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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 observe the effect of miRNA targeted silencing P65 gene on in vitro adhesion and invasion of human triple-negative breast cancer (TNBC) MDA-MB-231 cells and its possible mechanism. Methods: Three specific miRNAs (P65miRNA-1, P65miRNA-2 and P65miRNA-3) targeting P65 gene were designed and transfected into MDA-MB-231 cells. The expression of P65 protein was detected by Western blotting. Follow-up experiment. Adhesion and invasion of MDA-MB-231 cells before and after P65miRNA transfection were detected by in vitro adhesion assay and Transwell chamber assay. The expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 mRNA in MDA-MB-231 cells was detected by RT-PCR. The expression of MMP- Enzyme activity of MMP-9. Results: The recombinant plasmids P65miRNA-1, P65miRNA-2 and P65miRNA-3 were successfully constructed. The expression of P65 protein was inhibited more than 80% in MDA-MB-231 cells. P65miRNA-1 and P65miRNA-2 transfected MDA-MB-231 cells adhesion had no significant effect (P> 0.05), but significantly inhibited the invasion of MDA-MB-231 cells (0.371 ± 0.039,0.309 ± 0.046 vs 0.698 ± 0.065 , P <0.05). P65miRNA-1and P65miRNA-2transfected MMP-2 mRNA (P <0.05) and MMP-2 mRNA (P <0.05) (0.193 ± 0.013,0.371 ± 0.035 vs 1.206 ± 0.069,1.089 ± 0.057, P <0.05) and their activities were significantly down-regulated. CONCLUSIONS: The silencing of P65 by miRNA can inhibit the in vitro invasion of human TNBC MDA-MB-231 cells, which may be related to the inhibition of the expression and activity of MMP-2 and MMP-9.