RESUMEN
Soybean mosaic virus (SMV), one of the major viral diseases of Pinellia ternata (Thunb.) Breit., has had a serious impact on its yield and quality. The construction of viral infectious clones is a powerful tool for reverse genetics research on viral gene function and interaction between virus and host. To clarify the molecular mechanism of SMV infection in Pinellia ternata, it is particularly important to construct the SMV full-length cDNA infectious clone. Therefore, the infectious clone of Soybean mosaic virus Shanxi Pinellia ternata isolate (SMV-SXBX) was constructed in this study by Gibson in vitro recombination system, and the healthy Pinellia ternata leaves were inoculated by Agrobacterium infiltration, further through mechanical passage and RT-PCR, confirming that the 3' end of the SMV-SXBX infectious clone had a stable infectivity when it contained 56-nt of poly(A) tail. This method is not only convenient and efficient, but also avoids the instability of SMV infectious clones in Escherichia coli. The construction of SMV full-length infectious cDNA clones laid the foundation for further study on the molecular mechanism of SMV replication and pathogenesis.
Asunto(s)
Pinellia , Potyvirus , ADN Complementario , Pinellia/virología , Enfermedades de las Plantas/virología , Potyvirus/aislamiento & purificación , Potyvirus/metabolismoRESUMEN
Ku80 is involved in DNA double-strand breaks (DSBs) repair. Ku80 is overexpressed in lung cancer tissues, yet, molecular mechanisms have not been examined. We identified that miRNA, hsa-miR-526b, is bound to the 3'-UTR of Ku80 mRNA, thus decreasing Ku80 expression in NSCLC cells. Hsa-miR-526b was downregulated in NSCLC tissues compared with corresponding non-tumorous tissues, and its expression was inversely correlated with Ku80 upregulation. Overexpression of Ku80 and downregulation of hsa-miR-526b were associated with poor clinical outcomes of NSCLC patients. Hsa-miR-526b suppressed NSCLC cell proliferation, clonogenicity, and induced cell cycle arrest and apoptosis. Hsa-miR-526b inhibited xenografts and orthotopic lung tumor growth. Further, Ku80 knockdown in NSCLC cells suppressed tumor properties in vitro and in vivo similar to hsa-miR-526b overexpression. In agreement, Ku80 restoration partially reversed cell cycle arrest and apoptosis induced by hsa-miR-526b in NSCLC cells in vitro and in vivo. In addition, hsa-miR-526b overexpression or Ku80 knockdown increased p53 and p21CIP1/WAF1 expression. These findings reveal that hsa-miR-526b is a potential target in cancer therapy.
Asunto(s)
Antígenos Nucleares/biosíntesis , Carcinoma de Pulmón de Células no Pequeñas/genética , Proteínas de Unión al ADN/biosíntesis , Neoplasias Pulmonares/genética , MicroARNs/genética , Animales , Antígenos Nucleares/genética , Apoptosis/fisiología , Secuencia de Bases , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Ciclo Celular/fisiología , Proliferación Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Autoantígeno Ku , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , MicroARNs/metabolismo , Persona de Mediana Edad , Datos de Secuencia Molecular , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
AIM: To investigate the role of three K+ channels-delayed rectifier K+ channel (Kv), large-conductance Ca(2+)-activated K+ channel (BK(Ca)) and ATP-sensitive K+ channel (K(ATP)) in the regulation of the resting and contracting tone of rat bronchial smooth muscle (BSM). METHODS: By measuring the isometric tone of bronchial strips in vitro, the regulating effects of the three K+ channels on the tone of rat BSM were observed. RESULTS: (1) Kv blocker 4-aminopyridine (4-AP) caused concentration dependent contraction in resting bronchial strips, but BK(Ca) blocker tetraethylammonium (TEA) and K(ATP) blocker glibenclamide (Glib) had no such effects. (2) The bronchial epithelium had no effects on the contraction induced by 4-AP, but nifedipine, a Ca2+ channel blocker, significantly suppressed it. (3) Before or after treatment with 0.1 mmol/L histamine or 50 mmol/L KCl, administration of TEA (1 or 5 mmol/L) or 0.1 mmol/L 4-AP could significantly increase the contraction induced by histamine and KCl in bronchial strips. But Glib (10 micromol/L) had no effect on it. CONCLUSION: Not BK(Ca) and K(ATP) but Kv participated in the regulation of the resting tone in rat BSM. The closure of BK(Ca) or Kv increased the contracting tone induced by histamine or KCl in rat BSM in vitro, but K(ATP) had no such effect on it.