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1.
Biol Res ; 52(1): 52, 2019 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-31540582

RESUMO

BACKGROUND: Long noncoding RNAs (lncRNAs) have been reported to be associated with dermis process during burn wound healing. This study aimed to investigate the role of lncRNA X-inactive specific transcript (XIST) in human skin fibroblasts (HSF) and extracellular matrix (ECM) as well as the regulatory network of XIST/microRNA-29b-3p (miR-29b-3p)/collagen 1 alpha 1 (COL1A1). METHODS: The wound samples were collected from 25 patients with deep partial thickness burn at day 5 after burn. The thermal injured model was established using HSF cells. The expressions of XIST, miR-29b-3p and COL1A1 were measured by quantitative real-time polymerase chain reaction and western blot. ECM synthesis, cell proliferation and migration were detected by western blot, cell counting kit-8 and trans-well assays, respectively. The interaction between miR-29b-3p and XIST or COL1A1 was explored by bioinformatics analysis and luciferase reporter assay. RESULTS: The expressions of XIST and COL1A1 were enhanced but miR-29b-3p expression was decreased after thermal injury. XIST overexpression promoted ECM synthesis, cell proliferation and migration in thermal injured HSF cells. However, XIST knockdown played an opposite effect. miR-29b-3p overexpression inhibited ECM synthesis, cell proliferation and migration, which was reversed by XIST. COL1A1 silence suppressed ECM synthesis, cell proliferation and migration by miR-29b-3p targeting. Moreover, COL1A1 up-regulation weakened the effect of XIST silence on ECM synthesis and HSF cell function. CONCLUSION: XIST promoted ECM synthesis, cell proliferation and migration by sponging miR-29b-3p and targeting COL1A1 in HSF cells after thermal injury, indicating the promoting role of XIST in wound healing.


Assuntos
Queimaduras/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , RNA Longo não Codificante/metabolismo , Western Blotting , Queimaduras/genética , Movimento Celular , Proliferação de Células , Matriz Extracelular/genética , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , Reação em Cadeia da Polimerase em Tempo Real
2.
Life Sci ; 234: 116779, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31430452

RESUMO

Emerging evidence has revealed that microRNAs (miRNAs) play critical roles in keloid pathogenesis. However, potential molecular mechanism of keloid formation remains unclear. In the present study, our findings showed that miR-152-3p mRNA expression level was notably up-regulated in keloid tissues and keloid fibroblasts compared with that of normal skin tissues and normal skin fibroblasts, respectively. Furthermore, miR-152-3p inhibition remarkably suppressed cell proliferation, which was increased by miR-152-3p overexpression. Cell invasion was also significantly decreased by miR-152-3p inhibition, whereas was increased by miR-152-3p overexpression. The mRNA and protein expression levels of extracellular matrix components including type I collagen, type III collagen and fibronectin were decreased by miR-152-3p inhibition, but were increased by miR-152-3p overexpression. In addition, results of dual-luciferase reporter assay indicated that FOXF1 is a direct target of miR-152-3p. FOXF1 overexpression significantly inhibits cell proliferation, invasion, and extracellular matrix in keloid fibroblasts, and the suppressive effects of miR-152-3p mimic on these functions were notably partly reversed by FOXF1 overexpression. Taken together, these findings indicated that miR-152-3p regulates cell proliferation, invasion and extracellular matrix expression through targeting FOXF1 in keloid fibroblasts, suggesting that miR-152-3p is a novel and promising molecular target for keloid treatment.


Assuntos
Matriz Extracelular/patologia , Fibroblastos/patologia , Fatores de Transcrição Forkhead/genética , Queloide/patologia , MicroRNAs/genética , Regiões 3' não Traduzidas , Adolescente , Adulto , Movimento Celular , Proliferação de Células , Células Cultivadas , Matriz Extracelular/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Queloide/genética , Regulação para Cima , Adulto Jovem
3.
Life Sci ; 232: 116637, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31288014

RESUMO

Keloid is characterized by overactive fibroblasts. Forkhead box M1 (FOXM1) is transcription factor that plays important roles in the progression of fibrosis. However, the role of FOXM1 in keloid has not been elucidated. In the present study, we examined the expression levels of FOXM1 in clinical keloid tissue specimens and primary keloid fibroblasts (KFs). The results showed that FOXM1 levels were significantly increased in both keloid tissues and KFs. To further investigate the biological functions of FOXM1, FOXM1 was knocked down in KFs by transfection with small interfering RNA targeting FOXM1 (si-FOXM1). Knockdown of FOXM1 inhibited transforming growth factor-ß1 (TGF-ß1)-induced cell proliferation and migration of KFs. Besides, the increased expressions of collagen (coll I), connective tissue growth factor (CTGF), and α-smooth muscle actin (α-SMA) in TGF-ß1-induced KFs were suppressed by si-FOXM1 transfection. Furthermore, TGF-ß1-induced increase in p-Smad2 and p-Smad3 expressions was attenuated by FOXM1 knockdown. These data indicated that knockdown of FOXM1 inhibited TGF-ß1-induced KFs activation and extracellular matrix (ECM) accumulation, which was attributed to the inhibition of TGF-ß1/Smad pathway.


Assuntos
Proteína Forkhead Box M1/deficiência , Queloide/metabolismo , Actinas/metabolismo , Adolescente , Adulto , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Colágeno/metabolismo , Colágeno Tipo I/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/metabolismo , Proteína Forkhead Box M1/genética , Proteína Forkhead Box M1/metabolismo , Técnicas de Silenciamento de Genes/métodos , Humanos , Queloide/genética , Masculino , Fosforilação , RNA Interferente Pequeno/genética , Transdução de Sinais , Proteína Smad2/antagonistas & inibidores , Proteína Smad2/metabolismo , Proteína Smad3/antagonistas & inibidores , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta1/metabolismo
4.
Tumour Biol ; 41(5): 1010428319847081, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31122159

RESUMO

Type 1 collagen is an important part of the extracellular matrix and changes in its metabolism and distribution are essential in breast cancer induction and progression. Serum concentrations of type 1 collagen synthesis (aminoterminal propeptide (PINP)) and degradation markers (carboxyterminal telopeptide (ICTP)) have previously been studied in early and metastatic breast cancer, but no data are available on specific breast cancer subtypes. We assayed 662 preoperative serum samples for PINP and ICTP and 109 postoperative serum samples for ICTP. The results were linked to prospectively collected clinical data and the cases were divided into breast cancer subtypes for survival analyses. The concentrations of both pre- and postoperative ICTP serum levels increased linearly from ductal in situ carcinoma to stage I-II tumors, stage III tumors, and finally to those with concomitant primary metastases (preoperative ICTP, p = 0.009; postoperative ICTP, p = 0.016). High-preoperative ICTP levels were associated with better breast cancer-specific survival in connection with luminal-B-like (HER2-negative) tumors (p = 0.017), which was confirmed in Cox regression analysis (relative risk = 3.127; 95% confidence interval = 1.081-9.049, p = 0.035), when T-class (relative risk = 4.049; 95% confidence interval = 1.263-12.981; p = 0.019) and nodal status (relative risk = 3.896; 95% confidence interval = 1.088-13.959; p = 0.037) were included in the analysis. In patients with triple-negative breast cancer, a high-preoperative ICTP level was a significant predictor of local relapse-free survival in univariate (p = 0.0020) and multivariate analyses (relative risk = 13.04; 95% confidence interval = 1.354-125.5; p = 0.026; for T-class, relative risk = 2.128 and 95% confidence interval = 0.297-15.23; p = 0.452; for N-class, relative risk = 0.332 and 95% confidence interval = 0.033-3.307; p = 0.347). A preoperatively elevated serum ICTP level appears to be an important marker of better prognosis in triple-negative breast cancer and luminal-B-like (HER2-negative) subtypes.


Assuntos
Biomarcadores Tumorais/sangue , Colágeno Tipo I/sangue , Neoplasias de Mama Triplo Negativas/sangue , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Intervalo Livre de Doença , Matriz Extracelular/genética , Feminino , Humanos , Masculino , Estadiamento de Neoplasias , Fragmentos de Peptídeos/sangue , Peptídeos/sangue , Período Pré-Operatório , Pró-Colágeno/sangue , Prognóstico , Modelos de Riscos Proporcionais , Receptor ErbB-2/genética , Resultado do Tratamento , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia
5.
Curr Pharm Biotechnol ; 20(6): 517-524, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31057106

RESUMO

BACKGROUND: The study aimed to investigate the effects of the active ingredient, nimodipine, on chondrocyte proliferation and extracellular matrix (ECM) structures in cartilage tissue cells. METHODS: Chondrocyte cultures were prepared from tissues resected via surgical operations. Nimodipine was then applied to these cultures and molecular analysis was performed. The data obtained were statistically calculated. RESULTS: Both, the results of the (3-(4,5 dimethylthiazol2-yl)-2,5-diphenyltetrazolium (MTT) assay and the fluorescence microscope analysis [a membrane permeability test carried out with acridine orange/ propidium iodide staining (AO/PI)] confirmed that the active ingredient, nimodipine, negatively affects the cell cultures. CONCLUSION: Nimodipine was reported to suppress cellular proliferation; chondroadherin (CHAD) and hypoxia-inducible factor-1 alpha (HIF-1α) expression thus decreased by 2.4 and 1.7 times, respectively, at 24 hrs when compared to the control group (p < 0.05). Furthermore, type II collagen (COL2A1) expression was not detected (p < 0.05). The risk that a drug prescribed by a clinician in an innocuous manner to treat a patient by relieving the symptoms of a disease may affect the proliferation, differentiation, and viability of other cells and/or tissues at the molecular level, beyond its known side effects or adverse events, should not be forgotten.


Assuntos
Bloqueadores dos Canais de Cálcio/toxicidade , Proliferação de Células/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Nimodipina/toxicidade , Cartilagem/efeitos dos fármacos , Cartilagem/patologia , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/patologia , Colágeno Tipo II/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/patologia , Proteínas da Matriz Extracelular/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Pessoa de Meia-Idade , Cultura Primária de Células
6.
Biomed Res Int ; 2019: 3638469, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31058187

RESUMO

Eosinophil asthma is characterized by the infiltration of eosinophils to the bronchial epithelium. The toxic cationic protein released by eosinophils, mainly major basic protein (MBP), is one of the most important causative factors of epithelium damage. Poly-L-Arginine (PLA) is a kind of synthetic cationic polypeptides, which is widely used to mimic the effects of MBP on epithelial cells in vitro. However, little is known about the changes of differentially expressed genes (DEGs) and transcriptome profiles in cationic protein stimulated epithelial cells. In this study, we compared the expression of DEGs and transcriptome profiles between PLA-treated airway epithelial cells NCI-H292 and control. The results showed that there were a total of 230 DEGs, of which 86 were upregulated and 144 were downregulated. These DEGs were further analyzed using gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The results showed that the upregulated DEGs were involved in cholesterol synthesis, protein binding, and composition of cellular membranes, mainly enriched in metabolic and biosynthesis pathways. While downregulated DEGs were implicated in cell adhesion, extracellular matrix (ECM) composition and cytoskeleton and were enriched in ECM pathway. In conclusion, our research provided the mechanism of the cationic polypeptides acting on the airway epithelial cells on the basis of transcriptomic profile, and this could be regarded as important indications in unveiling the pathologic role of natural cationic proteins in the damage to epithelial cells of asthmatics.


Assuntos
Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Pulmão/metabolismo , Transcriptoma/genética , Cátions/farmacologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Colesterol/genética , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/genética , Redes Reguladoras de Genes/genética , Humanos , Pulmão/efeitos dos fármacos , Peptídeos/farmacologia , Sequenciamento Completo do Exoma
7.
J Mol Histol ; 50(3): 285-294, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30993430

RESUMO

The aim of this study was to ascertain whether, like many cell types in cartilaginous tissues if type XI collagen was a pericellular component of annulus fibrosus (AF) cells and chondrocytes. Fine fibrillar networks were visualised which were perlecan, HS (MAb 10E4) and type XI collagen positive. Heparitinase-III pre-digestion abolished the type XI collagen and 10E4 localisation in these fibrillar assemblies demonstrating a putative HS mediated interaction which localised the type XI collagen. Type XI collagen was confirmed to be present in the Heparitinase III treated AF monolayer media samples by immunoblotting. Heparitinase-III generated ΔHS stub epitopes throughout these fibrillar networks strongly visualised by MAb 3-G-10. Monolayers of murine hip articular chondrocytes from C57BL/6 and Hspg2 exon 3 null mice also displayed pericellular perlecan localisations, however type XI collagen was only evident in the Wild type mice. Perlecan was also immunolocalised in control and murine knee articular cartilage from the two mouse genotypes subjected to a medial meniscal destabilisation procedure which induces OA. This resulted in a severe depletion of perlecan levels particularly in the perlecan exon 3 null mice and was consistent with OA representing a disease of the pericellular matrix. A model was prepared to explain these observations between the NPP type XI collagen domain and HS chains of perlecan domain-I in the pericellular matrix of AF cells which likely contributed to cellular communication, tissue stabilization and the regulation of extracellular matrix homeostasis.


Assuntos
Anel Fibroso/efeitos dos fármacos , Colágeno Tipo XI/genética , Proteoglicanas de Heparan Sulfato/genética , Animais , Anel Fibroso/metabolismo , Anel Fibroso/patologia , Cartilagem Articular/crescimento & desenvolvimento , Cartilagem Articular/metabolismo , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Éxons/genética , Matriz Extracelular/genética , Proteínas da Matriz Extracelular/genética , Homeostase/genética , Disco Intervertebral/crescimento & desenvolvimento , Disco Intervertebral/metabolismo , Camundongos , Polissacarídeo-Liase/farmacologia
8.
Cancer Sci ; 110(6): 1959-1973, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31004547

RESUMO

Activation of transforming growth factor ß (TGF-ß) combined with persistent hypoxia often affects the tumor microenvironment. Disruption of cadherin/catenin complexes induced by these stimulations yields aberrant extracellular matrix (ECM) production, characteristics of epithelial-mesenchymal transition (EMT). Hypoxia-inducible factors (HIF), the hallmark of the response to hypoxia, play differential roles during development of diseases. Recent studies show that localization of cadherin/catenin complexes at the cell membrane might be tightly regulated by protein phosphatase activity. We aimed to investigate the role of stabilized HIF-1α expression by protein phosphatase activity on dissociation of the E-cadherin/ß-catenin complex and aberrant ECM expression in lung cancer cells under stimulation by TGF-ß. By using lung cancer cells treated with HIF-1α stabilizers or carrying doxycycline-dependent HIF-1α deletion or point mutants, we investigated the role of stabilized HIF-1α expression on TGF-ß-induced EMT in lung cancer cells. Furthermore, the underlying mechanisms were determined by inhibition of protein phosphatase activity. Persistent stimulation by TGF-ß and hypoxia induced EMT phenotypes in H358 cells in which stabilized HIF-1α expression was inhibited. Stabilized HIF-1α protein expression inhibited the TGF-ß-stimulated appearance of EMT phenotypes across cell types and species, independent of de novo vascular endothelial growth factor A (VEGFA) expression. Inhibition of protein phosphatase 2A activity abrogated the HIF-1α-induced repression of the TGF-ß-stimulated appearance of EMT phenotypes. This is the first study to show a direct role of stabilized HIF-1α expression on inhibition of TGF-ß-induced EMT phenotypes in lung cancer cells, in part, through protein phosphatase activity.


Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Fator de Crescimento Transformador beta1/farmacologia , Animais , Hipóxia Celular , Linhagem Celular , Linhagem Celular Transformada , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Estabilidade Proteica , Interferência de RNA , Ratos
9.
Methods Mol Biol ; 1952: 21-31, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30825162

RESUMO

The gene expression of the extracellular matrix macromolecules is critical in the analysis of various pathologies. The use of a RT-PCR directly on a fixed tissue enables the recognition of the real expressing cells for any ECM molecules together with the tissue localization. The method here described is easy to perform using the same material as for common immunostaining and the same primers used for quantitative RT-PCR. Moreover, the used primers, designed with a final amplicon that spans the exon-exon junction, allow to detect the cDNA but not the gDNA sequences.


Assuntos
Proteínas da Matriz Extracelular/genética , Expressão Gênica , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Linhagem Celular , DNA Complementar/genética , Matriz Extracelular/genética , Humanos
10.
PLoS Pathog ; 15(3): e1007618, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30870530

RESUMO

RsaE is a conserved small regulatory RNA (sRNA) which was previously reported to represent a riboregulator of central carbon flow and other metabolic pathways in Staphylococcus aureus and Bacillus subtilis. Here we show that RsaE contributes to extracellular (e)DNA release and biofilm-matrix switching towards polysaccharide intercellular adhesin (PIA) production in a hypervariable Staphylococcus epidermidis isolate. Transcriptome analysis through differential RNA sequencing (dRNA-seq) in combination with confocal laser scanning microscopy (CLSM) and reporter gene fusions demonstrate that S. epidermidis protein- and PIA-biofilm matrix producers differ with respect to RsaE and metabolic gene expression. RsaE is spatiotemporally expressed within S. epidermidis PIA-mediated biofilms, and its overexpression triggers a PIA biofilm phenotype as well as eDNA release in an S. epidermidis protein biofilm matrix-producing strain background. dRNA-seq and Northern blot analyses revealed RsaE to exist as a major full-length 100-nt transcript and a minor processed species lacking approximately 20 nucleotides at the 5'-end. RsaE processing results in expansion of the mRNA target spectrum. Thus, full-length RsaE interacts with S. epidermidis antiholin-encoding lrgA mRNA, facilitating bacterial lysis and eDNA release. Processed RsaE, however, interacts with the 5'-UTR of icaR and sucCD mRNAs, encoding the icaADBC biofilm operon repressor IcaR and succinyl-CoA synthetase of the tricarboxylic acid (TCA) cycle, respectively. RsaE augments PIA-mediated biofilm matrix production, most likely through activation of icaADBC operon expression via repression of icaR as well as by TCA cycle inhibition and re-programming of staphylococcal central carbon metabolism towards PIA precursor synthesis. Additionally, RsaE supports biofilm formation by mediating the release of eDNA as stabilizing biofilm matrix component. As RsaE itself is heterogeneously expressed within biofilms, we consider this sRNA to function as a factor favoring phenotypic heterogeneity and supporting division of labor in S. epidermidis biofilm communities.


Assuntos
Matriz Extracelular/genética , Pequeno RNA não Traduzido/metabolismo , Staphylococcus epidermidis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , Matriz Extracelular/fisiologia , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/genética , Óperon/genética , Fenótipo , Polissacarídeos Bacterianos/genética , Polissacarídeos Bacterianos/metabolismo , Pequeno RNA não Traduzido/genética , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/metabolismo , Staphylococcus/genética , Staphylococcus epidermidis/metabolismo
11.
Int J Mol Sci ; 20(6)2019 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-30884785

RESUMO

Fibrosis is characterized by excessive deposition of the extracellular matrix and develops because of fibroblast differentiation during the process of inflammation. Various cytokines stimulate resident fibroblasts, which differentiate into myofibroblasts. Myofibroblasts actively synthesize an excessive amount of extracellular matrix, which indicates pathologic fibrosis. Although initial fibrosis is a physiologic response, the accumulated fibrous material causes failure of normal organ function. Cardiac fibrosis interferes with proper diastole, whereas pulmonary fibrosis results in chronic hypoxia; liver cirrhosis induces portal hypertension, and overgrowth of fibroblasts in the conjunctiva is a major cause of glaucoma surgical failure. Recently, several reports have clearly demonstrated the functional relevance of certain types of histone deacetylases (HDACs) in various kinds of fibrosis and the successful alleviation of the condition in animal models using HDAC inhibitors. In this review, we discuss the therapeutic potential of HDAC inhibitors in fibrosis-associated human diseases using results obtained from animal models.


Assuntos
Fibrose/tratamento farmacológico , Inibidores de Histona Desacetilases/uso terapêutico , Inflamação/tratamento farmacológico , Diferenciação Celular/efeitos dos fármacos , Matriz Extracelular/genética , Matriz Extracelular/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Fibrose/genética , Histona Desacetilases/genética , Humanos , Hipertensão Portal/tratamento farmacológico , Hipertensão Portal/genética , Hipertensão Portal/patologia , Inflamação/genética , Inflamação/patologia , Modelos Animais , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/patologia
12.
Med Sci Monit ; 25: 1952-1959, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30873956

RESUMO

BACKGROUND MicroRNAs (miRNAs) have emerged as central regulators of many processes. MiRNA-34 (miR-34) functions as a well-known tumor suppressor. The aim of this study was to investigate the mechanisms underlying how miR-34 participates in vascular disorders. MATERIAL AND METHODS Three miR-34 family members (miR-34a, miR-34b, and miR-34c) were overexpressed or silenced in human vascular smooth muscle cells (VSMCs) and umbilical vein endothelial cells (UVECs), respectively, before the proliferation and apoptosis of cells were detected by using Cell Counting Kit-8 assay and Annexin V- fluorescein isothiocyanate/propidium iodide flow cytometry. The protein expression of apoptosis biomarkers was detected by western blot. Dual-luciferase reporter assay was performed to determine the candidate target of miR-34, and enzyme-linked immune sorbent assay was used to evaluate the effect of miR-34 on the expression of the target gene. RESULTS Overexpression of miR-34 family members repressed proliferation and promoted apoptosis of VSMCs and UVECs, whereas miR-34 knockdown led to the opposite results. In addition, miR-34a inhibited the expression of alpha-1 antitrypsin (AAT), a serine protease inhibitor that suppresses the degradation of extracellular matrix, through a miR-34-binding site within the 3'-UTR of AAT. CONCLUSIONS MiR-34 promoted apoptosis of VSMC and UVEC cells by inhibiting AAT expression. This finding provides an update on the understanding of the clinical value of miR-34, which might assist to uncover novel and effective therapeutic strategies for the treatment of vascular diseases.


Assuntos
MicroRNAs/genética , MicroRNAs/fisiologia , Doenças Vasculares/genética , Regiões 3' não Traduzidas/genética , Apoptose/genética , Movimento Celular/genética , Proliferação de Células/genética , China , Matriz Extracelular/genética , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiologia , Neoplasias/genética
13.
Int J Mol Sci ; 20(3)2019 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-30709061

RESUMO

Hyaline cartilage is a tissue of very low regenerative capacity because of its histology and limited nutrient supply. Cell-based therapies have been spotlighted in the regeneration of damaged cartilage. Dental pulp stem cells (DPSCs) are multipotent and are easily accessible for therapeutic purposes. In human gastrointestinal tracts, Enterococcus faecium is a naturally occurring commensal species of lactic acid bacteria. In this work, the human DPSCs were differentiated into chondrocytes using a chondrogenic differentiation medium with or without L-15 extract. We observed that chondrogenic differentiation improved in an E. faecium L-15 extract (L-15)-treated DPSC group via evaluation of chondrogenic-marker mRNA expression levels. In particular, we found that L-15 treatment promoted early-stage DPSC differentiation. Cells treated with L-15 were inhibited at later stages and were less likely to transform into hypertrophic chondrocytes. In L-15-treated groups, the total amount of cartilage extracellular matrix increased during the differentiation process. These results suggest that L-15 promotes chondrogenic differentiation, and that L-15 may be used for cartilage repair or cartilage health supplements. To our knowledge, this is the first report demonstrating the beneficial effect of L-15 treatment on chondrogenic differentiation.


Assuntos
Condrogênese , Meios de Cultura/farmacologia , Polpa Dentária/citologia , Enterococcus faecium/crescimento & desenvolvimento , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Sistema Livre de Células , Células Cultivadas , Meios de Cultura/química , Polpa Dentária/efeitos dos fármacos , Enterococcus faecium/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Marcadores Genéticos , Humanos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos
14.
Pain ; 160(4): 932-944, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30763288

RESUMO

Chronic pain is a debilitating and poorly treated condition whose underlying mechanisms are poorly understood. Nerve injury and inflammation cause alterations in gene expression in tissues associated with pain processing, supporting molecular and cellular mechanisms that maintain painful states. However, it is not known whether transcriptome changes can be used to reconstruct a molecular pathophysiology of pain. In the current study, we identify molecular pathways contributing to chronic pain states through the analysis of global changes in the transcriptome of dorsal root ganglia, spinal cord, brain, and blood in mouse assays of nerve injury- and inflammation-induced pain. Comparative analyses of differentially expressed genes identified substantial similarities between 2 animal pain assays and with human low-back pain. Furthermore, the extracellular matrix (ECM) organization has been found the most commonly regulated pathway across all tested tissues in the 2 animal assays. Examination of human genome-wide association study data sets revealed an overrepresentation of differentially expressed genes within the ECM organization pathway in single nucleotide polymorphisms most strongly associated with human back pain. In summary, our comprehensive transcriptomics analysis in mouse and human identified ECM organization as a central molecular pathway in the development of chronic pain.


Assuntos
Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Inflamação/genética , Inflamação/patologia , Neuralgia/genética , Neuralgia/patologia , Animais , Modelos Animais de Doenças , Feminino , Adjuvante de Freund/toxicidade , Redes Reguladoras de Genes/genética , Estudos de Associação Genética , Testes Genéticos , Humanos , Inflamação/induzido quimicamente , Camundongos , Camundongos Endogâmicos BALB C , Medição da Dor , Polimorfismo de Nucleotídeo Único/genética , RNA Mensageiro/metabolismo , Transcriptoma/fisiologia
15.
Biomed Res Int ; 2019: 6159490, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30809544

RESUMO

Miniature pigs, a valuable alternative model for understanding human tooth development, have deciduous teeth from all four tooth families that are replaced once by permanent molars. The extracellular matrix (ECM) supports cells and maintains the integrity of tooth germs during tooth development. However, details on the role of the ECM in tooth development are poorly understood. Here, we performed long noncoding RNA (lncRNA) and messenger RNA (mRNA) expression profiles in the ECM components of deciduous tooth germs by RNA sequencing in miniature pigs. From the early cap to the late bell stages, we identified 4,562 and 3,238 differentially expressed genes (DEGs) from E40 to E50 and E50 to E60, respectively. In addition, a total of 1,464 differentially expressed lncRNAs from E40 to E50 and 969 differentially expressed lncRNAs from E50 to E60 were obtained. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that DEGs were enriched significantly for multiple signaling pathways, especially for the ECM pathway. We then outlined the detailed dynamic gene expression profiling of ECM components during deciduous molar development. Comparison of the cap and bell stages revealed that the structure and functions of the ECM dynamically changed. The ECM-related genes, including THBS1, COL4A5, COL4A6, COL1A1, CHAD, TNR, GP1BA, and ITGA3, were significantly changed, and some were shown to enrich during the bell stage development. Finally, we outlined the coexpression of lncRNAs and ECM properties during tooth development. We showed that the interplay of key lncRNAs could change ECM processes and influence the ECM establishment of tooth patterns to accomplish full tooth formation. These results might provide information to elucidate the regulation network of the lncRNA and ECM in tooth development.


Assuntos
Matriz Extracelular/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Dente Decíduo/crescimento & desenvolvimento , Animais , Regulação da Expressão Gênica no Desenvolvimento/genética , Redes Reguladoras de Genes/genética , Humanos , Dente Molar/crescimento & desenvolvimento , Suínos , Porco Miniatura , Germe de Dente/crescimento & desenvolvimento , Dente Decíduo/metabolismo
16.
Nano Lett ; 19(4): 2280-2290, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30775927

RESUMO

Cancer cell invasion through physical barriers in the extracellular matrix (ECM) requires a complex synergy of traction force against the ECM, mechanosensitive feedback, and subsequent cytoskeletal rearrangement. PDMS microchannels were used to investigate the transition from mesenchymal to amoeboid invasion in cancer cells. Migration was faster in narrow 3 µm-wide channels than in wider 10 µm channels, even in the absence of cell-binding ECM proteins. Cells permeating narrow channels exhibited blebbing and had smooth leading edge profiles, suggesting an ECM-induced transition from mesenchymal invasion to amoeboid invasion. Live cell labeling revealed a mechanosensing period in which the cell attempts mesenchymal-based migration, reorganizes its cytoskeleton, and proceeds using an amoeboid phenotype. Rho/ROCK (amoeboid) and Rac (mesenchymal) pathway inhibition revealed that amoeboid invasion through confined environments relies on both pathways in a time- and ECM-dependent manner. This demonstrates that cancer cells can dynamically modify their invasion programming to navigate physically confining matrix conditions.


Assuntos
Citoesqueleto/efeitos dos fármacos , Mesoderma/efeitos dos fármacos , Invasividade Neoplásica/genética , Neoplasias/genética , Fenômenos Biomecânicos , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Citoesqueleto/genética , Dimetilpolisiloxanos/química , Dimetilpolisiloxanos/farmacologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/genética , Humanos , Mesoderma/patologia , Invasividade Neoplásica/patologia , Neoplasias/patologia , Nylons/química , Nylons/farmacologia
17.
Life Sci ; 221: 274-283, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30797016

RESUMO

AIMS: Lower back pain is often associated with intervertebral disc degeneration (IDD), which results from a decrease in nucleus pulposus (NP) cells and an imbalance between the degradation and synthesis of extracellular matrix (ECM) components. Multiple microRNAs play crucial roles in the modulation of NP cell apoptosis and matrix degradation. miR-145 is an important microRNA related to degenerative diseases such as osteoarthritis. Here, the effect of miR-145 in IDD was elucidated. The aim of this study was to explore the role and mechanism of miR-145 in the apoptosis of NP cells and in matrix metabolism in NP cells. MATERIALS AND METHODS: Real-time PCR, western blotting and flow cytometry analysis were used to observe the effect of miR-145 on NP cell apoptosis in the absence or presence of oxidative stress. Cell transfection, loss-of-function experiments using an ADAM17 inhibitor or lentiviral shADAM17, immunofluorescence, real-time PCR and western blotting were performed to demonstrate the role and mechanism of miR-145 in NP cell matrix metabolism. KEY FINDINGS: miR-145 attenuated NP cell apoptosis in the absence and presence of oxidative stress. Moreover, miR-145 overexpression increased and miR-145 suppression decreased matrix synthesis. ADAM17, which is expressed in degenerative discs, is the target of miR-145. ADAM17 gene suppression with lentiviral shRNA or an inhibitor enhanced matrix synthesis in NP cells. In addition, siADAM17 reversed the matrix degradation induced by miR-145 inhibition. SIGNIFICANCE: miR-145 suppresses apoptosis and promotes ECM synthesis in NP cells. miR-145 is thus a potential therapeutic microRNA for IDD.


Assuntos
MicroRNAs/genética , MicroRNAs/metabolismo , Núcleo Pulposo/metabolismo , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Animais , Apoptose/genética , Células Cultivadas , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Humanos , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/metabolismo , Ratos , Ratos Sprague-Dawley
18.
Tissue Cell ; 56: 107-113, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30736898

RESUMO

Limited self-restorative ability of the cartilage has necessitated the use of cell and tissue engineering based therapies. Recent advances in the isolation, expansion and characterization of articular cartilage derived chondroprogenitors(CPs) has gained popularity in its role for cartilage repair. Platelet rich plasma (PRP) is a reliable biological scaffold for in-vitro and in-vivo studies with reported therapeutic applications in cartilage and bone pathologies. The aim of this study was to evaluate whether human allogeneic PRP could serve as a biological scaffold for chondroprogenitors (CPs) in cartilage repair. CPs were isolated from the superficial layer of three osteoarthritic knee joints by fibronectin adhesion assay and characterized using flow cytometric analysis. Allogeneic citrated blood was harvested from three subjects to obtain PRP. CPs at a concentration of one million cells per ml were gelled with PRP using calcium chloride. The PRP-CP scaffolds were subjected for adipogeneic, osteogenic, chondrogeneic differentiation and processed for post differentiation-staining studies (Oil Red O, Von Kossa, Alcian blue staining), immunofluorescence (collagen II) and live dead assays (Calcein AM-Ethidium Homodimer). We show that PRP was able to sustain CP cell viability and differentiate towards adipogenic, osteogenic and chondrogenic lineage under appropriate culture conditions. We also noted positive extracellular matrix production in PRP-CP scaffolds cultured without chondrogenic supplementation. Our results suggest that PRP could be a promising bio-active scaffold due to its synergistic effect in supporting cell proliferation, maintaining cell viability and favoring extracellular matrix production. PRP can be used as biological scaffold for the delivery of CPs in cartilage healing.


Assuntos
Cartilagem Articular/crescimento & desenvolvimento , Condrogênese/genética , Plasma Rico em Plaquetas/citologia , Tecidos Suporte , Diferenciação Celular/genética , Condrócitos/citologia , Matriz Extracelular/genética , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Plasma Rico em Plaquetas/metabolismo
19.
Tissue Cell ; 56: 71-78, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30736907

RESUMO

The interactions between cells and the extracellular matrix (ECM) play a major role in normal and pathological conditions. The ECM can modulate several biological functions including cell proliferation, adhesion, differentiation and survival through its interactions with cell receptors. Laminins are one of the most important glycoproteins present in basement membranes, a type of ECM. The pattern of expression of its different isoforms depends on the spatiotemporal organization of each tissue. While integrins are the most studied laminin receptors, other non-integrin laminin receptors are also involved. This review focuses on two particular non-integrin laminin receptors in the epithelial context: dystroglycan and 37/67 laminin receptor (37/67LR). Dystroglycan is a two-subunit protein discovered in the muscle as part of the dystrophin-associated glycoprotein complex. This protein can also be found in many epithelia where its roles are variable. The 37/67LR is a still incompletely understood laminin receptor that is important to regulate intestinal epithelial cell function and could be involved in various pathological conditions.


Assuntos
Proliferação de Células/genética , Distroglicanas/genética , Laminina/genética , Receptores de Laminina/genética , Membrana Basal/metabolismo , Diferenciação Celular/genética , Células Epiteliais/metabolismo , Epitélio/metabolismo , Matriz Extracelular/genética , Humanos , Integrinas/genética
20.
Nat Commun ; 10(1): 845, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30783087

RESUMO

Cell metabolism is strongly influenced by mechano-environment. We show here that a fraction of kindlin-2 localizes to mitochondria and interacts with pyrroline-5-carboxylate reductase 1 (PYCR1), a key enzyme for proline synthesis. Extracellular matrix (ECM) stiffening promotes kindlin-2 translocation into mitochondria and its interaction with PYCR1, resulting in elevation of PYCR1 level and consequent increase of proline synthesis and cell proliferation. Depletion of kindlin-2 reduces PYCR1 level, increases reactive oxygen species (ROS) production and apoptosis, and abolishes ECM stiffening-induced increase of proline synthesis and cell proliferation. In vivo, both kindlin-2 and PYCR1 levels are markedly increased in lung adenocarcinoma. Ablation of kindlin-2 in lung adenocarcinoma substantially reduces PYCR1 and proline levels, and diminishes fibrosis in vivo, resulting in marked inhibition of tumor growth and reduction of mortality rate. Our findings reveal a mechanoresponsive kindlin-2-PYCR1 complex that links mechano-environment to proline metabolism and signaling, and suggest a strategy to inhibit tumor growth.


Assuntos
Adenocarcinoma de Pulmão/metabolismo , Proteínas do Citoesqueleto/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Proteínas de Neoplasias/metabolismo , Prolina/biossíntese , Células A549 , Adenocarcinoma de Pulmão/patologia , Animais , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Proteínas do Citoesqueleto/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , Proteínas de Membrana/genética , Camundongos Transgênicos , Mitocôndrias/metabolismo , Proteínas Musculares/genética , Proteínas de Neoplasias/genética , Pirrolina Carboxilato Redutases/genética , Pirrolina Carboxilato Redutases/metabolismo
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