Dexamethasone (Dex) is a prescribed medicine for the patients with recurrent and persistent inflammatory diseases, cancers, and autoimmune diseases . Excessive and long-term Dex exposure can lead to osteoporosis [2,3] or even osteonecrosis . Dex treatment invitro induces osteoblast apoptosis and necrosis [, , , ]. Our group [8,9] is dedicated to exploring the underlying mechanisms of Dex-induced osteoblast injury, and to developing possible intervention strategies [, , , ].
Long non-coding RNAs (LncRNAs) are a large family of RNA molecules over 200 nucleotide [, , ]. LncRNAs can alter protein expression and/or functions in a number of different ways [, , ]. Myc is a key transcriptional regulator with roles in various aspects of cell behaviors, including cell survival, proliferation, and apoptosis-resistance [13,14]. It has been proposed that Myc is important for osteoblast cell survival . A very recent study shows that LncRNA EPIC1 (“Lnc-EPIC1”, ENSG00000224271) physically interacts with Myc through Lnc-EPIC1's 129–283 nucleotide region . Silencing of Lnc-EPIC1 by targeted siRNA inhibited Myc-mediated expression to its target genes, including CDC20, and CDC45 and CCNA2 . Myc depletion abolished Lnc-EPIC1's actions . Here, our study shows that Lnc-EPIC1 expression protects human osteoblasts from Dex via regulation of Myc.
Chemicals and reagents
Dexamethasone (Dex), puromycin, MTT (3-[4,5-dimethylthylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) and JC-1 dye were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies of tubulin, cyclophilin D (CyPD), cytochrome C (Cyto-C), adenine nucleotide translocator-1 (ANT1), voltage-dependent anion channel (VDAC) and rabbit/mouse IgG-horseradish peroxidase (IgG-HRP) were provided by Santa Cruz Biotechnology (Santa Cruz, CA). All other antibodies were provided by Cellular Signaling Tech
Lnc-EPIC1 overexpression protects OB-6 osteoblastic cells from dexamethasone
We first tested the potential effect of dexamethasone (Dex) on Lnc-EPIC1 expression. In OB-6 human osteoblastic cells, Dex treatment (1 μM, for 24 h) increased the Lnc-EPIC1 expression by over one fold (Fig.1A). These results indicate that Lnc-EPIC1 might play a role in Dex-induced cytotoxicity. To test this hypothesis, a lentiviral Lnc-EPIC1 expression construct (“LV-EPIC1”, see Methods) was transfected to OB-6 cells. After selection by puromycin, two lines of stable OB-6 cells with LV-EPIC1
Sustained and excessive Dex exposure can result in osteoporosis [2,3] or even osteonecrosis , which is partially due to apoptosis of osteoblasts. Decreased number of osteoblasts, inhibited osteoblastogenesis and increased osteoblast cell apoptosis/necrosis are detected in the bones of Dex-taking patients [2,3]. Treatment with Dex to the cultured osteoblasts invitro could induce cell apoptosis and necrosis [28,29]. Our group [8,9] is focusing on the pathological mechanisms of Dex-induced
The study was supported by the National Natural Science Foundation of China (81171712). Grants from Natural Science Foundation of Jiangsu Province (BK20151213) and Innovation Project of Jiangsu Province (BK 201423 and BK20160340). The authors have no conflict of interests.
MAFG-driven osteosarcoma cell progression is inhibited by a novel miRNA miR-4660
2021, Molecular Therapy - Nucleic Acids
Osteosarcoma (OS) is the most common primary bone malignancy in the adolescent population. MAFG (v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog G) forms a heterodimer with Nrf2 (NF-E2-related factor 2), binding to antioxidant response element (ARE), which is required for Nrf2 signaling activation. We found that MAFG mRNA and protein expression is significantly elevated in human OS tissues as well as in established and primary human OS cells. In human OS cells, MAGF silencing or knockout (KO) largely inhibited OS cell growth, proliferation, and migration, simultaneously inducing oxidative injury and apoptosis activation. Conversely, ectopic overexpression of MAFG augmented OS cell progression invitro. MicroRNA-4660 (miR-4660) directly binds the 3′ untranslated region (UTR) of MAFG mRNA in the cytoplasm of OS cells. MAFG 3′ UTR luciferase activity and expression as well as OS cell growth were largely inhibited with forced miR-4660 overexpression but augmented with miR-4660 inhibition. Invivo, MAGF short hairpin RNA (shRNA) or forced overexpression of miR-4660 inhibited subcutaneous OS xenograft growth in severe combined immunodeficient mice. Furthermore, MAFG silencing or miR-4660 overexpression inhibited OS xenograft in situ growth in proximal tibia of the nude mice. In summary, MAFG overexpression-driven OS cell progression is inhibited by miR-4660. The miR-4660-MAFG axis could be novel therapeutic target for human OS.
LncRNA EPIC1 promotes tumor angiogenesis via activating the Ang2/Tie2 axis in non-small cell lung cancer
2021, Life Sciences
Non-small cell lung cancer (NSCLC) is considered a highly fatal tumor. Importantly, angiogenesis is critical for tumor progression. Long non-coding RNAs (lncRNAs), which are untranslatable, control cell functions through different pathways. lncRNA EPIC1 has been reported to promote cell viability, cell cycle progression, and invasion. However, the relationship between EPIC1 and tumor angiogenesis remains an enigma. We explored the role of EPIC1 in tumor angiogenesis in NSCLC.
First, EPIC1 expression was analyzed using the GEPIA database and was further verified using qPCR in tumor tissues from patients with NSCLC and NSCLC cell lines. Next, EPIC1 function was detected using loss-of-function and gain-of-function assays. Moreover, EdU staining, flow cytometry, and channel formation assays were performed to assess HUVEC proliferation and channel the formation in the NSCLC-HUVEC transwell co-culture system.
EPIC1 expression was significantly upregulated in NSCLC tissues and cell lines. Furthermore, the overexpression of EPIC1 in NSCLC cells stimulated HUVEC channel formation and proliferation by activating Ang2/Tie2 signaling, and the opposite results were obtained when EPIC1 was silenced in NSCLC cells. The density of new blood vessels was simultaneously increased by EPIC1 overexpression in vivo, using CAM angiogenesis model and a nude mouse tumor model. Finally, all these experimental findings could be established in the samples from patients with NSCLC. We postulate that EPIC1 promotes tumor angiogenesis by activating the Ang2/Tie2 axis in NSCLC.
Elucidating the molecular and cellular mechanisms of EPIC1 in tumor angiogenesis provides a novel perspective on NSCLC clinical therapy.
Dysregulation of cPWWP2A-miR-579 axis mediates dexamethasone-induced cytotoxicity in human osteoblasts
2019, Biochemical and Biophysical Research CommunicationsSee Also2,3,7,8-Tetrachlorodibenzo-p-dioxin differentially suppresses angiogenic responses in human placental vein and artery endothelial cellsA synergistic negative effect of gestational smoke-exposure and small litter size on rat placental efficiency, vascularisation and angiogenic factors mRNA expression - Document
Citation Excerpt :
We show that relative miR-579 expression is significantly increased in the necrotic femoral head tissues (P < 0.05 vs. its levels in normal femoral head tissues, Fig. 1B). To explore the role of DEX on cPWWP2A expression in human osteoblasts, OB-6 osteoblastic cells [21,22] were treated with DEX (1 μM, the concentration was determined based on previous studies [10,11,14]). qPCR results confirmed that DEX downregulated cPWWP2A expression in a time-dependent manner (Fig. 1C).
Dexamethasone (DEX) induces significant cytotoxicity to human osteoblasts. cPWWP2A is recently-indentified novel circular RNA (circRNA), acting as an endogenous sponge of microRNA-579 (miR-579). The present study tested the expression and potential functions of the cPWWP2A-miR-579 axis in DEX-treated osteoblasts. We show that cPWWP2A is downregulated in the necrotic femoral head tissues of DEX-taking human patients as well as in DEX-treated human osteoblasts. In OB-6 osteoblastic cells and primary human osteoblasts ectopic overexpression of cPWWP2A potently inhibited DEX-induced miR-579 accumulation, cell death, apoptosis and programmed necrosis. Silencing miR-579, by targeted siRNAs, also attenuated DEX-induced cytotoxicity in human osteoblasts. Significantly, mimicking DEX-induced actions, cPWWP2A silencing or forced miR-579 overexpression induced significant cytotoxicity in human osteoblasts. Further analyses demonstrated that miR-579's targets, including SIRT1 and PDK1 (phosphoinositide-dependent protein kinase 1), were downregulated in DEX-treated osteoblasts. Their levels were decreased as well in the necrotic femoral head tissues of DEX-taking human patients. Taken together we show that dysregulation of the cPWWP2A-miR-579 axis is involved in DEX-induced cytotoxicity in human osteoblasts.
Dexamethasone-induced cytotoxicity in human osteoblasts is associated with circular RNA HIPK3 downregulation
2019, Biochemical and Biophysical Research Communications
Citation Excerpt :
Medium LDH percentage (vs. total LDH), indicating cell death intensity, was calculated [12,26]. Apoptosis assays, including the caspase-3 activity assay, the Histone DNA ELISA assay, and Annexin V FACS, were described in detail in our previous studies [12,28,29]. With mitochondrial depolarization JC-1 dye aggregates to form the green monomers .
Dexamethasone (DEX) exerts potent cytotoxicity against cultured human osteoblasts. The current study examined the role of the circular RNA HIPK3 (circHIPK3) in the mechanism of cell death. We found that circHIPK3 expression was downregulated in DEX-treated human osteoblasts and circHIPK3 levels decreased in human necrotic femoral head tissues. In OB-6 osteoblastic cells and primary human osteoblasts ectopic overexpression of circHIPK3 potently suppressed DEX-induced apoptosis and programmed necrosis. Conversely, knockdown of circHIPK3by targeted siRNAs enhanced DEX-induced cytotoxicity in human osteoblasts. We further observed that microRNA-124 (miR-124), a key miRNA sponged by circHIPK3, accumulated following DEX treatment in OB-6 cells and primary osteoblasts. Confirming the role of miR-124 in DEX-induced cytotoxicity, miR-124 inhibitor attenuated cell death in human osteoblasts. Conversely, forced overexpression of miR-124 mimicked DEX-induced actions and induced cytotoxicity in human osteoblasts. We conclude that DEX-induced cytotoxicity in human osteoblasts is associated with circHIPK3 downregulation.
Silencing FOXO1 attenuates dexamethasone-induced apoptosis in osteoblastic MC3T3-E1 cells
2019, Biochemical and Biophysical Research Communications
Dexamethasone (DEX), a widely used glucocorticoid with strong anti-inflammatory and immunosuppressive activities, has been reported to induce apoptosis in osteoblasts, but the underlying mechanisms are still not comprehensively investigated. FOXO1 plays an important role in the regulation of cell proliferation and apoptosis. Our study aims to explore the role of FOXO1 in DEX-induced apoptosis of osteoblastic MC3T3-E1 cells through bioinformatics and experiments. We first employed bioinformatics to identify DEX-related genes and revealed their functions by GO enrichment analysis including FOXO1 associated biological processes. Expression level of FOXO1 was validated by GEO data. Then, experiments were performed to verify the hypothesis. CCK8 was used to detect cell viability and apoptosis was detected by flow cytometry. SiRNA was used to silence FOXO1 and western-blot was employed to detect protein expression. Results demonstrated DEX-related genes involved in cell proliferation, apoptosis and angiogenesis and FOXO1 was a regulator of apoptosis. DEX could up-regulate FOXO1 expression, inhibit cell viability, promote apoptosis. SiRNA-FOXO1 could attenuate DEX-induced apoptosis in MC3T3-E1. These findings suggested DEX could affect some vital biological processes of MC3T3-E1 and FOXO1 played an essential role in DEX-induced apoptosis in MC3T3-E1.
Long non-coding RNA EPIC1 inhibits viability and invasion of osteosarcoma cells by promoting MEF2D ubiquitylation
2019, International Journal of Biological Macromolecules
Long non-coding RNAs (lncRNAs) can modulate gene expression through different mechanisms, but the fundamental molecular mechanism behind EPIC1 and osteosarcoma (OS) was poorly understood.
Bone tumor tissues and the matched normal tissues were obtained from 36 OS patients who received tumor resection from 2014 to 2018. The expression of EPIC1 and MEF2D was determined by quantitative real-Time PCR and western blotting. Cell viability and invasion were evaluated by MTT assay and transwell assay. The animal xenograft model was also established.
EPIC1 was down-regulated, but MEF2D was up-regulated in OS tissues and OS cell lines. Overexpression of EPIC1 inhibited cell viability and invasion of OS cells. Targeting relationship between EPIC1 and MEF2D was confirmed by RNA pull-down and RNA immunoprecipitation (RIP). The MEF2D protein binding to ubiquitin was significantly increased in OS cells overexpressing EPIC1. The co-transfection with pcDNA-EPIC1 and pcDNA-MEF2D rescued the inhibition of cell viability and invasion caused by the overexpression of EPIC1. Overexpression of EPIC1 suppressed tumor growth in the OS xenograft model.
Our findings indicated that overexpression of EPIC1 inhibited cell viability and invasion of OS cells by promoting MEF2D ubiquitylation, which provided innovative lncRNA and protein targets for treating OS.
Early emergence of de novo EGFR T790M gatekeeper mutations during erlotinib treatment in PC9 non-small cell lung cancer cells
Biochemical and Biophysical Research Communications, Volume 503, Issue 2, 2018, pp. 710-714
The emergence of the T790M gatekeeper mutation in the Epidermal Growth Factor Receptor (EGFR) gene is an important mechanism that can lead to the acquired resistance to EGFR-targeted tyrosine kinase inhibitors such as erlotinib or gefitinib. These drugs have been used in treating a subset of non-small cell lung cancer (NSCLC) patients harboring EGFR activating mutations. Here we investigated the paths leading to the acquisition of the T790M mutation by establishing an erlotinib resistant PC9 cell model harboring ectopically introduced EGFR cDNA. We detected the emergence of T790M mutation within the EGFR cDNA in a subset of erlotinib resistant PC9 cell models through Sanger sequencing and droplet digital PCR-based methods, demonstrating that T790M mutation can emerge via de novo events following treatment with erlotinib. In addition, we show that the de novo T790M bearing erlotinib resistant PC9 cells are sensitive to the 3rd generation EGFR-targeted drug, WZ4002. Furthermore, GFP-based competition cell proliferation assays reveal that PC9 cells ectopically expressing EGFR mutant become more rapidly resistant to erlotinib than parental PC9 cells through the acquisition of the T790M mutation. Taken together, we believe that our findings expand upon the previous notion of evolutionary paths of T790M development, providing an important clue to designing a therapeutic strategy to overcome drug resistance.
C1 Titanium Cables Combined with C2 Pedicle Screw-Rod Fixation for Atlantoaxial Instability Not Suitable for Placement of C1 Screws
World Neurosurgery, Volume 120, 2018, pp. e453-e456
We sought to develop a new posterior fusion technique composed of bilateral C1 titanium cables and C2 pedicle screw-rods for treatment of atlantoaxial instability not suitable for C1 screw placement.
A study was conducted of 18 patients with atlantoaxial instability who had C1 broken screw trajectory or anatomic anomalies. All patients underwent posterior fixation with bilateral C1 titanium cables and C2 pedicle screws. The follow-up period was a minimum 1 year. Clinical outcomes measurements included visual analog scale score for neck pain assessment, the American Spinal Injury Association Impairment Scale and Japanese Orthopaedic Association score for neurologic status and function. According to preoperative computed tomography (CT) reconstruction and CT angiography, the patients selected in this study were not suitable for C1 screw placement. Postoperative plain radiographs and CT reconstruction were performed to evaluate the reduction, bony fusion, and implant position. All outcomes were evaluated at each follow-up.
The average clinical follow-up period was 24 months (range 12–36 months). All patients had complete neck pain relief at postoperative 6 months. Their neurologic symptoms had improved significantly at 1-year follow-up. Radiologic outcomes indicated good bony fusion and construction stability in all patients without implant failure at the last follow-up. No neural or vascular complications related to this technique were observed.
Posterior atlantoaxial fixation using C1 titanium cables and C2 pedicle screw-rod construct appears to be an effective and safe technique for treatment of atlantoaxial instability, which could be an alternative method for cases unsuitable for C1 screw placement when using C1-C2 screw fixation.
Stable Fe deficient Sr2Fe1−δMoO6 (0.0⩽δ⩽0.10) compound
Journal of Alloys and Compounds, Volume 601, 2014, pp. 245-250
The structural, magnetic and magneto-transport properties of bulk ceramic double perovskite Sr2Fe1−δMoO6 (δ=0.0, 0.02, 0.05 and 0.10) samples were systematically investigated. The rietveld fitted X-ray diffraction patterns shows the tetragonal symmetry with Fe deficiency up to δ=0.10, which suggested that the crystal structure of the Sr2FeMoO6 (SFMO) does not change so much with Fe deficiency. However, the lattice parameters found to be slightly increased in higher Fe deficiency δ=0.10. The Scanning Electron Microscopy and energy dispersive X-ray spectroscopy analysis reveals that there is no considerable change in grain size, morphology and compositions with Fe deficiency. The saturation magnetization (Ms) showed the decreasing–increasing–decreasing behavior with increasing Fe deficiency. The low field magnetoresistance found to be decreased by increasing Fe deficiency. The observed result shows that the little amount of Fe deficiency provide the structure stability by varying the Fe/Mo ion valences, but it harmful and should be avoided to achieve the better magnetoresistive properties in pristine SFMO compound.
Expression of Matrix Metalloproteinase-9, Pituitary Tumor Transforming Gene, High Mobility Group A 2, and Ki-67 in Adrenocorticotropic Hormone–Secreting Pituitary Tumors and Their Association with Tumor Recurrence
World Neurosurgery, Volume 113, 2018, pp. e213-e221
Matrix metalloproteinase-9 (MMP-9), pituitary tumor transforming gene (PTTG), and high mobility group A 2 (HMGA2) play important roles in the tumorigenesis of adrenocorticotrophic hormone (ACTH)-secreting pituitary tumors, but their associations with tumor recurrence after transsphenoidal adenomectomy remain unclear. The aim of the study was to investigate the immunohistochemical expression profiles of MMP-9, PTTG, HMGA2, and Ki-67 in recurrent and nonrecurrent ACTH-secreting pituitary tumors and to identify their associations with tumor behavior and recurrence status.
A retrospective study was performed including 55 patients with sporadic Cushing's disease with long-term remission after transsphenoidal adenomectomy. Fifty-five ACTH-secreting pituitary tumor specimens and 2 normal pituitary glands were collected. After an intensive follow-up (33–59 months, mean 41.8 months), patients were divided into 2 groups based on their recurrence status: thenonrecurrent group (n= 28) and the recurrent group (n= 27). The expression of MMP-9, PTTG, HMGA2, and Ki-67 in each sample was examined and quantified by immunohistochemistry. The association between MMP-9, PTTG, HMGA2, and Ki-67 expression and clinicopathologic characteristics and tumor recurrence were evaluated.
There was a significantly increased expression of MMP-9 in the recurrent group compared with the nonrecurrent group (P= 0.022), and this was strongly associated with the recurrence-free interval (P= 0.007, correlation coefficient.= –0.354). PTTG, HMGA2, and Ki-67 expression were not significantly different between the recurrent group and the nonrecurrent group. No expression of MMP-9, PTTG, HMGA2, or Ki-67 was detected in the 2normal pituitary glands.
ACTH-secreting pituitary tumors with greater levels of MMP-9 were associated with a greater recurrence rate and a shorter recurrence-free interval. MMP-9 could be a valuable tool for predicting recurrence of ACTH-secreting pituitary tumors.
Triptolide suppresses growth and hormone secretion in murine pituitary corticotroph tumor cells via NF-kappaB signaling pathway
Biomedicine & Pharmacotherapy, Volume 95, 2017, pp. 771-779
Triptolide is a principal diterpene triepoxide from the Chinese medical plant Tripterygium wilfordii Hook. f., whose extracts have been utilized in dealing with diverse diseases in traditional Chinese medicine for centuries. Recently, the antitumor effect of triptolide has been found in several pre-clinical neoplasm models, but its effect on pituitary corticotroph adenomas has not been investigated so far. In this study, we are aiming to figure out the antitumor effect of triptolide and address the underlying molecular mechanism in AtT20 murine corticotroph cell line. Our results demonstrated that triptolide inhibited cell viability and colony number of AtT20 cells in a dose- and time-dependent pattern. Triptolide also suppressed proopiomelanocortin (Pomc) mRNA expression and extracellular adrenocorticotropic hormone (ACTH) secretion in AtT20 cells. Flow cytometry prompted that triptolide leaded to G2/M phase arrest, apoptosis program and mitochondrial membrane depolarization in AtT20 cells. Moreover, dose-dependent activation of caspase-3 and decreased Bcl2/Bax proportion were observed after triptolide treatment. By western blot analysis we found that triptolide impeded phosphorylation of NF-κB p65 subunit and extracellular signal-regulated kinase (ERK), along with reduction of cyclin D1, without any impact on other NF-κB related protein expression like total p65, p50, IκB-α, p-IκB-α. Furthermore, the mouse xenograft model revealed the inhibition of tumor growth and hormone secretion after triptolide administration. Altogether this compound might be a potential pharmaceutical choice in managing Cushing’s disease.
Hydrogen sulfide promotes lipopolysaccharide-induced apoptosis of osteoblasts by inhibiting the AKT/NF-κB signaling pathway
Biochemical and Biophysical Research Communications, Volume 524, Issue 4, 2020, pp. 832-838
Apoptosis of osteoblasts plays a crucial role in osteomyelitis. Hydrogen sulfide (H2S) levels are increased in the pathophysiological processes of osteomyelitis. However, the effect of H2S on the apoptosis of osteoblasts remains unclear. To investigate the specific role of H2S in osteoblast apoptosis, MC3T3-E1 and hFOB cells were treated with NaHS or Na2S, a donor of H2S, and lipopolysaccharide (LPS), during osteomyelitis. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, flow cytometry analysis, western blotting, immunofluorescence, polymerase chain reaction, and Alizarin red staining were performed to examine the effects of H2S on osteoblast cell apoptosis, cell osteogenic differentiation, and AKT kinase (AKT)/nuclear factor kappa B (NF-κB) signaling. Hydrogen sulfide increased cell apoptosis, and inhibited the proliferation and osteogenic differentiation of osteoblast cells impaired by LPS. H2S increased apoptosis through upregulation of the FAS ligand (FASL) signaling pathway. H2S-induced apoptosis was alleviated using a FAS/FASL signaling pathway inhibitor. Treatment with NaHS also increased cell apoptosis by downregulating AKT/NF-κB signaling. In addition, treatment with an AKT signaling pathway activator decreased apoptosis and reversed the inhibitory effects of H2S on osteogenic differentiation. Hydrogen sulfide promotes LPS-induced apoptosis of osteoblast cells by inhibiting AKT/NF-κB signaling.
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