MDM2对人乳腺癌细胞上皮间质转化的影响及其机制研究

目的 探讨鼠双微粒体2（MDM2）在上皮间质转化（EMT）过程中的作用及其分子机制。方法 采用Western blotting检测人乳腺癌MCF-7、MDA-MB-231和MDA-MB-435细胞株中MDM2及EMT相关标记分子（E-cadherin、N-cadherin及Vimentin）水平；分别于MCF-7细胞中瞬时转染MDM2空质粒pcmv、MDM2过表达质粒pcmv-MDM2，MDA-MB 231细胞中瞬时转染针对MDM2三个不同靶点的干扰质粒36h后观察细胞形态，并采用Western blotting和免疫荧光检测EMT相关标记分子及MDM2的表达情况；采用Western blotting及实时定量PCR检测在MCF-7、MDA-MB-231细胞过表达MDM2后对促EMT转录因子Snail1、Twist 蛋白和mRNA水平的影响。结果 MCF-7细胞过表达MDM2后形态由鹅卵石形变为纺锤形，E-cadherin蛋白随MDM2表达水平的上调表达降低；MDA-MB-231细胞敲低MDM2后形态由长梭形变为卵圆形，Vimentin、N-cadherin蛋白随MDM2表达水平的下调表达依次降低；MCF-7及MDA-MB-231细胞过表达MDM2后Snail1、Twist的蛋白及mRNA水平均升高。结论 在乳腺癌细胞株中MDM2可促进EMT发生，其可能通过上调转录因子Snail1和Twist表达来实现。

Influences of MDM2 on epithelial mesenchymal transition of human breast cancer cells and its molecular mechanism

Objective To explore the influence of murine double minute 2 （ MDM2） on epithelial mesenchymal transition （ EMT） of human breast cancer cells and its molecular mechanism. Methods Western blotting method was performed to examine the protein expressions of MDM2 and EMT-related proteins （ E-cadherin, N-cadherin and Vimentin） in human breast cancer cell lines MCF-7, MDA-MB-231 and MDA-MB-435. The optical microscope was used to observe the morphological changes at 36th hour after transiently transfection of MCF-7 cells with empty plasmid （ pcmv） or MDM2 overexpression plasmid （ pcmv-MDM2） and MDA-MB-231 cells with three different interference plasmids. Meanwhile, the expressions of MDM2 and EMT related molecular markers were measured by Western blotting and immunofluorescence assays, respectively. The Western blotting and real-time quantitative PCR were used to evaluate the protein and mRNA level changes of Snail1 and Twist in MCF-7, MDA-MB-231 cells after overexpression of MDM2. Results MCF-7 cells displayed decreased protein levels of E-cadherin and its morphology changed from tightly packed colonies to spindle-shaped growth after overexpression of MDM2. MDA-MB-231 cells exhibited reduced levels of Vimentin and N-cadherin and its morphology changed from fibroblast-like growth to cobblestone-like colonies after the knockdown of MDM2. Both the protein and mRNA levels of Snail1 and Twist increased after overexpression of MDM2 in MCF-7 and MDA-MB-231 cells. Conclusion MDM2 can promote the EMT in human breast cancer cells with the possible meachism of up-regulating the expressions of Snail1 and Twist.