目的：探讨miR-9-5p在乳腺癌恶性生物学行为中发挥的作用及其可能的调控机制。方法：利用OncomiR在线数据库分析miR-9-5p在乳腺癌组织与正常乳腺组织中表达的差异，qPCR检测乳腺癌细胞系与正常乳腺细胞中miR-9-5p表达水平。基于靶基因预测软件TargetScan分析ONECUT2（one cut homeobox 2）可能是miR-9-5p的作用靶基因，双荧光素酶报告实验验证两者的靶向关系。向MDA-231细胞中分别转染miR-9-5p mimic、ONECUT2 siRNA及相应对照，qPCR及WB实验检测转染对MDA-231细胞中干性基因NOTCH1、NANOG和Y染色体性别决定区（sex-determing region of Y chromosome，SRY）-盒转录因子9（SRY-box transcription factor 9，SOX9）表达水平的影响，BrdU法、Annexin Ⅴ流式细胞术、MTS实验分别检测转染对细胞增殖、凋亡和化疗耐药的影响，ALDEFLUOR染色流式细胞术检测miR-9-5p及靶基因 ONECUT2 对肿瘤干细胞化特征的影响。建立NSG 小鼠乳腺癌化疗模型 ，体内实验进一步验证 ONECUT2 对肿瘤干性化及化疗抵抗等肿瘤恶性生物学行为的影响。结果：miR-9-5p 在乳腺癌组织（P=0.007）及乳腺癌 MDA-231 细胞系（P=0.0005）中呈现显著高表达，并与乳腺癌患者不良预后呈正相关（P=0.0016）。miR-9-5p 可靶向负调控 ONECUT2，进而增加 ALDH+MDA-231 细胞比例（P=0.0006），上调干性NOTCH1、NANOG和SOX9蛋白表达，并增强乳腺癌细胞抗凋亡能力（P=0.0003）及其对多西他赛（DTX）和多柔比星（DOXO）化疗的耐受性 ；然而 miR-9-5p/ONECUT2 轴未能显著影响 MDA-231 细胞的增殖能力（P>0.05）。与对照组相比 ，MDA-231/ONECUT2组小鼠接受DTX治疗后，移植瘤体积较对照组显著缩小（P<0.05），瘤组织中NOTCH1、SOX9蛋白和ABC转运蛋白的mRNA和蛋白表达水平均显著降低（P<0.05或P<0.01）。结论：乳腺癌组织中高表达的miR-9-5p通过靶向ONECUT2诱导乳腺癌干细胞化及抗凋亡能力，增强了其对化学治疗的抵抗性。

Objective: To explore the role of miR-9-5p in the biological behaviors of breast cancer cells and its possible regulatory mechanism. Methods: online OncomiR database was used to analyze the differential expression of miR-9-5p in breast cancer tissues andnormal breast tissues. qPCR was used to detect the miR-9-5p expression in breast cancer cell lines and normal breast cells. Based on target gene prediction software TargetScan, ONECUT2 (one cut homeobox 2) was predicted to be the target gene of miR-9-5p. Dual luciferase reporter system was used to validate the relationship between miR-9-5p and its promising target gene ONECUT2. MDA-231 cells were transfected with miR-9-5p mimic, ONECUT2 siRNAs as well as the corresponding control sequences. The protein and mRNA levels of stemness-associated gene NOTCH1, NANOG and SOX9 (SRY (sex-determing region of Y chromosome)-Box transcription Factor 9) were detected by WB and qPCR. The effect of transfection on proliferation, apoptosis and chemo-resistance of cells was detected by BrdU method,Annexin Ⅴ method and MTS Assay, respectively. The ALDEFLUOR experiment was used to detect the effects of miR-9-5p and its target gene ONECUT2 on tumor stemness. NSG mouse breast cancer chemotherapy model was established, and the in vivo experiments further verified the effect of ONECUT2 on tumor malignant biological behaviors, such as cell stemness and chemo-resistance. Results: miR-9-5p was highly expressed in breast cancer tissues (P=0.007) and breast cancer MDA-231 cell line (P=0.0005), and was positively correlated with the poor prognosis of breast cancer patients (P=0.0016). Compared to control group, miR-9-5p could target and negatively regulate ONECUT2 expression, further increase ALDH+ cell population (P=0.0006), as well as increase the expressions of stemness-associated genes NOTCH1,NANOG and SOX9. Besides, miR-9-5p increased the anti-apoptosis ability (P=0.0003) and chemo-resistance of MDA-231 cells; however,miR-9-5p/ONECUT2 exerted no significant effect on the proliferation ability of MDA-231 cells (P>0.05). Compared with the control group,the volume of xenografts in mice of MDA-231/ONECUT2 group after DTX chemotherapy was significantly lower than that in the control group (P<0.05), and the protein expressions of NOTCH1, SOX9 and the mRNA expression of ABC transporter in the transplanted tumor tissues were significantly reduced (P<0.05 or P<0.01). Conclusions: The highly expressed miR-9-5p in breast cancer induces tumor stemness and anti-apoptotic ability by targeting ONECUT2 and enhances its resistance to chemotherapy.

天津市教委科研计划资助项目（No. 2019KJ185）