RESUMO
The aim of this study was to investigate the effect of lncRNA KCNQ1OT1 on HCC and to explore the possible underlying mechanisms. The expression levels of KCNQ1OT1, miR-149 and S1PR1 were detected by qRT-PCR assay. A dual luciferase reporter assay was used to detect the interaction between KCNQ1OT1 and miR-149, as well as miR-149 and S1PR1. The interaction between KCNQ1OT1 and miR-149 was further investigated by RNA pull-down assay. Wound healing assays and Transwell assays were carried out to determine cell migration and invasion. A xenograft tumour assay was used to validate the role of KCNQ1OT1 in vivo. KCNQ1OT1 and S1PR1 were significantly increased, but miR-149 was decreased in HCC cells. Luciferase reporter assays and RNA pull-down assays revealed that KCNQ1OT1 directly targeted miR-149. In addition, miR-149 bound to the 3'-UTR of S1PR1. Knockdown of KCNQ1OT1 or overexpression of miR-149 inhibited the invasion and migration of HCC cells. However, suppression of miR-149 could abrogate the effect of KCNQ1OT1 knockdown on the invasion and migration abilities of HCC cells. In vivo assays showed that KCNQ1OT1 knockdown suppressed tumour growth. This work suggests that lncRNA KCNQ1OT1 might act as a potential therapeutic target in HCC.
Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Animais , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular , Humanos , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Nus , Invasividade Neoplásica , TransfecçãoRESUMO
The present study was designed to test whether Rho-kinase (ROCK) specific inhibitor fasudil (HA-1077) could contribute to migration and vasculogenesis of endothelial cells differentiated from rat bone marrow mesenchymal stem cells (rBMSCs) in vitro. rBMSCs were separated by gradient centrifugation on lymphocytes separation medium from bone marrow of Sprague-Dawley rats, and were cultured, purified and expanded in vitro. Cells of passage 2 to 3 were induced to differentiate into endothelial lineage cells by HG-DMEM plus EGM-2. These cells were identified as endothelial cells with positive factor VIII and Ulex europaeus agglutinin-1 expressions and DiI-Ac-LDL uptake. HA-1077 and VEGF synergistically promoted cell migration, especially in response to transwell chamber assay. When the cells were cultured on ECMatrix™, they showed cellular protrusions and/or cords of aligned cells resembling primitive capillary-like structures at 8 to 12 h of incubation. HA-1077 promoted cell migration and formation of capillary-like tubes. The length of the total capillary tubes was longer than that in the control group (P<0.05). When the cells were exposed to a combination of VEGF and HA-1077, the number of the capillary-like networks and the stability of tube increased. The results obtained suggest that HA-1077 can promote migration and vasculogenesis of endothelial cells differentiated from rBMSCs in vitro.
