敲低MSX2对胰腺癌PANC-1细胞上皮-间充质转化的影响

摘要

目的：研究MSX2基因在胰腺癌PANC-1细胞的上皮-间充质转化（EMT）/间充质-上皮转化（MET）转变中的作用。方法通过脂质体2000将含有MSX2干扰序列的质粒（共3种，分别命名为shMSX2-1、shMSX2-2和shMSX2-3，转染空质粒的阴性对照组NC）转染PANC-1细胞，利用Real-time RT-PCR、Western blot等技术检测MSX2在基因和蛋白水平的表达，检测干扰效率，并选取干扰效率最高的一组用于后续试验；进而检测EMT相关上皮标志分子E-cadherin、间质标志分子Vimentin在基因和蛋白水平的表达变化；采用CCK-8法检测干扰前后细胞的增殖能力变化，Transwell和划痕试验比较细胞的侵袭转移能力变化。结果3组干扰质粒中shMSX2-1的干扰效率最高，CCK-8试验结果显示敲低MSX2可明显抑制PANC-1细胞增殖能力，与对照组相比差异有显著性（P<0.05）；敲低MSX2可明显抑制PANC-1细胞的侵袭转移，差异有统计学意义（P<0.05）；EMT相关分子E-cadherin表达升高，而Vimentin表达降低，且与对照组相比均有统计学意义（P<0.05）；倒置显微镜下观察敲低MSX2后细胞形态由松散的长梭形变成紧密连接的椭圆形。RT-PCR发现敲低MSX2后转录因子twist和snail表达降低（P<0.05），而slug和zeb1表达变化无差异（P>0.05）。结论敲低MSX2可抑制胰腺癌PANC1细胞的增殖、侵袭转移能力，并且上皮标志分子E-cadherin表达升高，间质标志分子Vimentin表达降低，有发生MET的趋势，MSX2可能通过twist、snail发挥其抑制作用。%Objective To investigate the effect of MSX2 interference on epithelial-mesenchymal transitions (EMT) of pancreatic cancer cell line PANC-1. Methods Three vectors containing short hairpin RNAs (shRNAs) of MSX2 (shMSX2-1, shMSX2-2, and shMSX2-3) and the empty vector (negative control) were transfected separately into PANC-1 cell line with Lipofectamine2000. Real-time RT-PCR and Western blotting were used to observe changes in the expressions of MSX2, E-cadherin, and vimentin in the cells. CCK-8 assay was used to assess the changes in the cell growth, and wound scratch assay and Transwell assay were employed to evaluate the cell invasion and metastasis after the transfection. Results Among the 3 shRNA, shMSX2-1 showed the highest interference efficiency. MSX2 knockdown by the specific shRNA of MSX2 significantly increased E-cadherin expressions, lowered vimentin expressions, and suppressed the invasion, metastasis and proliferation of the cells (P<0.05). MSX2 knockdown also resulted in morphological changes of the cells into cobblestone-like cells in close contact. RT-PCR results revealed significantly reduced mRNA expressions of the transcription factors snail and twist (P<0.05) without affecting slug and zeb1 expressions in the cells with MSX2 knockdown. Conclusion MSX2 knockdown can reverse EMT and induce MET in PANC1 cells, in which process the transcription factors snail and twist may play a role.