Original Article
Biomedical
MicroRNA-21 Modulates Chemosensitivity of Breast Cancer Cells to Doxorubicin by Targeting PTEN

https://doi.org/10.1016/j.arcmed.2011.06.008Get rights and content

Background and Aims

Ovexpression of microRNA-21 (miR-21) is found in various human cancers. Our aim is to investigate the association of miR-21 expression with the sensitivity of breast cancer cells to doxorubicin (ADR).

Methods

The half maximal inhibitory concentration (IC50) value of ADR in resistant MCF-7/ADR or parental MCF-7 cells was determined by MTT assay. TaqMan RT-PCR or Western blot assay was performed to detect the expression of mature miR-21 and tumor suppressor gene (PTEN) protein. MCF-7 or MCF-7/ADR cell line was transfected with miR-21mimic or inhibitor. The IC50 value of ADR was determined. Flow cytometry and TUNEL assays were performed to analyze apoptosis. The activity of caspase-3 was analyzed.

Results

The IC50 of ADR in MCF-7 and MCF-7/ADR cells was 0.21 ± 0.05 and 16.5 ± 0.08 μmol/L, respectively. We showed that upregulation of miR-21 in MCF-7/ADR cells was concurrent with downregulation of PTEN protein. MiR-21 mimic or inhibitor could obviously affect the sensitivity of breast cancer cells to ADR. Moreover, miR-21 inhibitor could enhance caspase-3-dependent apoptosis in MCF-7/ADR cells. Overexpression of PTEN could mimic the same effects of miR-21 inhibitor in MCF-7/ADR cells and PTEN-siRNA could increase the resistance of MCF-7 cells to ADR. MiR-21 inhibitor could increase PTEN protein expression and the luciferase activity of a PTEN 3′ untranslated region-based reporter construct in MCF-7/ADR cells. PTEN-siRNA could partially reverse the increased chemosensitivity of MCF-7/ADR cells induced by miR-21 inhibitor.

Conclusions

Dysregulation of miR-21 plays critical roles in the ADR resistance of breast cancer, at least in part via targeting PTEN.

Introduction

Breast cancer is one of the leading causes of cancer death in females worldwide. Approximately 1,200,000 patients were newly diagnosed with breast cancer annually worldwide and about 465,000 will die from the disease (1). In China, about 40,000 patients die from breast cancer every year. Despite the use of multiple therapies including chemotherapy, the tumor does not actively respond to the therapies (2). Therefore, a better understanding of the molecular mechanisms involved in breast cancer formation and progression will be helpful for identification of sensitive tumor markers so as to develop novel chemotherapeutic drugs and preventive strategies for breast cancer therapy.

MicroRNAs (miRNAs), a class of small noncoding RNAs first reported in 1993, are posttranscriptional regulators of gene expression and have been shown to be involved in cell differentiation, survival and apoptosis (3). Aberrant posttranscriptional regulation of mRNAs by miRNAs can lead to oncogenesis with increased cell proliferation, decreased apoptosis, and enhanced metastatic potential of affected cells (4). Thus, dysregulation of miRNAs could play critical roles in tumorigenesis and tumor progression. However, their associations with chemosensitivity of human tumors are still not elucidated systematically. MiR-21 is a miRNA, which has been reported to be overexpressed in many human malignancies and acts as an oncogene related to proliferation, apoptosis, and metastasis 5, 6, 7. Asgaga et al. showed that circulating miR-21 is a potential diagnostic and prognostic biomaker in breast cancer (8). Huang and colleagues reported that high expression of miR-21 indicates a more aggressive phenotype for breast cancer patients (9). Additionally, other groups showed that miR-21 could affect invasion and metastasis of breast cancer 10, 11. To the best of our knowledge, there have been no reports about the expression of miR-21 and sensitivity of breast cancer cells to ADR.

In the present study we provide the first evidence that miR-21 regulates ADR resistance of breast cancer cells, at least in part, by targeting the tumor suppressor gene PTEN. These data showed that miR-21 targeting therapy may be a potential strategy for chemosensitization of breast cancer.

Section snippets

Cell Culture

Human breast cancer cell line MCF-7 and its doxorubicin-resistant variant MCF-7/ADR (obtained from Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai, China) were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (Gibco BRL, Grand Island, NY) in a humidified atmosphere containing 5% CO2 at 37°C. To maintain the ADR-resistance phenotype, ADR (with final concentration of 1.0 μmol/L) was added to the culture media for MCF-7/ADR cells.

Construction of Plasmid Vectors

Previously, the

Expression of miR-21 and PTEN Protein in MCF-7 and Paired MCF-7/ADR Cells

First, MTT assay was performed to determine the IC50 value of ADR in sensitive MCF-7 and resistant MCF-7/ADR cells. As shown in Figure 1A, the IC50 of ADR in MCF-7 and MCF-7/ADR cells was 0.21 ± 0.05 and 16.5 ± 0.08 μmol/L, respectively (p <0.01). Then, TaqMan RT-PCR assay was performed to detect the expression of miR-21 in breast cancer cell line (MCF-7) and paired ADR-resistant breast cancer cell line (MCF-7/ADR). Compared with that in MCF-7 cell line, the relative level of miR-21 expression

Discussion

Aberrant expression of miRNAs has been reported to be involved in tumor progression, metastasis and chemoradioresistance (12). Among them, miR-21 was one of the first miRNAs detected in the human genome and is the only miRNA known to be overexpressed in a variety of human cancers. Previously, much evidence has shown that miR-21 is found to be correlated with prognosis of tumor patients, indicating that it may be used as a molecular marker for predicting patient prognosis 13, 14. In addition,

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (No. 30973477), the Natural Science Foundation of Jiangsu Province (No. BK2010590), the Jiangsu Provincial Personnel Department “the Great of Six Talented Man Peak” Project (No. 09-B1-021 and 2010-IB10), Qing Lan Project 2010 of Jiangsu Province Department of Education, and the Foundation of Nanjing Medical University (09NMUZ21).

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    These authors contributed equally to this work.

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