乳腺癌细胞中MRJ稳定干扰导致MMP-9表达增强和细胞骨架重排  

siRNA-mediated Stably Knockdown of MRJ Up-regulates Expression of MMP-9 and Leads to Cytoskeleton Rearrangement in Breast Cancer

作　　者：彭娜娜[1] 庄红芹[1] 王耀[2] 华子春[1] 

机构地区：[1]南京大学医药生物技术国家重点实验室,南京210093 [2]新南威尔士大学圣乔治医学院

出　　处：《中国细胞生物学学报》2013年第9期1334-1341,共8页Chinese Journal of Cell Biology

基　　金：科技部(批准号:2012CB967004;2012AA020304;2008BAI51B01);江苏省科技厅(批准号:BK2011228;BZ2011048)资助的课题~~

摘　　要：应用构建稳定乳腺癌细胞系的方法研究MRJ干扰后上调乳腺癌细胞中基质金属蛋白酶MMP-9的表达,并影响细胞骨架的重排。为了探讨MRJ(S)与MMP-9和细胞骨架的关系,将MDAMB-231细胞转染干扰质粒pRNAT-U6.1/neo-MRJsi,用G418(Zeocin)(600μg/mL)筛选得到MRJ(S)稳定干扰的细胞系,命名为MDA-MB-231/MRJsi。利用Q-PCR和Western blot检测MDA-MB-231/MRJsi细胞和野生型细胞之间MRJ mRNA水平和蛋白水平的差异。利用鬼笔环肽染色和anti-α-tublin的免疫荧光分析观察MDA-MB-231/MRJsi细胞系中微丝和微管的变化。同时,利用明胶酶谱法检测MDA-MB-231和MDA-MB-231/MRJsi细胞系中基质金属蛋白酶MMP-9的酶活性变化并进一步利用Q-PCR检测MRJ干扰后细胞内MMP-9的mRNA水平差异。结果表明,成功构建MRJ稳定干扰的MDA-MB-231/MRJsi稳定细胞系,与野生型MDA-MB-231细胞比较,MDA-MB-231/MRJsi细胞系中MRJ mRNA水平下降了50%(P<0.05),MRJ(S)蛋白表达水平下调70%;细胞骨架微丝和微管发生重排;MMP-9酶活性增强;MMP-9的mRNA水平显著上升(P<0.01),与其酶活性水平变化一致。该文MDA-MB-231/MRJsi稳定细胞系提供了一个研究MRJ(S)与乳腺癌发生发展的细胞模型;MRJ(S)可以影响MDA-MB-231细胞中MMP-9的表达并引发细胞骨架重排。MRJ knockdown stable cell line of breast cancer MDA-MB-231 cells were constructed and screened with plasmid of MRJ siRNA. The MRJ knockdown stable cell line was used to investigate the effects of MRJ on cytoskeleton rearrangement and the expression of matrix metalloproteinase 9 （MMP-9）. We transfected the MDA-MB-231 cells with the plasmid of MRJ siRNA （pRNAT-U6.1/neo-MRJsi） and used 600μg/mL G418 to screen the stable knockdown cell line. The selected cell line was then subjected to Real-time PCR and Westernblot assay to quantitate the mRNA level and protein level. Then immunnofluorence was used to investigate the distribution of microtubes and microfilaments using anti-a-tublin antibody and phalloidin dyes, respectively. Moreover, the enzyme activity of MMP-9 was measured using gelatin zymography and its mRNA level was detected using quantitative Real-time PCR. The results showed that we successfully established MDA-MB-231/ MRJsi stable cell line that knockdowned MRJ expression and named it MDA-MB-231/MRJsi cell line. The gene expression of MRJ in MDA-MB-231/MRJsi cells reduced 50% compared to the wild type MDA-MB-231 cells （P〈0.05） and the protein level reduced 70%. Moreover, the cytoskeleton proteins in MDA-MB-231/MRJsi cells were redistributed when compared to wild type MDA-MB-231 cells. Furthermore, the enzyme activity of MMP-9 was enhanced after MRJ suppression. In addition, the mRNA level of MMP-9 was increased significantly （P〈0.01） which was consistant with its enzyme activity. In conclusion, the establishment of stable MDA-MB-23 I/MRJsi cell line is a fundamental cell model for further research of MRJ function in oncogenesis and development of breast cancer.

关 键 词：乳腺癌 MRJ 细胞骨架 基质金属蛋白酶 

分 类 号：R737.9[医药卫生—肿瘤]