RESUMO
Regulation of microRNAs (miRNA) has been extensively investigated in diseases; however, little is known about the roles of miRNAs in cleidocranial dysplasia (CCD). The aim of the present study was to investigate the potential involvement of miRNAs in CCD. In vitro site-directed mutagenesis was performed to construct three mutant Runx2 expression vectors, which were then transfected into LS8 cells and MC3T3-E1 cells, to determine the impact on amelogenesis and osteogenesis, respectively. miRCURY LNA miRNA microarray identify miR-185-5p as a miRNA target commonly induced by all three Runx2 mutants. Real-time quantitative PCR was applied to determine the expression of miR-185-5p and Dlx2 in samples. Dual-luciferase reporter assays were conducted to confirm Dlx2 as a legitimate target of miR-185-5p. The suppressive effect of miR-185-5p on amelogenesis and osteogenesis of miR-185-5p was evaluated by RT-PCR and western blot examination of Amelx, Enam, Klk4, and Mmp20 gene and protein expression, and by Alizarin Red stain. We found that mutant Runx2 suppressed amelogenesis and osteogenesis. miR-185-5p, induced by Runx2, suppressed amelogenesis and osteogenesis. Furthermore, we identified Dlx2 as direct target of miR-185-5p. Consistently, Dlx2 expression was inversely correlated with miR-185-5p levels. This study highlights the molecular etiology and significance of miR-185-5p in CCD, and suggests that targeting miR-185-5p may represent a new therapeutic strategy in prevention or intervention of CCD.
Assuntos
Amelogênese/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Proteínas de Homeodomínio/genética , MicroRNAs/genética , Mutação , Osteogênese/genética , Fatores de Transcrição/genética , Ameloblastos/metabolismo , Ameloblastos/patologia , Amelogenina/genética , Amelogenina/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Displasia Cleidocraniana/genética , Displasia Cleidocraniana/metabolismo , Displasia Cleidocraniana/patologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Células HEK293 , Proteínas de Homeodomínio/metabolismo , Humanos , Calicreínas/genética , Calicreínas/metabolismo , Luciferases/genética , Luciferases/metabolismo , Metaloproteinase 20 da Matriz/genética , Metaloproteinase 20 da Matriz/metabolismo , Camundongos , MicroRNAs/metabolismo , Modelos Biológicos , Osteoblastos/metabolismo , Osteoblastos/patologia , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
Both benign prostatic hyperplasia (BPH) and prostate cancer (PC) are common diseases for men around the world. Both serine/threonine protein kinase MST4 (MST4) and serine/threonine kinase 25 (STK25) belong to the Ste20-like kinases and interact with programmed cell death 10 (PDCD10) which is closely linked to cancer diseases. To clarify the roles of MST4, STK25 and PDCD10 in prostate carcinogenesis, we examined MST4, STK25 and PDCD10 expression in tissue microarray blocks containing 110 cores of BPH and 160 cores of PC immunohistochemically and evaluated their correlation with clinicopathological findings. MST4 was not expressed in all the BPH cases and expressed in 38.7% of PC cases (P < 0.0001). STK25 expression was found in 77.3% of BPH cases and 93.1% of PC cases (P < 0.0001). PDCD10 staining was considered weak in 82 (74.5%) and strong in 28 (25.5%) of BPH cases. However, in prostate cancer cases, PDCD10 staining was weak in 95 (59.4%) and strong in 65 (40.6%) (P < 0.05). PDCD10 and STK25 immunostaining were associated with age in prostatic hyperplasia cases (P < 0.05). The staining intensity for STK25 was significantly greater in Gleason grades 3-5 (47.1% of such cases staining strongly) compared with other grades of prostate cancer (only 26.5% of these cases staining strongly; P < 0.05). Our results suggest that MST4, STK25 and PDCD10 are unregulated in prostate cancer and may play roles in prostate tumorigenesis. MST4 may be a helpful marker for identifying prostate cancer.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Hiperplasia Prostática/diagnóstico , Neoplasias da Próstata/diagnóstico , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Diagnóstico Diferencial , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Regulação para Cima , Adulto JovemRESUMO
Maintenance of FOXP3 protein expression is crucial for differentiation and maturation of regulatory T (Treg) cells, which play important roles in immune homeostasis and immune tolerance. We demonstrate here that PDCD5 interacts with FOXP3, increases acetylation of FOXP3 in synergy with Tip60 and enhances the repressive function of FOXP3. In PDCD5 transgenic (PDCD5tg) mice, overexpression of PDCD5 enhanced the level of FOXP3 protein and percentage of CD4(+)CD25(+)FOXP3(+) cells. Naïve CD4(+) T cells from PDCD5tg mice were more sensitive to TGF-ß-induced Treg polarization and expansion. These induced Tregs retained normal suppressive function in vitro. Severity of experimentally-induced autoimmune encephalomyelitis (EAE) in PDCD5tg mice was significantly reduced relative to that of wild-type mice. The beneficial effect of PDCD5 likely resulted from increases of Treg cell frequency, accompanied by a reduction of the predominant pathogenic Th17/Th1 response. Activation-induced cell death enhanced by PDCD5 was also linked to this process. This is the first report revealing that PDCD5 activity in T cells suppresses autoimmunity by modulating Tregs. This study suggests that PDCD5 serves as a guardian of immunological functions and that the PDCD5-FOXP3-Treg axis may be a therapeutic target for autoimmunity.
