南方医科大学学报

南方医科大学学报 ›› 2019, Vol. 39 ›› Issue (03): 304-.doi: 10.12122/j.issn.1673-4254.2019.03.08

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沉默RRM1 可逆转乳腺癌细胞MCF-7/R对紫杉醇的耐药性

田楠楠，周磊，杨丹妮，吴焕贤，马韵词，吕琳，吴少瑜   

  • 出版日期:2019-03-20 发布日期:2019-03-20

Silencing RRM1 gene reverses paclitaxel resistance in human breast cancer cell line MCF- 7/R by inducing cell apoptosis

  • Online:2019-03-20 Published:2019-03-20

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摘要： 目的阐明沉默核苷酸还原酶M1亚基（RRM1）对乳腺癌耐药细胞MCF-7/R逆转耐药的作用。方法通过高浓度紫杉醇诱 导耐药株MCF-7/R；运用Kaplan-Meier Plotter绘制RRM1基因的生存曲线；通过siRNA沉默MCF-7/R细胞中RRM1基因表达， 并用Western blot 和qRT-PCR方法检测蛋白和基因水平的表达，筛选出高效特异的si-RRM1 序列；将该si-RRM1 序列转染 MCF-7/R细胞，筛选得到能稳定抑制RRM1基因表达的细胞株MCF-7/R/siRNA；四甲基偶氮唑盐（MTT）法和5-乙炔基-2’脱氧 尿嘧啶核苷（EdU）染色法检测RRM1沉默后MCF-7/R细胞增殖能力的变化；流式细胞术检测细胞周期和凋亡，并观察周期、凋 亡相关蛋白的变化；构建裸鼠皮下移植瘤模型，观察沉默RRM1后给予紫杉醇治疗对裸鼠体内抑瘤效果的影响。结果生存曲 线分析显示RRM1基因表达与乳腺癌患者的生存率呈负相关（P=0.000）；MCF-7/R细胞中证实RRM1蛋白和mRNA表达水平 较MCF-7细胞均显著升高（P<0.01）；si-RRM1序列筛选中，转染si-RRM1-04组细胞的RRM1蛋白和mRNA表达量降低最为显 著（P<0.001）；沉默RRM1后，MCF-7/R细胞对紫杉醇的敏感性显著增加，细胞晚期凋亡比例明显升高（P<0.001），同时降低Akt 蛋白的磷酸化并抑制凋亡蛋白Bcl-2 的表达，促进p53 蛋白表达水平的增加（P<0.001）；裸鼠实验显示，与si-NC组相比，沉默 RRM1后给予紫杉醇治疗可显著抑制裸鼠体内肿瘤的生长（P<0.001）。结论沉默RRM1可通过诱导细胞凋亡增加提高MCF- 7/R细胞化疗敏感性，逆转乳腺癌紫杉醇化疗耐药。

Abstract: Objective To investigate the effects of ribonucleotide reductase catalytic subunit M1 (RRM1) gene silencing on drug resistance of human breast cancer cell line MCF-7/R. Methods We established a paclitaxel-resistant breast cancer MCF-7 cell line (MCF-7/R) by exposing the cells to high-concentration paclitaxel in a short time. Small interfering RNAs (siRNAs) targeting RRM1 were designed to silence RRM1 expression in human breast cancer MCF-7/R cells. MTT assay was used to detect the IC50 values and the sensitivity to paclitaxel in the cells with or without siRNA transfection. The changes in the proliferative activity of MCF7 and MCF-7/R cells following RRM1 gene silencing were evaluated using EdU assay. Flow cytometry was used to analyze the cell apoptosis and cell cycle changes. We assessed the effect of RRM1 gene silencing and paclitaxel on the tumor growth in a nude mouse model bearing subcutaneous xenografts with or without siRNA transfection. Results We detected significantly higher expressions of RRM1 at both the mRNA and protein levels in the drug-resistant MCF- 7/R cells than in the parental MCF-7 cells (P<0.01). Transfection with the specific siRNAs significantly reduced the expression of RRM1 in MCF-7/R cells (P<0.05), which showed a significantly lower IC50 value of paclitaxel than the cells transfected with the negative control siRNA (P<0.05). RRM1 silencing significantly inhibited the proliferation (P<0.01) and enhanced the apoptosis- inducing effect of paclitaxel in MCF-7/R cells (P<0.001); RRM1 silencing also resulted in obviously reduced Akt phosphorylation, suppressed Bcl-2 expression and promoted the expression of p53 protein in MCF-7/R cells. In the tumor-bearing nude mice, the volume of subcutaneously transplanted tumors was significantly smaller in MCF-7/R/siRNA+ PTX group than in the other groups (P<0.001). Conclusion RRM1 gene silencing can reverse paclitaxel resistance in human breast cancer cell line MCF-7/R by promoting cell apoptosis.