RESUMO
Microtubules are polymers of α/ß-tubulin, with microtubule organization being regulated by microtubule-associated proteins (MAPs). Herein, we describe a novel role for the epithelial gene repressor, zinc finger E-box-binding homeobox 1 (ZEB1), that "switches" from a chromatin-associated protein during interphase, to a MAP that associates with α-, ß- and γ-tubulin during mitosis. Additionally, ZEB1 was also demonstrated to associate with γ-tubulin at the microtubule organizing center (MTOC). Using confocal microscopy, ZEB1 localization was predominantly nuclear during interphase, with α/ß-tubulin being primarily cytoplasmic and the association between these proteins being minimal. However, during the stages of mitosis, ZEB1 co-localization with α-, ß-, and γ-tubulin was significantly increased, with the association commonly peaking during metaphase in multiple tumor cell-types. ZEB1 was also observed to accumulate in the cleavage furrow during cytokinesis. The increased interaction between ZEB1 and α-tubulin during mitosis was also confirmed using the proximity ligation assay. In contrast to ZEB1, its paralog ZEB2, was mainly perinuclear and cytoplasmic during interphase, showing some co-localization with α-tubulin during mitosis. Considering the association between ZEB1 with α/ß/γ-tubulin during mitosis, studies investigated ZEB1's role in the cell cycle. Silencing ZEB1 resulted in a G2-M arrest, which could be mediated by the up-regulation of p21Waf1/Cip1 and p27Kip1 that are known downstream targets repressed by ZEB1. However, it cannot be excluded the G2/M arrest observed after ZEB1 silencing is not due to its roles as a MAP. Collectively, ZEB1 plays a role as a MAP during mitosis and could be functionally involved in this process.
Assuntos
Cromatina/genética , Proteínas Associadas aos Microtúbulos/genética , Mitose/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Pontos de Checagem do Ciclo Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Citocinese/genética , Humanos , Proteínas Associadas aos Microtúbulos/química , Ligação Proteica/genética , Fuso Acromático/genética , Tubulina (Proteína)/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/químicaRESUMO
Numerous microRNAs (miRNAs) have been identified as aberrantly expressed in osteosarcoma (OS). miRNAs serve important roles in the pathogenesis of OS as oncogenes or tumor suppressors. Recent studies revealed that miR7085p (miR708) was dysregulated in various types of human cancer; however, its roles and underlying molecular mechanisms in OS remain unknown. Therefore, the present study aimed to determine miR708 expression in OS, investigate the roles of miR708 in the progression of OS and reveal the potential mechanisms involved. It was demonstrated using reverse transcriptionpolymerase chain reaction that miR708 was downregulated in OS tissues and cell lines. Cell Counting Kit8 and Transwell assays revealed that miR708 overexpression suppressed the proliferation and invasion of OS cells in vitro. Additionally, zinc finger Ebox binding homeobox 1 (ZEB1) was validated as a direct target gene of miR708 in OS cells. ZEB1 was upregulated in OS tissues; elevated ZEB1 expression was negatively correlated with the levels of miR708 expression. Rescue experiments indicated that ZEB1 reintroduction significantly counteracted the inhibitory effects of miR708 overexpression on the proliferation and invasion of OS cells. The findings may improve understanding of the roles of miR708 in the development of OS, and suggest that miR708 may be a potential novel therapeutic target in the treatment of patients with this disease.
Assuntos
Proliferação de Células , MicroRNAs/metabolismo , Osteossarcoma/patologia , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Regiões 3' não Traduzidas , Adulto , Antagomirs/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Movimento Celular , Regulação para Baixo , Feminino , Humanos , Masculino , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Pessoa de Meia-Idade , Osteossarcoma/metabolismo , Alinhamento de Sequência , Adulto Jovem , Homeobox 1 de Ligação a E-box em Dedo de Zinco/química , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
Long non-coding RNAs (lncRNAs) are involved in the progression of several diseases. The interactions among lncRNAs, microRNA (miRNAs) or their targeting genes are reported to play crucial roles in the development of diseases. LINC00657 is observed to be upregulated in several cancers. However, the biological role of LINC00657 in neuropathic pain progress is unclear. Hence, in our study, we aimed to investigate the function of LINC00657 in neuropathic pain development. A chronic constriction injury (CCI) rat model was established, and we found that LINC00657 was greatly increased in CCI rats associated with a decrease of miR-136. Inhibition of LINC00657 suppressed neuropathic pain via alleviating mechanical and thermal hyperalgesia. In addition, miR-136 overexpression can also inhibit the neuropathic pain development. MiR-136 was predicted to serve as a miRNA target of LINC00657, and dual-luciferase reporter assay confirmed the correlation between LINC00657 and miR-136. Moreover, we observed that the decrease of LINC00657 was able to inhibit the neuroinflammation of CCI rats by targeting expression of cyclooxygenase-2, tumor necrosis factor-α and interleukin-1ß while miR-136 inhibitors reversed this phenomenon. Next, by using bioinformatics analysis, ZEB1 was predicted as a direct target of miR-136, and miR-136 could negatively modulate ZEB1 expression. Besides these, ZEB1 was remarkably increased in the CCI rats. Knockdown of ZEB1 can inhibit neuropathic pain development, while miR-136 inhibitors can reverse it. In conclusion, it was implied that LINC00657 can induce the neuropathic pain development via regulating miR-136/ZEB1 axis.
