下调paxillin高表达对结直肠癌细胞SW480细胞信号转导及超微结构的影响  

SiRNA-mediated silencing of paxillin down-regulates ERK1/2 signaling and alters cell ultrastructure in colorectal carcinoma cell line SW480

作　　者：郑建伟[1] 尹洪芳[2] 汪欣[1] 刘玉村[1] 万远廉[1] 朱静[3] 

机构地区：[1]北京大学第一医院普外科,4北京市100034 [2]北京大学第一医院病理科,北京市100034 [3]北京大学第一医院外科实验室,北京市100034

出　　处：《世界华人消化杂志》2013年第9期754-760,共7页World Chinese Journal of Digestology

摘　　要：目的:探讨下调paxillin高表达对结直肠癌细胞SW480细胞信号转导及超微结构的影响.方法:设计两种siRNA片段并用其转染高表达paxillin的结直肠癌细胞SW480细胞系,以空质粒作为阴性对照,构建稳定转染细胞系.NC组为转染空质粒的细胞系,SW545组是稳定转染针对paxillin545-565靶基因位点的siRNA片段的细胞系,SW782组是稳定转染针对paxillin782-802靶基因位点的siRNA片段的细胞系.Western blot检测SW480、NC、SW545及SW7824组细胞系中paxillin、FAK、ERK1/2及AKT1/2/3的表达及特异位点磷酸化水平.利用培养细胞制备切片,透射电镜下观察几组细胞系的超微结构.结果:SW545细胞paxillin的高表达无明显改变,SW782细胞paxillin的高表达被显著下调,SW782细胞paxillin(Tyr118)磷酸化水平也显著降低.SW782细胞ERK1/2的表达水平无明显变化,但是ERK1/2(Thr202/Tyr204)磷酸化水平显著降低.SW782细胞AKT1/2/3及FAK的表达及特异位点磷酸化水平均无明显变化.与SW480细胞相比,NC组细胞无明显超微结构改变,但是SW782细胞却发生了显著的超微结构变化,具有明显增多的微绒毛、微丝微管束与溶酶体以及明显减少的线粒体.结论:下调paxillin高表达能够逆转SW480细胞典型的恶性转化细胞的超微结构特征,paxillin高表达可能通过磷酸化激活paxillin及ERK1/2信号转导通路从而在结直肠癌发生发展中起着非常重要的作用.AIM： To study the effect of silencing of paxillin overexpression on cell signaling and ultrastructure in colorectal carcinoma cell line SW480.METHODS： Using empty plasmid as a negative control, two siRNA fragments were transfected into a colorectal carcinoma cell line SW480 which overexpresses paxillin. Stably transfected cells were screened and three new cell lines NC, SW545 and SW782 were obtained, which carried the negative control, the siRNA targeting the site 545-565, and the siRNA targeting the site 782-802, respectively. The expression and site-specific phosphorylation of paxillin, FAK, ERK1/2 and AKT1/2/3 were examined in the four cell lines by Western blot. Specimens were prepared with cultured carcinoma cells to ob- serve cell ultrastructure by transmission electron microscopy.RESULTS： Paxillin overexpression in SW545 cells was not silenced at all, whereas silenced paxillin overexpression and remarkably reduced phosphorylation of paxillin （Tyr118） were observed in SW782 cells. Expression of AKT1/2/3 and FAK as well as their site-specific phosphor-ylation were substantially the same in the four cell lines. Although expression of ERK1/2 was substantially the same in the four cell lines, sig-nificantly reduced phosphorylation of ERK1/2 （Thr202/Tyr204） was observed in SW782 cells. There was no distinct ultrastructural difference between NC cells and SW480 cells, whereas dra-matic ultrastructural changes were observed in SW782 cells, such as much more microvilli, microfilament and microtubule bundles, lysosomes and much less mitochondria.CONCLUSION： Paxillin overexpression may play an important role in the malignant transformation of colorectal carcinoma cells, which is character-ized by dramatic ultrastructural changes that can be reversed by silencing paxillin overexpression. Activation of ERK1/2 signaling downstream of paxillin is indispensable for the malignant transformation of colorectal carcinoma cells.

关 键 词：桩蛋白 siRNA SW480细胞系 细胞信号转导 超微结构 

分 类 号：R735.3[医药卫生—肿瘤]