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

Nan Fang Yi Ke Da Xue Xue Bao. 2019 Mar 30;39(3):304-312. doi: 10.12122/j.issn.1673-4254.2019.03.08.
[Article in Chinese]

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).

Conclusions: RRM1 gene silencing can reverse paclitaxel resistance in human breast cancer cell line MCF-7/R by promoting cell apoptosis.

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。

Keywords: MCF-7; breast cancer; drug resistance; paclitaxel; ribonucleotide reductase catalytic subunit M1; small interfering RNA.

MeSH terms

  • Animals
  • Apoptosis
  • Breast Neoplasms*
  • Drug Resistance, Neoplasm
  • Gene Silencing
  • Humans
  • MCF-7 Cells
  • Mice
  • Mice, Nude
  • Paclitaxel
  • RNA, Small Interfering
  • Ribonucleoside Diphosphate Reductase
  • Ribonucleotide Reductases
  • Tumor Suppressor Proteins

Substances

  • RNA, Small Interfering
  • Tumor Suppressor Proteins
  • Ribonucleotide Reductases
  • RRM1 protein, human
  • Ribonucleoside Diphosphate Reductase
  • Rrm1 protein, mouse
  • Paclitaxel

Grants and funding

国家自然科学基金(81473246);广东省科技计划项目(C1040924);广东省医学科学技术研究基金(A2018189);广东省自然科学基金(2018A030313405)