目的：观察miRNA靶向沉默P65 基因对人三阴性乳腺癌（triplenegative breast cancer，TNBC）MDAMB231细胞体外黏附和侵袭的影响及其可能机制。方法：设计3对针对P65基因的特异性miRNA（P65miRNA1、P65miRNA2和P65miRNA3），转染MDAMB231细胞，Western blotting检测P65蛋白的表达，筛选沉默效果最好的miRNA进行后续实验。体外黏附实验和Transwell小室实验检测P65miRNA转染前后MDAMB231细胞的黏附和侵袭。RTPCR检测MDAMB231细胞基质金属蛋白酶2（matrix metalloproteinase2，MMP2）、MMP9 mRNA的表达水平，明胶酶谱法检测MDAMB231细胞中MMP2、MMP9的酶活性。结果：成功构建重组质粒P65miRNA1、P65miRNA2和P65miRNA3，前两者转染MDAMB231细胞后抑制P65蛋白表达80%以上。P65miRNA1和P65miRNA2转染对MDAMB231细胞的黏附无明显影响（P>0.05），但显著抑制MDAMB231细胞的侵袭（0.371±0.039、0.309±0.046 vs 0.698±0.065, P＜0.05）。P65miRNA1和P65miRNA2转染沉默P65表达后，MDAMB231细胞MMP2 mRNA（0.281±0.018、0.478±0.023 vs 1.056±0.072、1.128±0.059，P＜005）和MMP9 mRNA表达（0.193±0.013、0.371±0.035 vs 1.206±0.069、1.089±0.057，P＜0.05）及其活性均显著下调。结论：miRNA靶向沉默P65的表达能抑制人TNBC MDAMB231细胞的体外侵袭，其机制可能与抑制MMP2、MMP9的表达和活性有关。

Objective:To investigate the effect of targeting silence of P65 gene by miRNA on adhesion and invasion of human triplenegative breast cancer (TNBC) cell line MDAMB231 in vitro and its possible mechanism. Methods: Three pairs of miRNAs specifically targeting P65 gene (P65miRNA1, P65miRNA2 and P65miRNA3) were designed and transfected into MDAMB231 cells, the level of P65 protein was detected by Western blotting, and P65miRNA with best silencing result was selected and used in the following experiments. The adhesive and invasive abilities of MDAMB231 cells before and after transfection were measured by adhesion assay and Transwell assay, respectively. mRNA expression of matrix metalloproteinase2 (MMP2) and MMP9 were detected by RTPCR and the activities of MMP2 and MMP9 were examined by gelatin zymography assay. Results: P65miRNA1, P65miRNA2 and P65miRNA3 plasmids were successfully constructed, P65miRNA1 and P65miRNA2, but not P65miRNA3, tranfection strongly inhibited the expression of P65 protein in MDAMB231 cells (>80%). The adhesion of MDAMB231 cells was not significantly influenced by P65miRNA1 and P65miRNA2 transfection (P>0.05), while the invasive ability of MDAMB231 cells was significantly reduced after P65miRNA1 or P65miRNA2 transfection (0.371±0.039, 0.309±0.046 vs 0.698±0065, P<0.05). Besides, mRNA expression of MMP2 (0.281±0.018, 0.478±0.023 vs 1.056±0.072, 1.128±0.059, P<0.05) and MMP9 (0.193±0.013, 0.371±0.035 vs 1.206±0.069, 1.089±0.057, P<0.05) and activities of MMP2 and MMP9 in MDAMB231 cells after P65miRNA1 or P65miRNA2 transfection were significantly decreased. Conclusion: Targeting silence of P65 expression can inhibit in vitro invasion of TNBC MDAMB231cells, which is related to the downregulation of MMP2 and MMP9 expression and their activities.

河北省科学技术研究与发展计划项目资助（No. 10276167）