RESUMO
Previous studies have demonstrated that G-protein coupled receptor kinase interacting protein-1 (GIT1) and microRNAs (miRNAs) serve an important role in chondrocyte proliferation and migration. However, a limited number of studies conducted thus far have investigated the association between GIT1 and miRNAs. In the present study, putative miR195 binding sites in the GIT1 3'untranslated region were identified using common bioinformatic algorithms (miRanda, TargetScan, miRBase and miRWalk), and it was demonstrated that they may be involved in regulating GIT1 expression. Following transfection of miR195 mimics in chondrocytes, the expression of GIT1 was significantly reduced, whereas the expression was significantly increased following transfection with miR195 inhibitors. In addition, the results of the current study demonstrated that increased miR195 expression may downregulate chondrocyte proliferation and reduce cell migration. However, chondrocyte proliferation and migration was enhanced following suppression of miR195 expression. Furthermore, upon cotransfection of miR195 and GIT1 expression vectors, the inhibitory effect of miR195 on chondrocyte proliferation and migration was attenuated. Therefore, miR195 may affect chondrocyte proliferation and migration via targeted regulation of GIT1 expression. The results of the current study provide novel evidence for the regulatory mechanisms of miRNAs in bone and cartilage tissues, which may facilitate further research and provide a greater understanding of different osteoarticular diseases.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Movimento Celular , Proliferação de Células , Condrócitos/citologia , Regulação para Baixo , MicroRNAs/genética , Linhagem Celular , Condrócitos/metabolismo , HumanosRESUMO
Chondroblastoma (CBL) is a benign bone tumor occurring mostly in teenagers. Despite this, CBL can recur and metastasize after curettage, which may impede normal epiphysis. In search of a novel targeted therapy for CBL, we aimed at BMP-2, a factor critical for chondro-osteogenesis and chondrocyte proliferation. Two pathways upstream of BMP-2, the mTOR and HIF, were targeted with rapamycin (Rapa) and FM19G11 (FM), respectively. Using immunohistochemistry, we found BMP-2 was highly expressed in CBL tissues. CBL cells explanted and confirmed with higher BMP-2 level than normal cartilage. Protumorigenic effect of Rapa and FM on CBL cells were transduced via BMP-2. Combination of Rapa and FM conferred stronger inhibition of cell proliferation than either monotherapy and inhibited levels of chondro-osteogenic markers (Sox9, aggrecan, and type II collagen). To minimize the adverse effect of Rapa, we performed screening in essential amino acids and found leucine deprivation-sensitized CBL cells to Rapa. Combination treatment of low dose Rapa, FM, and leucine deprivation conferred compatible inhibitory effects on CBL cell proliferation, chondro-osteogenic potential, and tumorigenic capacity. We conclude that targeting BMP-2 using mTOR/HIF inhibition could potently curb the disease. Addition of low-leucine diet could lower the dose of rapamycin in chase for less toxicity.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Benzamidas/farmacologia , Proliferação de Células/efeitos dos fármacos , Condroblastoma/tratamento farmacológico , Sirolimo/farmacologia , Adolescente , Adulto , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Condrogênese/efeitos dos fármacos , Regulação para Baixo , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Fator 1 Induzível por Hipóxia/metabolismo , Leucina/metabolismo , Masculino , Osteogênese/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Técnicas de Cultura de Tecidos , Células Tumorais Cultivadas/efeitos dos fármacos , Adulto JovemRESUMO
To evaluate the different traits of mesenchymal stem cell (MSC) isolated from osteosarcoma (OS) and normal bone marrow (BM) induced by bone-morphogenetic protein-2 (BMP-2). MSCs from implanted osteosarcoma or femur bone marrow were isolated and cultured. Differentiation potency was verified and phenotypes were evaluated by flow cytometry. Increased or decreased expressions of BMP-2 were delivered by adenovirus and lentivirus vector, respectively. Expressions of VEGF, EMMPRIN, and MMP-9 were examined. Cell cycle, apoptosis, invasiveness, and proliferation assays were performed between the transfected groups and controls. Increased BMP-2 induced over-expression of VEGF, EMMPRIN, and MMP-9 in OS- and BM-MSCs both intra- and extra-cellularly. Decreased BMP-2 expression induced inhibition of the factors. Increased BMP-2 also induced less population of cells at G1 phase, more apoptotic cells, more cells that invade through Transwell membrane, and faster proliferation in OSMSC compared to those in BMMSC. BMP-2 induced higher expression of tumorigenic factors, which could be responsible for promoting the proliferation and aggressiveness of OSMSC over BMMSC.
