RESUMEN

Prolyl 4-hydroxylase beta subunit (P4HB) plays a vital role in bone formation. This study intends to clarify the role of P4HB in the therapeutic effect of Icariin (ICA) on osteoporosis. Herein, in vivo and in vitro models were constructed by performing ovariectomy (OVX) in rats and inducing osteogenic differentiation in bone marrow stem cells (BMSCs), respectively. Hematoxylin and eosin staining and micro-computed tomography analysis were performed to evaluate osteoporosis in OVX rats. Alizarin Red staining, alkaline phosphatase staining, and the ALP activity test were employed to assess osteogenesis. m6A dot blotting and methylated RNA immunoprecipitation were used to determine m6A modification. We found that P4HB was downregulated in bone tissues of patients with osteoporosis and OVX rats. P4HB facilitated osteogenic differentiation of BMSCs. What's more, ICA upregulated P4HB expression, promoted osteogenic differentiation of BMSCs, and alleviated osteoporosis in OVX rats, which were reversed by knocking down P4HB. ICA enhanced the stability and m6A modification of P4HB. METTL14 mediated m6A modification of P4HB mRNA. In addition, METTL14 knockdown overturned the promotive effects of ICA on P4HB m6A level and BMSC osteogenic differentiation. To sum up, ICA elevated the METTL14-mediated m6A modification of P4HB to facilitate BMSC osteogenic differentiation.

Asunto(s)

Diferenciación Celular , Flavonoides , Metiltransferasas , Osteogénesis , Ratas Sprague-Dawley , Animales , Osteogénesis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Ratas , Femenino , Flavonoides/farmacología , Metiltransferasas/metabolismo , Metiltransferasas/genética , Humanos , Osteoporosis/patología , Osteoporosis/metabolismo , Osteoporosis/genética , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacos , Ovariectomía , Regulación hacia Arriba/efectos de los fármacos , Procolágeno-Prolina Dioxigenasa/metabolismo , Procolágeno-Prolina Dioxigenasa/genética , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Células Cultivadas , Adenosina/análogos & derivados , Adenosina/metabolismo

RESUMEN

Background: Observational studies have identified a heightened risk of osteoporosis and fractures in patients with primary biliary cholangitis (PBC). However, conclusive evidence establishing a causal relationship between the two, and a clear mechanism explaining this association, remains elusive. Methods: We conducted a bidirectional two-sample Mendelian randomization (MR) analysis to investigate the causal relationship between PBC and osteoporosis. This analysis utilized five MR methods: inverse-variance weighted (IVW), MR-Egger, weighted median, weighted mode, and simple mode. Sensitivity analyses were performed, employing various models and testing methods, to assess the impact of heterogeneity and pleiotropy on the results and to confirm their robustness. Results: A causal relationship between PBC and osteoporosis risk was established through IVW analysis (OR: 1.049, 95%CI: 1.017-1.082, P=0.002). Three other MR analyses corroborated these findings. Conversely, osteoporosis was not found to causally affect PBC risk, as evidenced by IVW analysis (OR: 0.941, 95%CI: 0.783-1.129, P=0.511). Across all MR analyses, no heterogeneity or horizontal pleiotropy was detected among the instrumental variables (IVs). Furthermore, the leave-one-out analysis indicated that no single SNP disproportionately influenced the results, affirming the reliability of the bidirectional MR findings. Conclusion: This study establishes a positive causal relationship between PBC and the risk of osteoporosis, while no definitive causal link was found from osteoporosis to PBC. These findings offer new insights and guidance for managing bone health in PBC patients.

Asunto(s)

Fracturas Óseas , Cirrosis Hepática Biliar , Osteoporosis , Humanos , Cirrosis Hepática Biliar/epidemiología , Cirrosis Hepática Biliar/genética , Análisis de la Aleatorización Mendeliana , Reproducibilidad de los Resultados , Osteoporosis/epidemiología , Osteoporosis/genética

RESUMEN

BACKGROUND: The physical health and quality of life of the elderly are severely affected by osteoporosis (OP). METHODS: We explored the regulatory mechanism of ICA in vivo and in vitro by constructing OP rats and inducing osteogenic differentiation of BMSCs. First, we determined the expression of miR-335-5p in bone tissues of OP patients, bone tissues of OP rats, and osteogenic BMSCs by RT-qPCR. Alizarin red staining was employed to detect the formation of calcium nodules in the cells. MTT was used to detect cell viability. Finally, we detected the bone tissue changes in OP rats by overexpression of miR-335-5p or oral ICA. RESULTS: miR-335-5p was lowly expressed in bone tissues of OP patients and OP rats. ICA treatment reversed the inhibitory effect of miR-335-5p inhibitor on BMSCs matrix mineralization. Moreover, PTEN was verified to be a downstream effector of miR-335-5p. During ICA induction, overexpression of PTEN reversed the promotive effect of miR-335-5p mimics on the osteogenic differentiation of BMSCs. In vivo experiments also found that overexpression of miR-335-5p or ICA treatment improved the pathogenesis of OP in rats. CONCLUSION: ICA improved OP by up-regulating miR-335-5p to inhibit PTEN, thereby providing a new strategy for the prevention and treatment of OP.

Asunto(s)

Células Madre Mesenquimatosas , MicroARNs , Osteoporosis , Anciano , Animales , Células de la Médula Ósea/metabolismo , Diferenciación Celular , Células Cultivadas , Flavonoides , Humanos , Células Madre Mesenquimatosas/metabolismo , MicroARNs/metabolismo , Osteogénesis/genética , Osteoporosis/tratamiento farmacológico , Osteoporosis/genética , Osteoporosis/metabolismo , Calidad de Vida , Ratas

RESUMEN

p15INK4b (cyclin-dependent kinase inhibitor 2B, CDKN2B, p15), a cyclin-dependent kinase inhibitor (CKI) belonging to the INK4 family, plays an important role in hematopoiesis. Its expression level was positively related to the blockage effects of RUNX1b at the early stage. Experiments using human embryonic stem cell (hESC) lines with inducible p15 expression suggested that p15 overexpression can significantly decrease the proportion of KDR+ cells in S and G2-M stages 4 days after induction from day 0. Moreover, p15 overexpression from the early stage can decrease production of CD34highCD43- cells and their derivative populations, but not CD34lowCD43- cells. These effects were weakened if induction was delayed and disappeared if induction started after day 6. All these effects were counteracted by inhibition of TGF-ß signaling. TGF-ß1 stimulation elicited similar effects as p15 overexpression. RUNX1 overexpression and activation of the TGF-ß signaling pathway upregulate the expression of p15, which is partially responsible for blockade of hematopoiesis and relevant to a change in the cell cycle status. However, it is possible that other mechanisms are involved in the regulation of hematopoiesis.

Asunto(s)

Proteínas de Ciclo Celular , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Ciclo Celular , Puntos de Control del Ciclo Celular , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Hematopoyesis , Humanos , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Supresoras de Tumor

RESUMEN

Deficiency of P18 can significantly improve the self-renewal potential of hematopoietic stem cells (HSC) and the success of long-term engraftment. However, the effects of P18 overexpression, which is involved in the inhibitory effects of RUNX1b at the early stage of hematopoiesis, have not been examined in detail. In this study, we established inducible P18/hESC lines and monitored the effects of P18 overexpression on hematopoietic differentiation. Induction of P18 from day 0 (D0) dramatically decreased production of CD34highCD43- cells and derivative populations, but not that of CD34lowCD43- cells, changed the cell cycle status and apoptosis of KDR+ cells and downregulated the key hematopoietic genes at D4, which might cause the severe blockage of hematopoietic differentiation at the early stage. By contrast, induction of P18 from D10 dramatically increased production of classic hematopoietic populations and changed the cell cycle status and apoptosis of CD45+ cells at D14. These effects can be counteracted by inhibition of TGF-ß or NF-κB signaling respectively. This is the first evidence that P18 promotes hematopoiesis, a rare property among cyclin-dependent kinase inhibitors (CKIs).

Asunto(s)

Diferenciación Celular , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/biosíntesis , Regulación de la Expresión Génica , Células Madre Embrionarias Humanas/metabolismo , FN-kappa B/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Línea Celular , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/genética , Humanos , FN-kappa B/genética , Factor de Crecimiento Transformador beta/genética

RESUMEN

Eukaryotic inducible overexpression systems, including Tet-On and mifepristone-inducible systems, have been widely used to study gene functions by reverse genetics. Among the transposon systems reported to date, the piggyBac transposon system is one of the most efficient in cultured mammalian cells. Here, we report a piggyBac-based double-inducible system that combined the advantages of previous systems. To create this system, the trans- and cis-elements of the Tet-On and mifepristone-inducible systems were cloned into a piggyBac-based trans-vector and cis-vector, respectively. The coding regions of two splicing variants of RUNX1, RUNX1a and RUNX1b, were inserted into the cis-vector to test its ability to express foreign genes along with fluorescent marker proteins. Transgenic 293 T cells were established, and the system was tested by inducing expression of foreign genes with DOX and/or mifepristone; GFP and/or mCherry were used as reporter genes. The system efficiently and stringently induced expression of GFP/mCherry and their co-expressed genes without significant mutual interference, as determined by qRT-PCR and Western blot. This piggyBac-based double-inducible system represents a new genetic tool for studying gene functions and interactions in vitro and in vivo in almost all organisms.

Asunto(s)

Elementos Transponibles de ADN/genética , Vectores Genéticos/genética , Plásmidos/genética , Clonación Molecular , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Doxiciclina/farmacología , Regulación de la Expresión Génica/genética , Genes Reporteros/genética , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Humanos , Secuencias Reguladoras de Ácidos Nucleicos/genética

RESUMEN

CCEPR (cervical carcinoma expressed PCNA regulatory lncRNA) has been found to be upregulated and enhance cell proliferation in human cancers. However, the role of CCEPR in osteosarcoma remains to be discovered. In this study, we found CCEPR expression was elevated in osteosarcoma tissue specimens and cell lines compared with adjacent normal tissue specimens and osteoblast cell line, respectively. Meanwhile, osteosarcoma patients with advanced stage or tumor size greater than 8 cm had higher expression of CCEPR than patients with early stage or tumor size less than or equal to 8 cm, respectively. Survival analysis suggested that osteosarcoma patients with high CCEPR expression had a worse overall survival rate than those with low CCEPR expression. The in vitro study indicated that CCEPR positively regulated proliferating cell nuclear antigen (PCNA) expression in osteosarcoma cells and silencing of CCEPR inhibited osteosarcoma cell proliferation through decreasing PCNA expression. In conclusion, CCEPR is a potential prognostic predictor and functions as oncogenic long non-coding RNA (lncRNA) to regulate cell proliferation via PCNA in osteosarcoma.

Asunto(s)

Biomarcadores de Tumor/genética , Neoplasias Óseas/mortalidad , Osteosarcoma/mortalidad , Antígeno Nuclear de Célula en Proliferación/genética , ARN Largo no Codificante/genética , Neoplasias Óseas/genética , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Masculino , Osteosarcoma/genética , Pronóstico , Análisis de Supervivencia , Regulación hacia Arriba

RESUMEN

Osteoarthritis (OA) is the most common joint disease among late middle-aged or elderly people. The pathological process of OA mainly involves the degeneration of cartilage tissue and deficiency of joint function. Salvianolic acid B (Sal B) is the main active ingredient of Salvia miltiorrhiza Bge, which possesses anti-inflammatory, anti apoptotic and other pharmacological activities. In this study, primary chondrocytes were cultured to investigate the effects of Sal B on the inflammatory response and apoptosis of OA induced by IL-1ß, and to explore the possible mechanism. First, we determined the cytotoxicity of Sal B; the results showed that the cell activity of chondrocytes was not influenced by Sal B when the concentration was below 150 µM. Moreover, Sal B (40 and 80 µM) suppressed the expression of iNOS in OA chondrocytes induced by IL-1ß, and restrained the secretion of NO, IL-6, IL-17 and TNF-α in chondrocytes obviously. Sal B (40, 80 µM) significantly alleviated the inhibitory effect of cell activity stimulated by IL-1ß and up-regulated the expression of Col II and reduced the expression of Col X. Besides, Sal B down-regulated the expression level of Bax and promoted the expression of Bcl-2, showed a significant effect on promoting proliferation and inhibiting cell apoptosis. In addition, we found that IL-1ß significantly reduced the ratio of p-PI3K/PI3K, p-Akt/Akt induced the nuclear translocation of AKT and inhibited the activation of the PI3K/Akt signaling pathway. Finally, the PI3K inhibitor, LY-294002, was added in IL-1ß-induced chondrocytes. The results suggest that Sal B ameliorates IL-1ß induced inflammation and suppresses apoptosis in OA by activating the PI3K/Akt signaling pathway. Our study reveals the mechanism of Sal B acts on OA and may provide a basis for the treatment of OA with Sal B.

RESUMEN

RUNX1 is absolutely required for definitive hematopoiesis, but the function of RUNX1b/c, two isoforms of human RUNX1, is unclear. We established inducible RUNX1b/c-overexpressing human embryonic stem cell (hESC) lines, in which RUNX1b/c overexpression prevented the emergence of CD34+ cells from early stage, thereby drastically reducing the production of hematopoietic stem/progenitor cells. Simultaneously, the expression of hematopoiesis-related factors was downregulated. However, such blockage effect disappeared from day 6 in hESC/AGM-S3 cell co-cultures, proving that the blockage occurred before the generation of hemogenic endothelial cells. This blockage was partially rescued by RepSox, an inhibitor of the transforming growth factor (TGF)-ß signaling pathway, indicating a close relationship between RUNX1b/c and TGF-ß pathway. Our results suggest a unique inhibitory function of RUNX1b/c in the development of early hematopoiesis and may aid further understanding of its biological function in normal and diseased models.

Asunto(s)

Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Regulación del Desarrollo de la Expresión Génica , Hematopoyesis , Células Madre Hematopoyéticas/citología , Células Madre Embrionarias Humanas/citología , Mesodermo/citología , Antígenos CD34/análisis , Línea Celular , Técnicas de Cocultivo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Células Madre Hematopoyéticas/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Humanos , Mesodermo/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia Arriba

RESUMEN

OBJECTIVE: We conducted a comprehensive study to investigate the role of GSTM1, GSTTI and GSTP1 genetic variation involved in transport pathways in response to chemotherapy and clinical outcome of osteosarcoma. METHODS: A total of 146 patients were included in our study between January 2008 and December 2009. All the patients were followed up to death or January 2012. Genotyping of GSTM1, GSTT1 and GSTP1 was conducted in a 384-well plate format on the Sequenom MassARRAY platform. RESULTS: Sixty seven patients (45.9%) died during the follow-up period. The median age of patients was 14.2 years and ranged from 9.3 to 38.7 years. The median follow-up time was 29.6 months (range 5 to 60 months). Individuals with GSTP1 G/G genotype tended to live shorter than A/A genotype, and we found a significantly higher risk of death from osteosarcoma (adjusted HR=2.73, 95% CI=1.05-7.45). Individuals with the GSTP GG genotype were more likely to have a poor response to chemotherapy, with an OR of 2.73 (95%CI, 1.07-7.81). However, we did not find association of polymorphisms in GSTM1 and GSTT1 with response to chemotherapy and prognosis of osteosarcoma. CONCLUSION: Our study provides information for prediction of treatment outcome in clinical oncology. Due to the limited number of samples, the results of our study need to be confirmed by large sample size studies.