RESUMO
OBJECTIVE: We aimed at studying the role of the most deregulated miR-99a, identifying its downstream targets, and exploring the clinical potential of miR-99a and its target(s) in oral cancer. SUBJECTS AND METHODS: Following confirmation of miR-99a deregulation in nine oral lines and 26 pairwise clinical specimens, miR-99a-manipulated oral cancer cells were subjected to cell proliferation, migration, invasion, and in vivo murine metastasis assays. We characterized putative miR-99a target(s) using luciferase reporter assays and genetic manipulation. The inverse relation of miR-99a and its target(s) was examined in clinical specimens using real-time PCR and Western blot analysis. RESULTS: MiR-99a down-regulation was confirmed both in tested oral cancer cell lines and clinical specimens. Ectopic miR-99a expression inhibited oral cancer cell migration and invasion. Anti-miR-99a, silencing miR-99a functions, had the opposite effect. Myotubularin-related protein 3 (MTMR3) with one evolutionarily conserved seed region in the 3'-untranslated region was a novel miR-99a target. Depleting MTMR3 expression significantly reduced cell proliferation, migration, or invasion. There was an inverse expression of miR-99a and MTMR3 protein in oral cancer lines and clinical specimens. CONCLUSION: miR-99a repressed oral cancer cell migration and invasion partly through decreasing MTMR3 expression. MTMR3 may serve as a therapeutic target for oral cancer treatment.
Assuntos
MicroRNAs/fisiologia , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Proteínas Tirosina Fosfatases não Receptoras/antagonistas & inibidores , Proteínas Tirosina Fosfatases não Receptoras/biossíntese , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Neoplásica , Células Tumorais CultivadasRESUMO
BACKGROUND: Angiostatin, a circulating angiogenic inhibitor, is an internal fragment of plasminogen and consists of several isoforms, K1-3 included. We previously showed that K1-3 was the most potent angiostatin to induce E-selectin mRNA expression. The purpose of this study was to identify the mechanism responsible for K1-3-induced E-selectin expression and investigate the role of E-selectin in the anti-angiogenic action of K1-3. METHODS AND RESULTS: Quantitative real time RT-PCR and Western blotting analyses confirmed a time-dependent increase of E-selectin mRNA and protein induced by K1-3. Subcellular fractionation and immunofluorescence microscopy showed the co-localization of K1-3-induced E-selectin with caveolin 1 (Cav1) in lipid rafts in which E-selectin may behave as a signaling receptor. Promoter-driven reporter assays and site-directed mutagenesis showed that K1-3 induced E-selectin expression via promoter activation and AP1 and Ets-1 binding sites in the proximal E-selectin promoter were required for E-selectin induction. The in vivo binding of both protein complexes to the proximal promoter was confirmed by chromatin immunoprecipitation (ChIP). Although K1-3 induced the activation of ERK1/2 and JNK, only repression of JNK activation attenuated the induction of E-selectin by K1-3. A modulatory role of E-selectin in the anti-angiogenic action of K1-3 was manifested by both overexpression and knockdown of E-selectin followed by cell proliferation assay. CONCLUSIONS: We show that K1-3 induced E-selectin expression via AP1 and Ets-1 binding to the proximal E-selectin promoter (-356/+1), which was positively mediated by JNK activation. Our findings also demonstrate E-selectin as a novel target for the anti-angiogenic therapy.
Assuntos
Angiostatinas/fisiologia , Selectina E/genética , Proteína Proto-Oncogênica c-ets-1/fisiologia , Fator de Transcrição AP-1/fisiologia , Ativação Transcricional , Sítios de Ligação , Caveolina 1/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Selectina E/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Cinética , Microdomínios da Membrana/química , Regiões Promotoras Genéticas , Ligação Proteica , Isoformas de Proteínas/fisiologia , RNA MensageiroRESUMO
By using a slit plate, scanographic arterial studies can be performed with a tomographic attachment of an X-ray unit that is normally used for routine radiographic examinations. The expense is minimal and no structural changes are necessary.