RESUMO
Cardiac fibroblast (CF) differentiation plays a crucial role in cardiac fibrosis, which is a specific manifestation distinguishing pathological cardiac hypertrophy from physiological hypertrophy. The DNA-binding activity of paired box 6 (Pax6) has been shown to be oppositely regulated in physiological and pathological hypertrophy; however, it remains unclear whether Pax6 is involved in CF differentiation during cardiac fibrosis. We found that Pax6 is expressed in the heart of and CFs isolated from adult mice. Moreover, angiotensin II (Ang II) induced the downregulation of Pax6 mRNA and protein expression in fibrotic heart tissue and cardiac myofibroblasts. Pax6 knockdown in CFs promoted the expression of the myofibroblast marker α-smooth muscle actin (α-SMA) and the synthesis of the extracellular matrix (ECM) proteins collagen I and fibronectin. Furthermore, we validated the ability of Pax6 to bind to the promoter regions of Cxcl10 and Il1r2 and the intronic region of Tgfb1. Pax6 knockdown in CFs decreased CXC chemokine 10 (CXCL10) and interleukin-1 receptor 2 (IL-1R2) expression and increased transforming growth factor ß1 (TGFß1) expression, mimicking the effects of Ang II. In conclusion, Pax6 exerts an inhibitory effect on CF differentiation and ECM synthesis by transcriptionally activating the expression of the anti-fibrotic factors CXCL10 and IL-1R2 and repressing the expression of the pro-fibrotic factor TGFß1. Therefore, maintaining Pax6 expression in CFs is essential for preventing CF differentiation, and provides a new therapeutic target for cardiac fibrosis.
Assuntos
Diferenciação Celular/fisiologia , Miocárdio/citologia , Miocárdio/metabolismo , Fator de Transcrição PAX6/fisiologia , Angiotensina II/farmacologia , Animais , Cardiomegalia/etiologia , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Diferenciação Celular/genética , Quimiocina CXCL10/genética , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/biossíntese , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibrose , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Íntrons , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator de Transcrição PAX6/antagonistas & inibidores , Fator de Transcrição PAX6/genética , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Receptores Tipo II de Interleucina-1/genética , Fator de Crescimento Transformador beta1/genéticaRESUMO
Paired-box 6 (PAX6) is an important transcription factor required for the function of human neuroectodermal epithelial tissues. Previous studies have suggested that it is also expressed in several types of tumors and has an oncogenic role. However, little is known about its role in non-small cell lung cancer (NSCLC). Here, we found that PAX6 expression levels were upregulated in human lung cancer tissues and correlated with poor clinical outcomes. PAX6 overexpression significantly promoted NSCLC epithelial-to-mesenchymal transition (EMT) and metastasis, whereas its knockdown inhibited these processes. PAX6 is commonly correlated with EMT-mediated stem cell transformation, thereby inducing cisplatin resistance. Using the RT2 Profiler PCR Array, we found that WNT5A, EGFR, and ZEB2 were differentially regulated in response to PAX6 modulation. In addition, PAX6 directly bound to the promoter region of ZEB2. ZEB2 knockdown significantly reduced the expression and function of PAX6. ZEB2 was upregulated upon PAX6 overexpression and downregulated upon PAX6 knockdown, whereas E-cadherin expression negatively correlated with PAX6 levels. Moreover, p-PI3K and p-AKT were significantly enhanced by PAX6, which was reversed by the addition of the PI3K-AKT inhibitor, LY294002. These data suggest that PAX6 can mediate E-cadherin downregulation through the PI3K/AKT signaling pathway by directly binding the promoter region of ZEB2, thereby mediating cell migration, stem cell transformation, and cisplatin resistance; and ultimately, affecting survival in NSCLC patients.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/patologia , Fator de Transcrição PAX6/metabolismo , Transdução de Sinais , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Animais , Caderinas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Linhagem Celular Tumoral , Movimento Celular , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Regulação para Baixo/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/mortalidade , Camundongos , Camundongos Nus , Fator de Transcrição PAX6/antagonistas & inibidores , Fator de Transcrição PAX6/genética , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Homeobox 2 de Ligação a E-box com Dedos de Zinco/antagonistas & inibidores , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genéticaRESUMO
Glioblastoma (GBM) is the predominant and most fatal type of brain tumor in adults. The prognosis of GBM remains poor despite advances in surgery, chemotherapy and radiotherapy. It is common that patients with GBM exhibit innate or acquired resistance to temozolomide (TMZ), a standard chemotherapeutic agent for GBM, and a previous report demonstrated that miRNA233 (miR223) promotes the growth and invasion of GBM cells by targeting tumor suppressor paired box 6 (PAX6). The present study explored the effect of TMZ on miR223/PAX6 signaling in addition to the effect of miR223/PAX6 signaling on TMZ chemoresistance in human GBM cells. Luciferase reporter assays confirmed that miR223 directly targets PAX6 through binding to its 3'untranslated region. TMZ reduced the expression level of miR223 in a concentrationdependent manner in U251 and U118 GBM cells, which led to increased expression of PAX6. miR223 and/or PAX6 were overexpressed and knocked down in U251 and U118 cells, and the half maximal inhibitory concentration (IC50) of TMZ and cell proliferation under TMZ treatment were used as measures of TMZ chemoresistance. The results demonstrated that overexpression of miR-223 in GBM cells markedly decreased TMZ-induced inhibition of cell proliferation and increased TMZ IC50, which could be abolished by overexpression of PAX6. On the other hand, knocking down miR223 in GBM cells with antagomir significantly enhanced the inhibitory effect of TMZ on GBM cell proliferation and decreased the TMZ IC50, which could be abolished by knockdown of PAX6. In conclusion, the present study demonstrated that TMZ inhibits GBM cell proliferation by inhibiting the expression of miR223, which leads to increased expression of tumor suppressor PAX6. Overexpression of miR223 increases TMZ chemoresistance, while inhibition of miR223 with antagomir markedly decreases TMZ chemoresistance in GBM cells. The present study provided novel insight into the molecular mechanisms underlying the pharmacological effects, in addition to the chemoresistance, of TMZ for GBM.