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1.
BMC Med Genet ; 21(1): 11, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31918667

RESUMO

BACKGROUND: To investigated the role of miR-19b-3p in regulating bone marrow mesenchymal stem cell (BMSC) proliferation and osteoblast differentiation. METHODS: The expression of miR-19b-3p and lncRNA H19 were measured in postmenopausal osteoporosis patients and BMP-22 induced BMSCs using qRT-PCR. MiR-19b-3p mimic or inhibitor was transfected into BMP-2 induced BMSCs. Cell proliferation was measured by BrdU method. Protein expression of RUNX2 and COL1A1 were measured by western blot. PcDNA3.1-lncRNA H19 with or without miR-19b-3p mimic was transfected into BMP-2 induced BMSCs. RESULTS: The expression of miR-19b-3p was significantly up-regulated in postmenopausal osteoporosis patients and BMP-2 induced BMSCs. MiR-19b-3p overexpression dramatically elevated, while miR-19b-3p inhibition decreased cell proliferation of BMSCs. Additionally, protein expression levels of RUNX2 and COL1A1, as well as ALP activity were significantly promoted by miR-19b-3p mimic transfection and inhibited by miR-19b-3p inhibitor transfection. LncRNA H19 was obviously down-regulated in postmenopausal osteoporosis patients. H19 overexpression significantly decreased cell proliferation and differentiation by down-regulating miR-19b-3p. Moreover, the expression of miR-19b-3p was inhibited, while H19 elvated in 17ß-estradiol (E2) treated BMSCs in a dose-dependent manner. CONCLUSION: These data were the first to reveal the critical role of H19/miR-19b-3p in postmenopausal osteoporosis, and provided a new therapeutic target for OP.


Assuntos
MicroRNAs/genética , Osteogênese/genética , Osteoporose Pós-Menopausa/genética , RNA Longo não Codificante/genética , Proteína Morfogenética Óssea 2/genética , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Colágeno Tipo I/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Estradiol/administração & dosagem , Feminino , Humanos , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Osteoblastos/metabolismo , Osteoporose Pós-Menopausa/patologia
2.
J Orthop Res ; 38(1): 182-191, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31161610

RESUMO

Although several studies revealed a multifactorial pathogenesis of degenerative rotator cuff disorders, the impact and interaction of extrinsic variables is still poorly understood. Thus, this study aimed at uncovering the effect of patient- and pathology-specific risk factors that may contribute to degeneration of the rotator cuff tendons. Between 2015 and 2018, 54 patients who underwent arthroscopic shoulder surgery at three specialized shoulder clinics were prospectively included. Using tendon samples harvested from the macroscopically intact subscapularis (SSC) tendon, targeted messenger RNA expression profile analysis was performed in the first cohort (n = 38). Furthermore, histological analyses were conducted on tendon tissue samples obtained from a second cohort (n = 16). Overall, both study cohorts were comparable concerning patient demographics. Results were then analyzed with respect to specific extrinsic factors, such as patient age, body mass index, current as well as previous professions and sport activities, smoking habit, and systemic metabolic diseases. While patient age, sports-activity level, and preexisting rotator cuff lesions were considered to contribute most strongly to tendinopathogenesis, no further coherences were found. With regards to gene expression analysis, change in expression correlated most strongly with patient age and severity of the rotator cuff pathology. Further, chronic disorders increased overall gene expression variation. Taken together, our study provides further evidence that tendon degeneration is the consequence of a multifactorial process and pathological changes of the supraspinatus tendon affect the quality of SSC tendon and most likely vice versa. Therefore, the rotator cuff tendons need to be considered as a unit when managing rotator cuff pathologies. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society J Orthop Res 38:182-191, 2020.


Assuntos
Lesões do Manguito Rotador/etiologia , Manguito Rotador/patologia , Tendões/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Feminino , Humanos , Masculino , Metaloproteinase 2 da Matriz/genética , Pessoa de Meia-Idade , Estudos Prospectivos , Fatores de Risco , Adulto Jovem
3.
Gene ; 723: 144120, 2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31589964

RESUMO

PURPOSE: Matrix Gla protein (MGP) is a vitamin K-dependent, γ-carboxylated protein that was initially found to be a physiological inhibitor of ectopic calcifications affecting mainly cartilage and the vascular system. Mutations in the MGP gene were found to be responsible for a human pathology, the Keutel syndrome, characterized by abnormal calcifications in cartilage, lungs, brain and vascular system. MGP was recently implicated in tumorigenic processes such as angiogenesis and shown to be abnormally regulated in several tumors, including cervical, ovarian, urogenital and breast. This fact has triggered our interest in analyzing the expression of MGP and of its regulator, the transcription factor runt related transcription factor 2 (RUNX2), in colorectal cancer (CRC). METHODS: MGP and RUNX2 expression were analyzed in cancer and non-tumor biopsies samples from 33 CRC patients and 9 healthy controls by RT-qPCR. Consequently, statistical analyses were performed to evaluate the clinical-pathological significance of MGP and RUNX2 in CRC. MGP protein was also detected by immunohistochemical analysis. RESULTS: Showed an overall overexpression of MGP in the tumor mucosa of patients at mRNA level when compared to adjacent normal mucosa and healthy control tissues. In addition, analysis of the expression of RUNX2 mRNA demonstrated an overexpression in CRC tissue samples and a positive correlation with MGP expression (Pearson correlation coefficient 0.636; p ≤ 0.01) in tumor mucosa. However correlations between MGP gene expression and clinical-pathological characteristics, such as gender, age and pathology classification did not provide relevant information that may shed light towards the differences of MGP expression observed between normal and malignant tissue. CONCLUSIONS: We were able to associate the high levels of MGP mRNA expression with a worse prognosis and survival rate lower than five years. These results contributed to improve our understanding of the molecular mechanism underlying MGP deregulation in cancer.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Neoplasias Colorretais/patologia , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Regulação para Cima , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Análise de Sobrevida
4.
Mater Sci Eng C Mater Biol Appl ; 106: 110289, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31753386

RESUMO

This paper systematically investigates the biomedical performance of selective laser melted (SLM) porous Ti6Al4V ELI scaffolds for bone implantation through in vitro and in vivo experiments. Scaffolds with pore sizes of 500 µm, 600 µm and 700 µm and porosities of 60% and 70% were manufactured in order to explore the optimum pore size and porosity. Rat bone marrow mesenchymal stem cells (rBMMSCs) were used in the in vitro experiments. Cell Counting Kit-8, live/dead staining and scanning electron microscope were used to assess the cytotoxicity of the porous scaffolds. DNA content quantification was performed to investigate cell proliferation on the porous scaffolds. The osteogenic differentiation of cells was measured by alkaline phosphatase (ALP) activity and osteogenic gene expressions, including bone morphogenetic protein-2 (BMP-2), collagen type 1α1 (COL-1), osteocalcin (OCN), osteopontin (OPN) and runt-related transcription factor-2 (RUNX-2). The Sprague-Dawley (SD) rat models with distal femoral condyles defect were used in the in vivo experiments. Micro-CT analysis and histological analysis were performed after implantation surgery to reveal the bone ingrowth into the porous scaffolds. All in vitro data were analyzed by one-way ANOVA followed by Tukey post hoc tests, in vivo data were analyzed using Kruskall-Wallis ANOVA and Conover-Inman post-hoc test. Based on the in vitro and in vivo experiments, it is found that the porous scaffolds manufactured by SLM did not induce a cytotoxic effect. Among all the porous scaffolds, the scaffold with a pore size of 500 µm and porosity of 60% showed the best cell proliferation and osteogenic differentiation (in vitro experiments) and bone ingrowth (in vivo experiments).


Assuntos
Diferenciação Celular , Proliferação de Células , Osteogênese , Tecidos Suporte/química , Titânio/química , Animais , Células da Medula Óssea/citologia , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Fraturas Ósseas/terapia , Lasers , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/efeitos dos fármacos , Porosidade , Ratos , Ratos Sprague-Dawley , Engenharia Tecidual , Titânio/toxicidade , Microtomografia por Raio-X
5.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(12): 1179-1182, 2019 Dec 10.
Artigo em Chinês | MEDLINE | ID: mdl-31813142

RESUMO

OBJECTIVE: To analyze variants of RUNX2 gene in two pedigrees affected with cleidocranial dysplasia and provide prenatal diagnosis for them. METHODS: For the two probands, the coding sequences of the RUNX2 gene were analyzed with PCR and bidirectional Sanger sequencing. To verify the results, peripheral blood samples were collected from their parents and 100 healthy controls. For family 1, umbilical cord blood was also collected for prenatal genetic diagnosis. RESULTS: In family 1, the proband and the fetus both carried a heterozygous c.578G>C (p.Arg193Pro) mutation. For family 2, the proband was found to carry a heterozygous c.909C>A (p.Tyr303X) mutation. The same mutations were not found among their parents and 100 healthy controls. Neither mutation was reported previously. CONCLUSION: Variants of the RUNX2 gene probably underlie the cleidocranial dysplasia in both pedigrees. The results enabled prenatal diagnosis for the affected family.


Assuntos
Displasia Cleidocraniana/diagnóstico , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Displasia Cleidocraniana/genética , Éxons , Feminino , Humanos , Mutação , Linhagem , Gravidez , Diagnóstico Pré-Natal
6.
PLoS Pathog ; 15(12): e1008154, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31815961

RESUMO

Murine leukemia virus (MLV) integrase (IN) lacking the C-terminal tail peptide (TP) loses its interaction with the host bromodomain and extraterminal (BET) proteins and displays decreased integration at promoter/enhancers and transcriptional start sites/CpG islands. MLV lacking the IN TP via an altered open reading frame was used to infect tumorigenesis mouse model (MYC/Runx2) animals to observe integration patterns and phenotypic effects, but viral passage resulted in the restoration of the IN TP through small deletions. Mice subsequently infected with an MLV IN lacking the TP coding sequence (TP-) showed an improved median survival by 15 days compared to wild type (WT) MLV infection. Recombination with polytropic endogenous retrovirus (ERV), Pmv20, was identified in seven mice displaying both fast and slow tumorigenesis, highlighting the strong selection within the mouse to maintain the full-length IN protein. Mapping the genomic locations of MLV in tumors from an infected mouse with no observed recombination with ERVs, TP-16, showed fewer integrations at TSS and CpG islands, compared to integrations observed in WT tumors. However, this mouse succumbed to the tumor in relatively rapid fashion (34 days). Analysis of the top copy number integrants in the TP-16 tumor revealed their proximity to known MLV common insertion site genes while maintaining the MLV IN TP- genotype. Furthermore, integration mapping in K562 cells revealed an insertion preference of MLV IN TP- within chromatin profile states associated with weakly transcribed heterochromatin with fewer integrations at histone marks associated with BET proteins (H3K4me1/2/3, and H3K27Ac). While MLV IN TP- showed a decreased overall rate of tumorigenesis compared to WT virus in the MYC/Runx2 model, MLV integration still occurred at regions associated with oncogenic driver genes independently from the influence of BET proteins, either stochastically or through trans-complementation by functional endogenous Gag-Pol protein.


Assuntos
Carcinogênese , Vetores Genéticos/toxicidade , Leucemia Experimental , Infecções por Retroviridae , Infecções Tumorais por Vírus , Animais , Cromatina , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Modelos Animais de Doenças , Genes myc , Humanos , Integrases/metabolismo , Células K562 , Vírus da Leucemia Murina/genética , Camundongos , Camundongos Transgênicos , Integração Viral
7.
Cell Physiol Biochem ; 53(5): 832-850, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31703162

RESUMO

BACKGROUND/AIMS: Runt-related transcription factor 2 (Runx2) is a master regulator of osteogenic differentiation, but most of the direct downstream targets of RUNX2 during osteogenesis are unknown. Likewise, High-temperature requirement factor A1 (HTRA1) is a serine protease expressed in bone, yet the role of Htra1 during osteoblast differentiation remains elusive. We investigated the role of Htra1 in osteogenic differentiation and the transcriptional regulation of Htra1 by RUNX2 in primary mouse mesenchymal progenitor cells. METHODS: Overexpression of Htra1 was carried out in primary mouse mesenchymal progenitor cells to evaluate the extent of osteoblast differentiation. Streptavidin agarose pulldown assay, chromatin immunoprecipitation assay, and dual luciferase assay were carried out to investigate the interaction of RUNX2 protein at the Htra1 promoter during osteoblast differentiation. RESULTS: Overexpression of Htra1 increased the production of mineralized bone matrix, upregulating several osteoblast genes, such as Sp7 transcription factor (Sp7) and Alkaline phosphatase, liver/bone/kidney (Alpl). In addition, Htra1 upregulated osteogenesis-related signalling genes, such as Fibroblast growth factor 9 (Fgf9) and Vascular endothelial growth factor A (Vegfa). A series of experiments confirmed Htra1 as a direct RUNX2 transcriptional target. Overexpression of Runx2 resulted in the upregulation of Htra1 mRNA and protein. Chromatin immunoprecipitation and streptavidin agarose pull-down assays showed that RUNX2 binds a proximal -400 bp region of the Htra1 promoter during osteogenic differentiation. Dual luciferase assays confirmed that RUNX2 activates the proximal Htra1 promoter during osteogenic differentiation. Mutation of putative RUNX2 binding sites revealed that RUNX2 interacts with the Htra1 promoter at -252 bp and -84 bp to induce Htra1 expression. CONCLUSION: We demonstrate that Htra1 is a positive regulator of osteogenic differentiation, showing for the first time that Htra1 is a direct downstream target of RUNX2.


Assuntos
Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Fator 9 de Crescimento de Fibroblastos/metabolismo , Serina Peptidase 1 de Requerimento de Alta Temperatura A/genética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Osteogênese , Regiões Promotoras Genéticas , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismo
8.
Hereditas ; 156: 31, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31548836

RESUMO

Background: Cleidocranial dysplasia (CCD) is a rare autosomal dominant disorder mainly characterized by hypoplastic or absent clavicles, delayed closure of the fontanelles, multiple dental abnormalities, and short stature. Runt-related transcription factor 2 (RUNX2) gene variants can cause CCD, but are not identified in all CCD patients. Methods: In this study, we detected genetic variants in seven unrelated children with CCD by targeted high-throughput DNA sequencing or Sanger sequencing. Results: All patients carried a RUNX2 variant, totally including three novel pathogenic variants (c.722_725delTGTT, p.Leu241Serfs*8; c.231_232delTG, Ala78Glyfs*82; c.909C > G, p.Tyr303*), three reported pathogenic variants (c.577C > T, p.Arg193*; c.574G > A, p.Gly192Arg; c.673 C > T, p.Arg225Trp), one likely pathogenic variant (c.668G > T, p.Gly223Val). The analysis of the variant source showed that all variants were de novo except the two variants (c.909C > G, p.Tyr303*; c.668G > T, p.Gly223Val) inherited from the patient's father and mother with CCD respectively. Further bioinformatics analysis indicated that these variants could influence the structure of RUNX2 protein by changing the number of H-bonds or amino acids. The experimental result showed that the Gly223Val mutation made RUNX2 protein unable to quantitatively accumulate in the nucleus. Conclusions: The present study expands the pathogenic variant spectrum of RUNX2 gene, which will contribute to the diagnosis of CCD and better genetic counseling in the future.


Assuntos
Displasia Cleidocraniana/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Núcleo Celular , Criança , Pré-Escolar , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Masculino , Mutação , Linhagem
9.
Food Funct ; 10(9): 6184-6192, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31501830

RESUMO

Glucocorticoids (GCs) are widely used to treat a variety of autoimmune diseases, but long-term use can lead to osteoporosis. To elucidate the mechanism of osteoporosis caused by glucocorticoids and to find effective protective drugs/foods, osteoblasts treated by prednisolone acetate were studied and salvianolic acid B (Sal B) was added to osteoblasts. The results showed that Sal B increased the activity of ALP and stimulated the expression of ALP that had been suppressed by prednisolone acetate. To further study the mechanisms of the protective effect of Sal B on osteoblasts treated with prednisolone acetate, the effects of gene expression involved with bone formation and differentiation were studied. The results show that the mRNA and protein expression of Runx2, Osx, OCN, IGF-I, Col-I and HO-I was up-regulated by Sal B. In conclusion, by stimulating the osteoblast activity and the expression of genes related to bone formation and differentiation, Sal B had a protective effect on osteoblasts that had been treated with prednisolone acetate.


Assuntos
Benzofuranos/farmacologia , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Animais , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Osteoblastos/metabolismo , Prednisolona/efeitos adversos , Prednisolona/análogos & derivados , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacos
10.
J Biosci ; 44(4)2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31502565

RESUMO

Bone marrow mesenchymal stem cells (BMSCs) play an important role in the process of bone repair. The present study investigated the effect of 5-azacytidine (AZA) and trichostatin A (TSA) on BMSC behaviors in vitro. The role of WNT family member 5A (WNT5A)/WNT family member 5A (WNT7A)/beta-catenin signaling was also investigated. BMSCs were isolated from a steroid-induced avascular necrosis of the femoral head (SANFH) rabbit model. The third-generation of BMSCs was used after identification. The results revealed obvious degeneration and necrosis in the SANFH rabbit model. AZA, TSA and TSA + AZA increased BMSC proliferation in a time-dependent fashion. AZA, TSA and TSA + AZA induced the cell cycle release from the G0/G1 phase and inhibited apoptosis in BMSCs. AZA, TSA and TSA + AZA treatment significantly decreased caspase-3 and caspase-9 activities. The treatment obviously increased the activity and relative mRNA expression of alkaline phosphatase. The treatment also significantly up-regulated the proteins associated with osteogenic differentiation, including osteocalcin and runt-related transcription factor 2 (RUNX2), and Wnt/beta-catenin signal transduction pathway-related proteins beta-catenin, WNT5A and WNT7A. The relative levels of Dickkopf-related protein 1 (an inhibitor of the canonical Wnt pathway) decreased remarkably. Notably, TSA + AZA treatment exhibited a stronger adjustment ability than either single treatment. Collectively, the present studies suggest that AZA, TSA and TSA + AZA promote cell proliferation and osteogenic differentiation in BMSCs, and these effects are potentially achieved via upregulation of WNT5A/WNT7A/b-catenin signaling.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Necrose da Cabeça do Fêmur/tratamento farmacológico , Osteogênese/efeitos dos fármacos , Osteonecrose/tratamento farmacológico , Fosfatase Alcalina/genética , Animais , Azacitidina/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Necrose da Cabeça do Fêmur/induzido quimicamente , Necrose da Cabeça do Fêmur/patologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteonecrose/induzido quimicamente , Osteonecrose/patologia , Coelhos , Esteroides/toxicidade , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/genética
11.
Mol Med Rep ; 20(5): 4193-4201, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31545469

RESUMO

The present study aimed to investigate the role of lysosomal­associated transmembrane protein 5 (LAPTM5) in osteoclast differentiation induced by osteoblasts. The results demonstrated that the expression levels of LAPTM5 were downregulated following runt­related transcription factor 2 (RUNX2) silencing and upregulated following RUNX2 overexpression in ST2 cells. Chromatin immunoprecipitation analysis identified the binding of RUNX2 to the LAPTM5 promoter at the ­1176 to ­1171 position. Dual­luciferase reporter assays confirmed that RUNX2 directly activated the LAPTM5 gene. The concentration of receptor activator of nuclear factor­κB ligand (RANKL) protein in the cytoplasm and in the media was significantly increased following LAPTM5 knockdown. LAPTM5 silencing in ST2 cells enhanced osteoclastic differentiation of co­cultured RAW264.7 cells. The present study indicated that expression of LAPTM5 was regulated by the interaction of RUNX2 with its promoter region and that LAPTM5 was involved in the trafficking of RANKL. These findings suggested a possible coupling mechanism between osteogenesis and osteoclastogenesis in which RUNX2 may be involved in osteoclast differentiation through the regulation of the lysosome­associated genes that modulate RANKL expression.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Proteínas Imediatamente Precoces/genética , Proteínas de Membrana/genética , Osteoblastos/metabolismo , Ligante RANK/metabolismo , Ativação Transcricional , Animais , Biomarcadores , Linhagem Celular , Células Cultivadas , Técnicas de Cocultura , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Expressão Gênica , Regulação da Expressão Gênica , Proteínas Imediatamente Precoces/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , Transporte Proteico , Ligante RANK/genética
12.
Mol Med Rep ; 20(4): 3746-3754, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31485621

RESUMO

It has been previously reported that psoralen, one of the active ingredients in Psoralea corylifolia, could induce osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), suggesting its potential to treat osteoporosis. Additionally, runt­related transcription factor 2 (Runx2) is a transcription factor that plays vital roles in BMSC osteogenic differentiation. However, whether and how microRNAs (miRNAs/miRs) modulate osteogenic differentiation induced by psoralen have not yet been examined, to the best of the authors' knowledge. The present study aimed to identify the miRNA target genes that regulate osteogenic differentiation of BMSCs induced by psoralen. A Cell Counting Kit­8 assay and alizarin red staining were used to detect the viability and osteogenic differentiation of BMSCs, respectively, under treatment with psoralen. miRNA microarray analysis was performed to identify the differentially expressed miRNAs under treatment with psoralen. A bioinformatics analysis and a luciferase reporter assay were conducted to identify the targets of miR­488. Finally, the mechanisms of miR­488 in psoralen­induced BMSC osteogenic differentiation were investigated using overexpression or inhibition methods in vitro. Cell viability was elevated and osteogenic differentiation of BMSCs was improved under treatment with psoralen. miRNA microarray analysis and further validation by reverse transcription­quantitative PCR revealed that miR­488 was downregulated during psoralen­induced BMSC osteogenic differentiation. Bioinformatics analysis and experimental validation by a luciferase reporter assay identified Runx2 as a potential target of miR­488. Overexpression of miR­488 by transfection with miR­488 mimics markedly inhibited the expression of Runx2, Osterix and alkaline phosphatase, whereas, the inhibition of miR­488 expression by the miR­488 inhibitor promoted their expression compared with the control. Rescue assays demonstrated that Runx2 overexpression partially rescued the inhibitory effect of miR­488 on BMSC osteogenic differentiation. The present results suggested that miR­488 is a negative regulator of psoralen­induced BMSC osteogenic differentiation by targeting Runx2, providing a possible therapeutic target for osteoporosis.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/genética , Ficusina/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ficusina/química , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Psoralea/química , Ratos Sprague-Dawley
13.
J Exp Clin Cancer Res ; 38(1): 346, 2019 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-31395086

RESUMO

BACKGROUND: RUNX2 is a Runt-related transcription factor required during embryogenesis for skeletal development and morphogenesis of other organs including thyroid and breast gland. Consistent evidence indicates that RUNX2 expression is aberrantly reactivated in cancer and supports tumor progression. The mechanisms leading to RUNX2 expression in cancer has only recently began to emerge. Previously, we showed that suppressing the activity of the epigenetic regulators HDACs significantly represses RUNX2 expression highlighting a role for these enzymes in RUNX2 reactivation in cancer. However, the molecular mechanisms by which HDACs control RUNX2 are still largely unexplored. Here, to fill this gap, we investigated the role of different HDACs in RUNX2 expression regulation in breast and thyroid cancer, tumors that majorly rely on RUNX2 for their development and progression. METHODS: Proliferation assays and evaluation of RUNX2 mRNA levels by qRT-PCR were used to evaluate the effect of several HDACi and specific siRNAs on a panel of cancer cell lines. Moreover, ChIP and co-IP assays were performed to elucidate the molecular mechanism underneath the RUNX2 transcriptional regulation. Finally, RNA-sequencing unveiled a new subset of genes whose transcription is regulated by the complex RUNX2-HDAC6. RESULTS: In this study, we showed that Class I HDACs and in particular HDAC1 are required for RUNX2 efficient transcription in cancer. Furthermore, we found an additional and cell-specific function of HDAC6 in driving RUNX2 expression in thyroid cancer cells. In this model, HDAC6 likely stabilizes the assembly of the transcriptional complex, which includes HDAC1, on the RUNX2 P2 promoter potentiating its transcription. Since a functional interplay between RUNX2 and HDAC6 has been suggested, we used RNA-Seq profiling to consolidate this evidence in thyroid cancer and to extend the knowledge on this cooperation in a setting in which HDAC6 also controls RUNX2 expression. CONCLUSIONS: Overall, our data provide new insights into the molecular mechanisms controlling RUNX2 in cancer and consolidate the rationale for the use of HDACi as potential pharmacological strategy to counteract the pro-oncogenic program controlled by RUNX2 in cancer cells.


Assuntos
Comunicação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Regulação Neoplásica da Expressão Gênica , Histona Desacetilases/metabolismo , Neoplasias/etiologia , Neoplasias/metabolismo , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Humanos , Modelos Biológicos , Complexos Multiproteicos , Neoplasias/patologia , Ligação Proteica , RNA Interferente Pequeno/genética , Neoplasias da Glândula Tireoide/etiologia , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Transcrição Genética
14.
Life Sci ; 233: 116746, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31401313

RESUMO

AIM: Diabetes accelerates pro-atherogenic and pro-osteogenic phenotypes of vascular smooth muscle cells (VSMCs), an important process for vascular calcification. Reticulocalbin 2 (RCN2) is a candidate gene for atherosclerosis and involved in vascular remodeling in hypertension. However, the role of RCN2 in VSMCs calcification under diabetic conditions is unclear. MATERIALS AND METHODS: Expression of RCN2 and Runt-related transcription factor 2 (Runx2) in femoropopliteal arterial plaques was compared between type 2 diabetes mellitus (DM) and non-DM patients using immunohistochemical staining (IHCS). Human aortic VSMCs (HAVSMCs) were analyzed under RCN2 gene knockdown and overexpression conditions. Alizarin red staining and intracellular calcium deposition quantification were used to observe calcification induced in vitro under normal glucose or high glucose combined with ß-glycerol phosphoric acid conditions. The cells were investigated for gene modulation of osteogenic differentiation markers using Western blotting. KEY FINDINGS: The expression of RCN2 and Runx2 in femoropopliteal artery plaques was significantly higher in DM than in non-DM patients. In addition, a significant positive correlation was observed between RCN2 and Runx2 levels. RCN2 was highly expressed when HAVSMCs were treated with high glucose and the expression levels correlated with the calcification characteristics. RCN2 upregulated osteogenic transformation markers Runx2 and Osterix in HAVSMCs and downregulated contractile phenotype markers α-SMA and SM22α. SIGNIFICANCE: The results from this study indicate RCN2 is a major factor in mediating the calcification process of HAVSMCs in diabetic conditions. Thus, RCN2 may serve as a future therapeutic target for vascular calcification in diabetes.


Assuntos
Aterosclerose/complicações , Proteínas de Ligação ao Cálcio/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Músculo Liso Vascular/citologia , Osteogênese , Calcificação Vascular/etiologia , Proteínas de Ligação ao Cálcio/genética , Estudos de Casos e Controles , Diferenciação Celular , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Humanos , Músculo Liso Vascular/metabolismo , Calcificação Vascular/metabolismo , Calcificação Vascular/patologia
15.
Artif Cells Nanomed Biotechnol ; 47(1): 3569-3576, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31448639

RESUMO

Osteoporosis-related bone fracture and falls have a severe impact on patients' daily lives. Osteoblasts are bone-building cells that play a vital role in bone formation and remodeling. Imbalanced osteoblast differentiation could lead to osteoporosis. GPR39 is an orphan G protein-coupled receptor that mediates metabolic pathways. In this study, we show that GPR39 is expressed in MC3T3-E1 cells. Osteoblast differentiation culture media induces GPR39, suggesting that GPR39 is a differentiation-responsive factor. Activation of GPR39 using its selective agonist TC-G 1008 induces alkaline phosphatase (ALP), osteocalcin (OCN), and type I collagen (Col-I) expression, and increases cellular ALP activity and calcium deposition, implying that GPR activation promotes cells toward osteoblast differentiation. Treatment with TC-G 1008 also increases Runx-2 expression and AMPK activation. However, the inhibition of AMPK by Compound C abolished TC-G 1008-mediated ALP, OCN, and Col-I induction, and reduces ALP activity and cellular calcium deposition as well as Runx-2 induction. These data indicate that TC-G 1008-mediated GPR39 activation involves AMPK-mediated Runx-2 induction. In summary, our study uncovers a new role of GPR39 activation in osteoblast differentiation, implying that GPR39 could be a promising therapeutic target for osteoporosis.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Minerais/metabolismo , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Pirimidinas/farmacologia , Receptores Acoplados a Proteínas-G/agonistas , Sulfonamidas/farmacologia , Células 3T3 , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo III/metabolismo , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Fosforilação/efeitos dos fármacos
16.
Food Funct ; 10(9): 5350-5360, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31393485

RESUMO

Diabetic osteoporosis (DOP) is a systemic endocrine-metabolic osteopathy which has the characteristics of bone mineral density (BMD) reduction and bone microstructural destruction. Although anthocyanin-rich extract from black rice (AEBR) was reported to have a beneficial effect on diabetic rats, no studies have been performed on whether black rice anthocyanins are beneficial for diabetic osteoporosis. Therefore, in this study, a streptozotocin-induced diabetic rat model was established to investigate the protective effect of AEBR on diabetes-induced osteoporosis and its possible mechanism. AEBR at three doses (0.5, 1.0, and 2.0 g kg-1 d-1) were administered by oral gavage to diabetic rats for 8 weeks. The blood glucose, BMD, bone histomorphometry parameters, serum bone turnover biomarkers, bone marrow adipocyte numbers, as well as osteoprotegerin (OPG), runt-related transcription factor 2 (RUNX 2), and receptor activator of nuclear factor-κ B ligand (RANKL) protein expression in bone and serum were detected. The results indicated that AEBR dose-dependently decreased the blood glucose, increased the BMD, and decreased the serum bone turnover markers. The bone microstructure and osteoclast numbers in bone tissues returned to normal in the high AEBR dosage group; at the same time, the AEBR dose-dependently suppressed bone marrow adipogenesis. The RUNX 2 as well as the OPG/RANKL ratio in diabetic rats' bone tissues increased significantly in the AEBR treatment group. Our results indicate that AEBR administration can ameliorate bone loss caused by diabetes; this is mainly attributed to its inhibition of bone turnover, suppression of bone marrow adipogenesis, and up-regulation of RUNX 2 and the OPG/RANKL expression ratio.


Assuntos
Antocianinas/administração & dosagem , Complicações do Diabetes/tratamento farmacológico , Oryza/química , Osteoporose/tratamento farmacológico , Extratos Vegetais/administração & dosagem , Animais , Antocianinas/análise , Glicemia/metabolismo , Densidade Óssea/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Complicações do Diabetes/genética , Complicações do Diabetes/metabolismo , Complicações do Diabetes/fisiopatologia , Feminino , Humanos , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteoporose/genética , Osteoporose/metabolismo , Osteoporose/fisiopatologia , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Ligante RANK/genética , Ligante RANK/metabolismo , Ratos , Ratos Sprague-Dawley
17.
Chem Biol Interact ; 310: 108750, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31319076

RESUMO

Osteoporosis is a major health concern occurring to the aging adult population across the globe. Currently, there is an increasing demand for treatment of osteoporosis with plant-based medicines. In the present study, we report that heraclenin was extracted and purified from unripe fruit portion of Bael (Aegle marmelos Corr.) using silica gel column chromatography. The identification and characterization of heraclenin were carried out by UV-Vis, HPLC, LC-MS, NMR, FT-IR, and XRD analyses. The standardized purification method recorded a yield efficiency of 42% heraclenin microcrystals with 99% purity from bael fruit. SEM image revealed the shape of the purified compound to be an orthorhombic-sphenoid prism. Cytotoxicity studies indicated that heraclenin-treatment did not alter cell viability in mouse mesenchymal stem cells (mMSCs, C3H10T1/2). The mRNA expression of Runx2, a bone transcription factor was found to be stimulated by heraclenin in these cells. At the cellular level, heraclenin-treatment enhanced osteoblast differentiation and mineralization in mMSCs. Thus, these results suggested that heraclenin purified from bael fruit has an osteogenic effect, indicating its potential towards bone regeneration.


Assuntos
Aegle/química , Furocumarinas/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Furocumarinas/isolamento & purificação , Camundongos , Osteoblastos/citologia , RNA Mensageiro/metabolismo , Espectrofotometria
18.
EBioMedicine ; 46: 330-341, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31331833

RESUMO

BACKGROUND: There is increasing evidence supporting the impact of neoosteogenesis in the pathophysiology of chronic rhinosinusitis (CRS), especially in the recalcitrant group of patients. Runt-related transcription factor 2 (RUNX2), a member of the RUNX family, controls osteoblast differentiation and bone formation. However, the role and regulation of RUNX2 in CRS patients with neoosteogenesis remain unclear. The aim of the study is to determine the role of RUNX2 in neoosteogenesis of CRS patients. METHODS: Sinonasal bone and overlying mucosa samples were obtained from CRS patients with or without neoosteogenesis (n = 67) and healthy controls (n = 11). Double immunofluorescence, immunohistochemistry, and immunoblotting were used to evaluate RUNX2 expression in CRS patients with and without neoosteogenesis. In addition, the osteogenic activity of pro-inflammatory cytokines was examined by measuring alkaline phosphatase (ALP) activity and bone mineralisation in vitro. FINDINGS: RUNX2 was highly expressed in osteoblasts of CRS patients with neoosteogenesis compared with tissues from control subjects and those with CRS without neoosteogenesis. Mucosal extracts from CRS patients with neoosteogenesis showed increased RUNX2 expression and ALP activity in C2C12 cells, whereas those from patients without neoosteogenesis did not. Expression of interleukin (IL)-13 and IL-17A was upregulated in CRS patients with neoosteogenesis. ALP activity and Alizarin Red staining showed IL-13 and IL-17A dose-dependent osteoblast differentiation and mineralisation in vitro. INTERPRETATION: These findings suggested that IL-13- or IL-17A-induced RUNX2 contributed to new bone formation in CRS patients through its effect on the activity of osteoblasts. RUNX2 may be a novel target for preventing neoosteogenesis in CRS patients.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/genética , Interleucina-13/metabolismo , Interleucina-17/metabolismo , Osteogênese/genética , Rinite/etiologia , Rinite/metabolismo , Sinusite/etiologia , Sinusite/metabolismo , Adulto , Animais , Biomarcadores , Cálcio/metabolismo , Linhagem Celular , Citocinas/metabolismo , Suscetibilidade a Doenças , Feminino , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Interleucina-13/antagonistas & inibidores , Interleucina-17/antagonistas & inibidores , Masculino , Camundongos , Pessoa de Meia-Idade , Osteoblastos/metabolismo
19.
Colloids Surf B Biointerfaces ; 182: 110332, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31325776

RESUMO

Micro/nano-topography (MNT) is an important variable affecting osseointegration of bone biomaterials, but the underlying mechanisms are not fully understood. We probed the role of a AMOT130/YAP pathway in osteoblastic differentiation of bone marrow mesenchymal stems cultured on titanium (Ti) carrying MNTs. Ti surfaces with two well-defined MNTs (TiO2 nanotubes of different diameters and wall thicknesses) were prepared by anodization. Rat BMSCs were cultured on flat Ti and Ti surfaces carrying MNTs, and cell behaviors (i.e., morphology, F-actin development, osteoblastic differentiation, YAP localization) were studied. Ti surfaces carrying MNTs increased F-actin formation, osteoblastic gene expression, and protein AMOT130 production in BMSCs (all vs. flat Ti), and the surface carrying larger nantubes was more effective, confirming osteoblastic differentiation induced by MNTs. Elevation of the AMOT130 level (by inhibiting its degradation) increased the osteoblastic gene expression, F-actin formation, and nuclear localization of YAP. These show that, AMOT130/YAP is an important pathway mediating the translation of MNT signals to BMSC osteoblastic commitment, likely via the cascade: AMOT130 promotion of F-actin formation, increased YAP nuclear import, and activation of osteoblastic gene expression.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Mecanotransdução Celular/genética , Proteínas de Membrana/genética , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Titânio/farmacologia , Actinas/genética , Actinas/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Nanotubos/química , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/genética , Osteopontina/genética , Osteopontina/metabolismo , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Propriedades de Superfície , Titânio/química
20.
J Photochem Photobiol B ; 197: 111515, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31255939

RESUMO

An extraordinary arrangement of research is as yet going on in the area of orthopedic implants advancement to determine different issues being looked by the engineering today. In spite of a few detriments of the orthopedic metallic inserts, they keep on being utilized, essentially as a result of their unrivaled mechanical properties. We investigated the conceivable utilization of silicon carbide (SiC) as a nano-ceramic covering material of titanium (Ti)-based all out femoral substitution implants. The thought is to keep wear garbage arrangement from the delicate titanium exterior. Silicon carbide is a hard and firmly holding bio-ceramic surface substance, and in light of these physico-chemical properties, it isn't actually degradable, just like the case with apatite (HA). To improve cytocompatibility and osseous-integration, we deposited anodized titanium nanotubes (TiO2) inserts, by electrochemical deposition method (EDM), with silicon carbide (SiC) with apatite (SiC@HA). The deposition was affirmed by SEM, while phase composition properties were assessed by XRD. Calcium affidavit, osteocalcin creation, and articulation of bone genes were essentially higher in rodent osteoblast cell culture on SiC@HA-covered anodized titanium nanotubes than in cells cultured on uncoated anodized titanium nanotubes. Implantation into rodent femurs likewise demonstrated that the SiC@HA-covered substance had unrivaled osseous-integration movement in correlation with that of customary inserts, as evaluated by in vivo tomography and histology. Therefore, anodized titanium nanotubes covered with SiC@HA holds guarantee as an orthopedic implant substance.


Assuntos
Regeneração Óssea , Compostos Inorgânicos de Carbono/química , Materiais Revestidos Biocompatíveis/química , Durapatita/química , Nanopartículas/química , Compostos de Silício/química , Titânio/química , Animais , Regeneração Óssea/efeitos dos fármacos , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Adesão Celular/efeitos dos fármacos , Materiais Revestidos Biocompatíveis/farmacologia , Materiais Revestidos Biocompatíveis/uso terapêutico , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Fraturas do Fêmur/terapia , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteocalcina/metabolismo , Próteses e Implantes , Ratos
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