RESUMEN
Previous studies have revealed several targets of miR-10b, such as syndecan-1, HOXD10, TBX5, and E-cadherin. In this study, we aimed to assess whether Krüppel-like factor 4 (KLF4) is a target gene of miR-10b in gastric cancer (GC). Targeting of KLF4 by miR-10b was confirmed by dual-luciferase reporter assays. The expression levels of miR-10b and KLF4 mRNA in 5 different gastric cancer cell lines and 65 pairs of gastric cancer tissues were detected by Real-time PCR. In addition, KLF4 protein in gastric cancer cell lines and 30 GC tissues was measured by western blotting and immunochemistry, respectively. KLF4 is a direct target gene of miR-10b in GC, and its expression is reduced by miR-10b at both mRNA and protein levels. In addition, the expression level of miR-10b was tendentiously upregulated in GC tissues while the expression levels of KLF4 mRNA and protein were decreased in gastric cancer tissues compared with normal adjacent tissue. There was a dramatically inverse correlation between the expression levels of miR-10b and KLF4 mRNA in GC (r=-0.339, P=0.006). These findings indicate that miR-10b was upregulated in GC and may have a key role in GC pathogenesis and development through the downregulation of its target gene KLF4.
Asunto(s)
Carcinoma/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , MicroARNs/metabolismo , Neoplasias Gástricas/metabolismo , Regiones no Traducidas 3' , Sitios de Unión , Carcinoma/genética , Carcinoma/patología , Línea Celular Tumoral , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Genes Reporteros , Células HEK293 , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Luciferasas/genética , Luciferasas/metabolismo , MicroARNs/genética , ARN Mensajero/metabolismo , Transducción de Señal , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Transfección , Regulación hacia ArribaRESUMEN
Midkine (MK) is a multifunctional growth factor and has been discovered to play important roles in carcinogenesis. MK has been reported to localize to the nucleus and nucleolus, however, the data are not consistent and the signals responsible for the localization are unknown. Here we reported that human MK exclusively localized to the nucleus and nucleolus in HepG2 cells by using GFP as a tracking molecule. In order to identify the motifs required for the nuclear localization and nucleolar accumulation, point- and deletion-mutations were introduced and the corresponding subcellular localizations were analyzed. Data revealed that (i) K79R81, K86K87, and the C-terminal tail of MK constitute the nuclear localization determinant of MK, and (ii) the C-terminal tail is the key element controlling MK nucleolar accumulation though the N-terminal tail, K79R81, and K86K87 also contribute to this process. Taken together, our results provide the first documentation about the determinants required for MK nuclear and nucleolar localization.
