LATS1过表达对肺癌A549细胞增殖及周期的影响

通过过表达手段上调大肿瘤抑制因子1（1arge tumor suppressor gene 1,LATS1）基因在A549细胞中的表达,研究LATS1对A549细胞生长和细胞周期调控的作用。构建过表达LATS1基因的慢病毒载体,转染A549N胞株,采用RT-PCR和蛋白质印迹法检测转染后A549细胞中LATS1、YAPmRNA和蛋白的表达效率;流式细胞术检测细胞凋亡、周期情况：CCK-8检测细胞的增殖水平变化。结果发现,过表达LATS1慢病毒载体转染A549细胞株后,LATS1mRNA及蛋白表达水平高于未处理组及转染空载体组,YAPmRNA及蛋白表达水平低于未处理组及转染空载体组;过表达LATS1慢病毒转染后,A549细胞增殖率从第五天开始低于对照组（P〈0．05）,过表达组细胞G1期比例明显增高（P〈0．05）,凋亡率明显增加（P〈0．05）,差异均有统计学意义。以上结果提示,LATS1可通过下调YAP的表达水平促进A549细胞的凋亡,诱导G1期阻滞,降低细胞的增殖能力。     

Identification of Glypican3 as a novel GLUT4-binding protein

Insulin stimulates glucose uptake in fat and muscle primarily by stimulating the translocation of vesicles containing facilitative glucose transporters, GLUT4, from intracellular compartments to the plasma membrane. Although cell surface externalization of GLUT4 is critical for glucose transport, the mechanism regulating cell surface GLUT4 remains unknown. Using a yeast two-hybrid screening system, we have screened GLUT4-binding proteins, and identified a novel glycosyl phosphatidyl inositol (GPI)-linked proteoglycan, Glypican3 (GPC3). We confirmed their interaction using immunoprecipitation and a GST pull-down assay. We also revealed that GPC3 and GLUT4 to co-localized at the plasma membrane, using immunofluorescent microscopy. Furthermore, we observed that glucose uptake in GPC3-overexpressing adipocytes was increased by 30% as compared to control cells. These findings suggest that GPC3 may play roles in glucose transport through GLUT4.     

YAP蛋白T425A位点突变对YAP功能的影响

Hippo信号通路在个体发育与组织生长中发挥了关键的作用,YAP(Yes-associated protein)是该通路中主要的下游效应因子。已有的研究表明YAP活性与肿瘤的发生发展密切相关,然而关于YAP活性的调控机制目前还不是很清楚。本研究将YAP蛋白(NP_001123617)第425位苏氨酸(T)突变为丙氨酸(A),发现YAP T425A突变能减弱YAP的转录活性,并通过免疫荧光实验发现该位点的突变还能阻滞YAP进入细胞核。该位点对YAP活性的调控并不依赖于YAP在S127位点受到的磷酸化调控。同时本研究在细胞水平上检测到T425A位点突变能部分降低YAP对细胞迁移的促进作用。本研究主要揭示了YAP T425位点对YAP进出细胞核及其转录活性调控的重要作用,丰富了Hippo通路的调节机理和YAP在肿瘤发生发展中的作用研究。