathway. Western blotting, immunofluorescence, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were used to detect NP cell apoptosis, extracellular matrix (ECM) metabolism, and related protein expression changes in the Wnt/β-catenin signaling pathway. To investigate the role of SKI in vivo, a rat IDD model was established by needle puncture of the intervertebral disc. Rats were injected with lentivirus-coated si-SKI and evaluated by magnetic resonance imaging (MRI), and hematoxylin and eosin (HE) and safranin O staining. SKI expression positively correlated with the severity of human IDD. In the IL-1β-induced NP cell degeneration model, SKI expression increased significantly and reached a peak at 24 h. SKI knockdown protected against IL-1β-induced NP cell apoptosis and ECM degradation. LiCl treatment reversed the protective effects of si-SKI on NP cells. Furthermore, lentivirus-coated si-SKI injection partially reversed the NP tissue damage in the IDD model in vivo. SKI knockdown reduced NP cell apoptosis and ECM degradation by inhibiting the Wnt/β-catenin signaling pathway, ultimately protecting against IDD. Therefore, SKI may be an effective target for IDD treatment.

     

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.     

miR-26a attenuates cardiac apoptosis and fibrosis by targeting ataxia–telangiectasia mutated in myocardial infarction

Apoptosis and fibrosis play a vital role in myocardial infarction (MI) induced tissue injury. Although microRNAs have been the focus of many studies on cardiac apoptosis and fibrosis in MI, the detailed effects of miR-26a is needed to further understood. The present study demonstrated that miR-26a was downregulated in ST-elevation MI (STEMI) patients and oxygen-glucose deprivation (OGD)-treated H9c2 cells. Downregulation of miR-26a was closely correlated with the increased expression of creatine kinase, creatine kinase-MB and troponin I in STEMI patients. Further analysis identified that ataxia–telangiectasia mutated (ATM) was a target gene for miR-26a based on a bioinformatics analysis. miR-26a overexpression effectively reduced ATM expression, apoptosis, and apoptosis-related proteins in OGD-treated H9c2 cells. In a mouse model of MI, the expression of miR-26a was significantly decreased in the infarct zone of the heart, whereas apoptosis and ATM expression were increased. miR-26a overexpression effectively reduced ATM expression and cardiac apoptosis at Day 1 after MI. Furthermore, we demonstrated that overexpression of miR-26a improved cardiac function and reduced cardiac fibrosis by the reduced expression of collagen type I and connective tissue growth factor (CTGF) in mice at Day 14 after MI. Overexpression of miR-26a or ATM knockdown decreased collagen I and CTGF expression in cultured OGD-treated cardiomyocytes. Taken together, these data demonstrate a prominent role for miR-26a in linking ATM expression to ischemia-induced apoptosis and fibrosis, key features of MI progression. miR-26a reduced MI development by affecting ATM expression and could be targeted in the treatment of MI.     

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.     

MicroRNA-370 carried by M2 macrophage-derived exosomes alleviates asthma progression through inhibiting the FGF1/MAPK/STAT1 axis

Emerging evidence has suggested the functions of exosomes in allergic diseases including asthma. By using a mouse model with asthma induced by ovalbumin (OVA), we explored the roles of M2 macrophage-derived exosomes (M2Φ-Exos) in asthma progression. M2Φ-Exos significantly alleviated OVA-induced fibrosis and inflammatory responses in mouse lung tissues, as well as inhibited abnormal proliferation, invasion, and fibrosis-related protein production in platelet derived growth factor (PDGF-BB) treated primary mouse airway smooth muscle cells (ASMCs). The OVA administration in mice or the PDGF-BB treatment in ASMCs reduced the expression of miR-370, which was detected in M2Φ-Exos by miRNA sequencing. However, treating the mice or ASMCs with M2Φ-Exos reversed the inhibitory effect of OVA or PDGF-BB on miR-370 expression. We identified that the target of miR-370 was fibroblast growth factor 1 (FGF1). Downregulation of miR-370 by Lv-miR-370 inhibitor or overexpression of FGF1 by Lv-FGF1 blocked the protective roles of M2Φ-Exos in asthma-like mouse and cell models. M2Φ-Exos were found to inactivate the MAPK signaling pathway, which was recovered by miR-370 inhibition or FGF1 overexpression. Collectively, we conclude that M2Φ-Exos carry miR-370 to alleviate asthma progression through downregulating FGF1 expression and the MAPK/STAT1 signaling pathway. Our study may offer a novel insight into asthma treatment.     

Sprouty1 regulates neuritogenesis and survival of cortical neurons

In multicellular organisms, receptor tyrosine kinases (RTKs) control a variety of cellular processes, including cell proliferation, differentiation, migration, and survival. Sprouty (SPRY) proteins represent an important class of ligand-inducible inhibitors of RTK-dependent signaling pathways. Here, we investigated the role of SPRY1 in cells of the central nervous system (CNS). Expression of SPRY1 was substantially higher in neural stem cells than in cortical neurons and was increased during neuronal differentiation of cortical neurons. We found that SPRY1 was a direct target gene of the CNS-specific microRNA, miR-124 and miR-132. In primary cultures of cortical neurons, the neurotrophic factors brain-derived neurotrophic factor (BDNF) and Basic fibroblast growth factor (FGF2) downregulated SPRY1 expression to positively regulate their own functions. In immature cortical neurons and mouse N₂A cells, we found that overexpression of SPRY1 inhibited neurite development, whereas knockdown of SPRY1 expression promoted neurite development. In mature neurons, overexpression of SPRY1 inhibited the prosurvival effects of both BDNF and FGF2 on glutamate-mediated neuronal cell death. SPRY1 was also upregulated upon glutamate treatment in mature neurons and partially contributed to the cytotoxic effect of glutamate. Together, our results indicate that SPRY1 contributes to the regulation of CNS functions by influencing both neuronal differentiation under normal physiological processes and neuronal survival under pathological conditions.     

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.     

Bu-Shen-Huo-Xue-Fang modulates nucleus pulposus cell proliferation and extracellular matrix remodeling in intervertebral disk degeneration through miR-483 regulation of Wnt pathway

Intervertebral disk degeneration (IDD) has been widely considered as one of the main causes for low back pain, which can cause a severe impact to human health and huge economic burden to worldwide society. IDD pathogenesis can be affected by extensive degradation of extracellular matrix (ECM) and the hyperproliferation of nucleus pulposus (NP) cells. During the IDD process, expression of the ECM degradation enzymes matrix metalloproteinase and ADAMTS increases, whereas expression of ECM synthesis–related aggrecan and COL2A1 decreases. In addition, the Wnt signaling pathway is reportedly involved in the process of IDD. Bu-Shen-Huo-Xue-Fang (BSHXF), a Chinese traditional medicine formula that contains six Chinese traditional medicinal herbs, is widely used in the treatment of IDD. Herein, we obtained the serum containing BSHXF from BSHXF-fed rat and demonstrated that the BSHXF promoted NP cell proliferation and ECM synthesis through the Wnt signaling pathway. By using DIANA online tools and luciferase reporter gene assays, we confirmed that miR-483-3p and miR-23c regulated CTNNB1 and GSK3B, respectively, through direct targeting, thereby affecting the effect of BSHXF on NP cell proliferation and ECM synthesis through the Wnt signaling pathway. Taken together, we demonstrated the function and mechanism of BSHXF in regulating NP cell proliferation and ECM remodeling through the Wnt signaling pathway during IDD.