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AMPK activation is required for miR-25-induced cytoprotection in osteoblastic cells. Stable OB-6 cells with miR-25 were constructed with dominant negative AMPKα ( " dn-AMPKα " , T172A), AMPKα shRNA, or the scramble control shRNA ( " scr shRNA " ), expressions of listed proteins in these cells were tested by Western blots A. Above cells were treated with or without Dex (1 μM), cell viability (MTT assay, 24 hours, B., apoptosis intensity (Histone DNA ELISA assay, 24 hours, C. and ROS content (DCFH-DA fluorescent dye assay, 6 hours, D. were tested; NADPH activity in above cells was also shown (4 hours, E). Experiments in this figure were repeated three times, and similar results were obtained. " Ctrl " stands for untreated control group. *p<0.05 vs. " scr shRNA " cells.
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AMP-activated protein kinase (AMPK) activation could protect osteoblasts from dexamethasone (Dex). This study aims to provoke AMPK activation via microRNA downregulation of its negative regulator protein kinase C ζ (PKCζ). Results show that microRNA-25-5p (miR-25-5p) targets PKCζ's 3' untranslated regions (UTRs). Forced-expression of miR-25 downreg...
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GSK621 is a novel AMP-activated protein kinase (AMPK) activator. This study tested its potential cytoprotective effect in hydrogen peroxide (H2O2)-treated osteoblasts. In cultured MC3T3-E1 osteoblastic cells and primary murine osteoblasts, GSK621 significantly attenuated H2O2-induced cell death and apoptosis. AMPK activation was required for GSK621...
Citations
... MiR-122 may affect SANFH by regulating the SPRY2 gene [59]. miRNA-25-5p and miR-135b enhance the activity of nicotinamide adenine dinucleotide phosphate by activating the AMPK signaling pathway to counteract GC-induced oxidative stress in osteoblasts [60]. Upregulating miR-146a, WNT/FOXO, and Sirt1/NF-KB pathways stabilized osteoblast homeostasis to prevent SANFH [61]. ...
Osteonecrosis of the femoral head (ONFH) is a refractory orthopedic condition characterized by bone cell ischemia, necrosis, bone trabecular fracture, and clinical symptoms such as pain, femoral head collapse, and joint dysfunction that can lead to disability. The disability rate of ONFH is very high, which imposes a significant economic burden on both families and society. Steroid-associated osteonecrosis of the femoral head (SANFH) is the most common type of ONFH. However, the pathogenesis of SANFH remains unclear, and it is an urgent challenge for orthopedic surgeons to explore it. In this paper, the pathogenesis of SANFH and its related signaling pathways were briefly reviewed to enhance comprehension of the pathogenesis and prevention of SANFH.
... On the other hand, miR-25 is a member of miR-106b ~ 25 clusters, which includes miR-106b, miR-93 and miR-25, its role in osteoblasts/ osteoblastic cells has not been extensively studied; miR-25 expression is pro-survival in human osteoblastic cells 19 . ...
Resumen and their relationship with some variables in serum of patients with Osteoporosis MiRNA-133a y MiRNA-25 3p y su relación con algunas variables en suero de pacientes con Osteoporosis Osteoporosis is a chronic disease characterized by bone fragility that results in fractures and a variety of miRNAs are involved in osteoclast differentiation therefore, the current case control study aimed to estimate miRNA-133a and miRNA-25 3p in os-teoporotic patients and evaluate relationship of these miRNAs with some variables including (calcium, vitamin D, BMD, smoking , history of previous fracture and gender, this study conducted on fifty patients suffering from osteoporosis with age range between 50-88 years, other group consist of 45 healthy individuals with an age range between 55-87 years included in this study as a control group. Blood samples used to extraction of miRNA-133a and miRNA-25 3p from the serum of patients and healthy control as a biomarker for osteoporosis were quan-titated by using RT-PCR. Results: miR-133a fold change was significantly upregulated in serum of osteoporotic patients and highest in patients group compared with control group, miR-133a highly significant difference in miR-133 among study groups (P < 0.001); while no significant difference in miR-25 among study groups (P = 0.295); although the level of patients groups was higher than that of control group. Receiver operator characteristic (ROC) curve of miR-133 was carried out and cutoff value was >8.3 with sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and area under curve of 76%, 80%,82.6%, 72.7% and 0.815 (0.723-906), while (ROC) curve analysis and cutoff value of miR-25 was >1.32 with sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and Area under curve of 66%, 45%, 60%, 51.4% and 0.565 (0.444-685).Conclu-sion: miRNA-133a is high sensitivity and Specificity in this study which was bushed to using them as a biomarker for osteopo-rosis diagnosis. La osteoporosis es una enfermedad crónica caracterizada por la fragilidad ósea que resulta en fracturas y una variedad de miARN están involucrados en la diferenciación de los osteo-clastos, por lo tanto, el presente estudio de casos y controles tuvo como objetivo estimar miARN-133a y miARN-25 3p en pacientes osteoporóticos y evaluar la relación de estos miARN. con algunas variables que incluyen (calcio, vitamina D, DMO, tabaquismo, antecedentes de fracturas previas y sexo, este estudio se realizó en cincuenta pacientes que padecían os-teoporosis con rango de edad entre 50-88 años, otro grupo conformado por 45 individuos sanos con rango de edad entre 55-87 años incluidos en este estudio como grupo control. Las muestras de sangre utilizadas para la extracción de miARN-133a y miARN-25 3p del suero de pacientes y control sano como biomarcador de osteoporosis se cuantificaron median-te RT-PCR. Resultados: el cambio de miR-133a se incrementó significativamente en el suero de pacientes osteoporóticos y fue más alto en el grupo de pacientes en comparación con el grupo de control, miR-133a hi diferencia muy significativa en miR-133 entre los grupos de estudio (P <0,001); mientras que no hubo diferencias significativas en el miR-25 entre los grupos de estudio (P = 0,295); aunque el nivel de los grupos de pacientes fue superior al del grupo de control. Se realizó la curva de características del operador del receptor (ROC) de miR-133 y el valor de corte fue> 8,3 con sensibilidad, especifi-cidad, valor predictivo positivo (VPP), valor predictivo negativo (VPN) y área bajo la curva de 76%, 80%, 82,6 %, 72,7% y 0,815 (0,723-906), mientras que el análisis de la curva (ROC) y el valor de corte de miR-25 fue> 1,32 con sensibilidad, especi-ficidad, valor predictivo positivo (VPP), valor predictivo negativo (VPN) y Área bajo la curva de 66%, 45%, 60%, 51,4% y 0,565 (0,444-685). Conclusión: el miARN-133a es de alta sensibili-dad y especificidad en este estudio que se propuso utilizarlos como biomarcador para el diagnóstico de osteoporosis.
... PKCζ has been shown to negatively regulate AMPK activation in different cell types, including osteoblasts and muscle cells. 26,27 Meanwhile, other isoforms of PKC have been shown to negatively regulate AMPK activation via phosphorylation of its Ser485. 28 A previous study also showed that PKCζ activation by peroxynitrite regulates AMPK activity by increasing LKB1 phosphorylation. ...
This study explored the antioxidative effect of a specific wheat germ-derived peptide on high glucose-induced oxidative stress in vascular smooth muscle cells (VSMCs) and the underlying mechanisms. The peptide ADWGGPLPH was identified by LC-MS/MS. The effects of this peptide on the production of ROS and the expression of oxidative stress signaling proteins in VSMCs were determined. STZ-induced mice were utilized to confirm the anti-oxidative and anti-diabetic cardiovascular disease effects of this peptide in vivo. Results showed that ADWGGPLPH significantly prevented high glucose-induced cell proliferation, decreased intracellular ROS generation, stimulated AMPK activity, inhibited the PKCζ, AKT and Erk1/2 phosphorylation, and suppressed NOX4 protein expression. Additionally, ADWGGPLPH enhanced the antioxidant abilities and attenuated inflammatory cytokine generation in STZ-induced diabetic mice. Therefore, ADWGGPLPH prevents high glucose-induced oxidative stress in VSMCs by modulating the PKCζ/AMPK/NOX4 pathway, suggesting that the induction of AMPK activation may be a new therapeutic approach against diabetic cardiovascular diseases.
... In addition, AMPK knockdown or mutation abolished this miR-135b-induced cytoprotection against steroid treatment in osteoblastic cells, supporting a critical function of AMPK activation in the actions of miR-135b. Subsequently, they found that miR-25-5p level was increased in patients' necrotic femoral head tissues, targeting and downregulating PKCζ in osteoblastic cells and in turn activating AMPK signaling and protecting cells against the damage induced by steroids [40]. ...
Steroid-induced osteonecrosis of the femoral head (ONFH) is a severe orthopedic disease caused by the long-term administration of glucocorticoids. The main pathological feature of ONFH is the gradually progressive necrosis of bone cells and the bone marrow, ultimately resulting in structural changes or even complete collapse of the femoral head. However, the exact pathogenic mechanism of ONFH remains unknown. Noncoding RNAs (ncRNAs) have emerged as very powerful regulators of gene expression, functioning at both transcriptional and posttranscriptional levels in the pathogenesis of ONFH. Here, we review the current knowledge of the role of ncRNAs, including microRNAs, long noncoding RNAs, and circular RNAs, in the pathogenesis of steroid-induced ONFH. Further focus and validation of these associations can provide new insight into the pathogenic mechanisms at the molecular level to suggest targets for treatment and prevention.
... Recent studies have revealed that some miRNAs are critically involved in MLT-induced apoptosis in several cancers, such as breast [31] and colon cancers [32]. Among them, though miR-25 was known to promote proliferation in several cancers, such as lung cancer [33], prostate cancer [34], ovarian cancer [35], and colorectal cancer [36] as an oncogene, miR-25-5p was reported to suppress PKCζ as a tumor suppressor [37,38]. Also, among several target molecules, neural precursor cell expressed developmentally downregulated 9 (NEDD9), so called Cas-L (Crk-associated substrate L) or human enhancer of filamentation1 (HEF1), promotes proliferation [39] and metastasis [19,39] in breast cancer, colorectal cancer, and head and neck cancer [40,41]. ...
... Among them, miR-25-5p was found most upregulated by MLT+Ptero in HT29 cells. Previous studies revealed that miR-25-5p acted as a tumor suppressor and PKCζ negative regulator in colon cancers [32,37], whereas miR-25 promotes proliferation of non-small cell lung cancer cells [33] and colorectal cancers as an oncogene [32]. Here, concentration-dependent upregulation of miR-25-5P was validated in MLT+Ptero-treated HT29 cells by qRT-PCR. ...
The underlying interaction between melatonin (MLT) and daily fruit intake still remains unclear to date, despite multibiological effects of MLT. Herein, the apoptotic mechanism by co-treatment of MLT and pterostilbene (Ptero) contained mainly in grape and blueberries was elucidated in colorectal cancers (CRCs). MLT and Ptero co-treatment (MLT+Ptero) showed synergistic cytotoxicity compared with MLT or Ptero alone, reduced the number of colonies and Ki67 expression, and also increased terminal deoxynucleotidyl transferase dUTP nick end labeling- (TUNEL) positive cells and reactive oxygen species (ROS) production in CRCs. Consistently, MLT+Ptero cleaved caspase 3 and poly (ADP-ribose) polymerase (PARP), activated sex-determining region Y-Box10 (SOX10), and also attenuated the expression of Bcl-xL, neural precursor cell expressed developmentally downregulated protein 9 (NEDD9), and SOX9 in CRCs. Additionally, MLT+Ptero induced differentially expressed microRNAs (upregulation: miR-25-5p, miR-542-5p, miR-711, miR-4725-3p, and miR-4484; downregulation: miR-4504, miR-668-3p, miR-3121-5p, miR-195-3p, and miR-5194) in HT29 cells. Consistently, MLT +Ptero upregulated miR-25-5p at mRNA level and conversely NEDD9 overexpression or miR-25-5p inhibitor reversed the ability of MLT+Ptero to increase cytotoxicity, suppress colony formation, and cleave PARP in CRCs. Furthermore, immunofluorescence confirmed miR-25-5p inhibitor reversed the reduced fluorescence of NEDD9 and increased SOX10 by MLT+Ptero in HT29 cells. Taken together, our findings provided evidence that MLT+Ptero enhances apoptosis via miR-25-5p mediated NEDD9 inhibition in colon cancer cells as a potent strategy for colorectal cancer therapy.
... In cancer cells, miR-25 was shown to target the proapoptotic protein MOAP1 while its antiapoptosis protective effects in other cell types have been linked with activation of the PTEN/Akt and AMPK signaling pathways [26]. Other studies documented protective roles for miR-25 in osteoblast responses to dexamethasone [27]. ...
... For example, miR-25 activates AMPK in dexamethasone-treated osteoblasts by direct targeting of PKCζ and can activate PI3K/Akt signaling by targeting PTEN in liver cancer stem cells (new Refs. [21,27]). ...
Background and aims:
Vascular smooth muscle cells (VSMCs) are central components of atherosclerotic plaque. Loss of VSMCs through apoptotic cell death can cause fibrous cap thinning, necrotic core formation, and calcification that may destabilize plaque. Elevated glucocorticoid levels caused by psychological stress promote VSMC apoptosis and can exacerbate atherosclerosis in mice and humans. Changes in the levels of antiapoptosis microRNA-25 (miR-25) have been linked with heart disease, inflammation, VSMC phenotype, oxidative stress, and apoptosis. Here, we investigated the pathways and mechanisms of glucocorticoid-induced apoptosis of mouse VSMCs and the protective role of miR-25.
Methods:
Primary mouse VSMCs were cultured +/- corticosterone for 48 h. Apoptosis, ROS, apoptotic protein activities, miR-25, MOAP1, a miR-25 target, and p70S6 kinase were quantified at intervals. The roles of miR-25 were assessed by treating cells with lenti-pre-miR-25 and anti-miR-25.
Results:
VSMC apoptosis, caspase-3 activity, and Bax were increased by corticosterone, and cell death was paralleled by marked loss of miR-25. Protection was conferred by pre-miR-25 and exacerbated by anti-miR-25. Pre-miR-25 conferred reduced expression of the proapoptotic protein MOAP1, and the protective effects of pre-miR-25 were abrogated by overexpressing MOAP1. The antiapoptotic effects of miR-25 were paralleled by inhibition of the p70S6K pathway, a convergence target for the survival signaling pathways, and protection by pre-miR-25 was abrogated by the p70S6k inhibitor rapamycin.
Conclusions:
MicroRNA-25 blocks corticosterone-induced VSMC apoptosis by targeting MOAP1 and the p70S6k pathway. Therapeutic manipulation of miR-25 may reduce atherosclerosis and unstable plaque formation associated with chronic stress.
... Previous reports found that Dex caused cell growth inhibition and apoptosis in MC3T3-E1 cells (Fan et al. 2017;Li et al. 2012). To study the influence of p21 on Dex-induced cell death, knockdown experiments were performed by Cells were pre-incubated with IGF-1 (50 ng/mL) for 1 h, and then coincubated with 1 μM dexamethasone for 24 h. a Cell viability was determined by the MTT assay. ...
... Dex induced significant ROS production and oxidative stress in osteoblastic cells, which may contribute to subsequent cell death (Fan et al. 2017;Li et al. 2016;Lin et al. 2015;Suwanjang et al. 2016). Previous reports revealed that p21 effectively inhibits the generation of ROS in HepG2 cells (Deng et al. 2016). ...
Dexamethasone (Dex), a glucocorticoid with strong anti-inflammatory and immunosuppressive activities, has been shown to exhibit marked cytotoxicity and apoptosis in osteoblasts, but the underlying mechanisms have not yet been comprehensively investigated. P21Waf1/Cip1 (p21) plays a critical role in the regulation of cell cycle progression and apoptosis. The present study aims to investigate the role of p21 in Dex-induced apoptosis in osteoblastic MC3T3-E1 cells, and to explore its mechanisms. Results demonstrated that Dex-induced apoptosis decreased the phosphorylation of Akt in a concentration-dependent manner. Moreover, LY294002, an inhibitor of the PI3K/Akt pathway enhanced the Dex-induced apoptosis of osteoblasts. On the contrary, insulin-like growth factor-1 (IGF-1), an activator of PI3K/Akt, attenuated the apoptosis of Dex in MC3T3-E1 cells. The protein level of p21 was downregulated by shortening its half-life, which was associated with inhibition of the PI3K/Akt pathway by Dex. Furthermore, depletion of p21 by siRNA enhanced Dex-induced caspase-3 activation and ROS generation, and promoted apoptosis of MC3T3-E1 cells. In addition, suppression of p21 led to a reduction of Dex-induced upregulation of nuclear Nrf2 and heme oxygenase-1 (HO-1) protein levels. These findings demonstrate that p21 depletion promotes Dex-induced apoptosis of MC3T3-E1 cells by inhibiting the antioxidant Nrf2/HO-1 pathway, which highlights the anti-apoptotic effect of p21 in MC3T3-E1 cells.
... The OB-6 human osteoblastic cells were cultured and differentiated as described in our previous studies [37,38]. The isolation and primary culture of murine osteoblasts derived from the trabecular bone of C57/B6 mice were described previously [39][40][41][42], with the animal protocol approved by Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University. ...
MIND4-17 is a recently developed NF-E2-related factor 2 (Nrf2) activator, which uniquely causes Nrf2 disassociation from Keap1. Here, we showed that pretreatment with MIND4-17 significantly inhibited hydrogen peroxide (H2O2)-induced viability reduction of primary osteoblasts and OB-6 osteoblastic cells. Meanwhile, MIND4-17 inhibited both apoptotic and non-apoptotic osteoblast cell death by H2O2. MIND4-17 treatment induced Keap1-Nrf2 disassociation, causing Nrf2 stabilization, accumulation and nuclear translocation in osteoblasts, leading to transcription of several Nrf2-dependent genes, including heme oxygenase 1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), γ-glutamylcysteine synthetase modifier subunit (GCLM) and catalytic subunit (GCLC). Additionally, MIND4-17 largely attenuated H2O2-reactive oxygen species (ROS) production, lipid peroxidation and DNA damages. Nrf2 knockdown by targeted short hairpin RNA (shRNA) exacerbated H2O2-induced cytotoxicity in OB-6 osteoblastic cells, and nullified MIND4-17-mediated cytoprotection against H2O2. Meanwhile, Keap1 shRNA took over MIND4-17′s actions and protected OB-6 cells from H2O2. Together, MIND4-17 activates Nrf2 signaling and protects osteoblasts from H2O2.
... Recent studies have confirmed a cytoprotective function of AMPK under various stress conditions. In particularly, activated AMPK is capable of fighting oxidative stress [25,26]. AMPK inhibits reactive oxygen species (ROS) production, thus protecting cells from oxidative stress [27]. ...
The present study tested the potential effect of OSU53, a novel AMPK activator, against hydrogen peroxide (H2O2)-induced spinal cord neuron damages. Treatment with OSU53 attenuated H2O2-induced death and apoptosis of primary murine spinal cord neurons. OSU53 activated AMPK signaling, which is required for its actions in spinal cord neurons. The AMPK inhibitor Compound C or AMPKa1 siRNA almost abolished OSU53-mediated neuroprotection against H2O2. On the other hand, sustained-activation of AMPK by introducing the constitutive-active AMPKa1 mimicked OSU53's actions, and protected spinal cord neurons from oxidative stress. OSU53 significantly attenuated H2O2-induced reactive oxygen species production, lipid peroxidation and DNA damages in spinal cord neurons. Additionally, OSU53 increased NADPH content and heme oxygenase-1 mRNA expression in H2O2-treated spinal cord neurons. Together, we indicate that targeted-activation of AMPK by OSU53 protects spinal cord neurons from oxidative stress.
... First, we demonstrate that miR-200a ("-3p") putatively targets the 3'-UTR of Keap1 mRNA (at position 131-138) ( Figure 1A), as reported by other studies [28,29]. Next, the miR-200a expression vector (pSuper-GFP-puro) was established, which was transfected to OB-6 human osteoblastic cells [30,31]. Via puromycin selection, two stable OB-6 cell lines ("Line1/2") with the construct were established. ...
Treatment with dexamethasone in human osteoblasts leads to oxidative stress and cell injures. NF-E2-related factor 2 (Nrf2) is a key anti-oxidant signaling. We want to induce Nrf2 activation via microRNA-mediated silencing its suppressor Keap1. Our results show that microRNA-200a (“miR-200a”) expression depleted Keap1, causing Nrf2 protein stabilization in OB-6 osteoblastic cells. Reversely, the miR-200a anti-sense led to Keap1 upregulation and Nrf2 degradation. miR-200a expression activated Nrf2 signaling, which inhibited dexamethasone-induced reactive oxygen species production and OB-6 cell death/apoptosis. Keap1 shRNA also activated Nrf2 and protected OB-6 cells from dexamethasone. Importantly, miR-200a was in-effective in Keap1-silenced (by shRNA) OB-6 cells. In the primary human osteoblasts, Keap1 silence by targeted-shRNA or miR-200a protected cells from dexamethasone. Significantly, miR-200a level was decreased in necrotic femoral head tissues, which was correlated with Keap1 mRNA upregulation. Together, miR-200a expression activates Nrf2 signaling and protects human osteoblasts from dexamethasone.
