[摘要] 目的：探讨miR-520d 通过调控自噬逆转三阴性乳腺癌（TNBC）细胞化疗耐药的作用及分子机制。方法：以人TNBC细胞系MDA-MB-231 和MDA-MB-468 为亲本株细胞构建多西他赛（Doc）耐药细胞株MDA-MB-231/Doc 和MDA-MB-468/Doc，实验分为空白组（亲本细胞）、对照组（耐药细胞组）和过表达miR-520d 组。用qPCR检测空白组和耐药细胞组细胞中miR-520d 的表达水平，MTT实验检测过表达miR-520d 的耐药细胞对Doc 的敏感性，MDC染色后荧光显微镜观察细胞中自噬小体的发生情况、共聚焦显微镜观察过表达miR-520d 的耐药细胞中自噬相关蛋白LC3 阳性的细胞数。用荧光素酶报告基因实验验证miR-520d 与Beclin1 的靶向关系。用WB实验检测过表达miR-520 对细胞中自噬相关蛋白Beclin1 和LC3Ⅰ、LC3Ⅱ表达的影响。结果：TNBC耐药细胞中miR-520d 的表达水平明显低于空白组细胞（P<0.01）。过表达miR-520d 的耐药细胞对Doc的敏感性显著提高（P<0.01）、细胞的自噬活性明显降低（P<0.01）。荧光素酶报告基因实验证明Beclin1 是miR-520d 可能的靶分子。Doc 与miR-520d mimics 联合使用可降低TNBC耐药细胞中LC3-Ⅱ/Ⅰ比值和自噬蛋白Beclin1 的表达水平（均P<0.05）。结论：通过调控miR-520d 水平可能改变自噬蛋白Beclin1 表达，从而逆转TNBC细胞Doc化疗耐药性。

[Abstract] Objective: To investigate the role and molecular mechanism of miR-520d in reversing the chemoresistance of triple negative breast cancer (TNBC) by regulating autophagy. Methods: Docetaxel (Doc) resistant cell lines MDA-MB-231/Doc and MDA-MB-468/Doc were constructed by using human TNBC cell lines MDA-MB-231 and MDA-MB-468 as parental cells, and the cells were divided into blank group (parental cells), control group (drug-resistant group), and miR-520d over-expression group. The expression levels of miR-520d in cells of the blank and drug-resistant groups were detected by qPCR. The Doc-sensitivity of resistant cells over-expressing miR-520d was detected by MTT assay. After MDC staining, the generation of autophagosome in cells was observed under fluorescence microscopy; the number of miR-520d over-expressed resistant cells with positive LC3 expression was observed under confocal microscopy. The luciferase reporter gene assay was used to verify the targeting relationship between miR-520d and Beclin1. The effect of miR-520d mimics on the expression of autophagy-associated protein Beclin1, and LC3Ⅰ, LC3Ⅱ in cells was detected by WB assay.Results: The results of qPCR showed that the expression of miR-520d in the drug-resistant TNBC cells was significantly lower than that of normal cells (P<0.01). In drug-resistant cells over-expressing miR-520d, the Doc-sensitivity was significantly improved,while the autophagy activity was significantly reduced (all P<0.01). At the same time, luciferase experiments demonstrated that Beclin1 was a possible target molecule of miR-520d (P<0.05). WB results showed that the combination of docetaxel and miR-520d mimics reduced the LC3-II/I ratio and the expression of autophagy protein Beclin1 in drug-resistant TNBC cells (all P<0.05). Conclusion: The regulation of miR-520d levels may alter the expression of autophagy protein Beclin1, thereby reversing Doc chemotherapy resistance in TNBC cells.

国家自然科学基金资助项目（No.81703881）