MicroRNA-34a suppresses the breast cancer stem cell-like characteristics by downregulating Notch1 pathway

Abstract

MicroRNAs play pivotal roles in cancer stem cell regulation. Previous studies have shown that microRNA-34a (miR-34a) is downregulated in human breast cancer. However, it is unknown whether and how miR-34a regulates breast cancer stem cells. Notch signaling is one of the most important pathways in stem cell maintenance and function. In this study, we verified that miR-34a directly and functionally targeted Notch1 in MCF-7 cells. We reported that miR-34a negatively regulated cell proliferation, migration, and invasion and breast cancer stem cell propagation by downregulating Notch1. The expression of miR-34a was negatively correlated with tumor stages, metastasis, and Notch1 expression in breast cancer tissues. Furthermore, overexpression of miR-34a increased chemosensitivity of breast cancer cells to paclitaxel (PTX) by downregulating the Notch1 pathway. Mammosphere formation and expression of the stemness factor ALDH1 were also reduced in the cells treated with miR-34a and PTX compared to those treated with PTX alone. Taken together, our results indicate that miR-34a inhibited breast cancer stemness and increased the chemosensitivity to PTX partially by downregulating the Notch1 pathway, suggesting that miR-34a/Notch1 play an important role in regulating breast cancer stem cells. Thus miR-34a is a potential target for prevention and therapy of breast cancer.

Keywords: Breast cancer stem cell; Notch1; drug resistance; miR-34a; paclitaxel.

Figure 1

MicroRNA-34a (MiR-34a) directly targets and functionally suppresses Notch1 in MCF-7 cells. (a) 3′-UTR wild-type (wt) and mutated (mut) reporter constructs of Notch1. (b) MiR-34a expression (exp) levels evaluated by quantitative RT-PCR. (c, d) Notch1 mRNA and protein expression levels were evaluated 48 h after transfection. (e) Luciferase reporter assays in MCF-7 cells. Cotransfection of wt/mut Notch1 3′-UTR with miRNAs. (f) Hes-1 mRNA expression level was evaluated 48 h after transfection. *P < 0.05, compared to the control (miR-Ctrl).

Figure 2

MicroRNA-34a (MiR-34a) expression is downregulated in breast cancer tissues and negatively correlated with tumor stage, metastasis, and expression of Notch1. (a) MiR-34a expression (exp) in 45 cases of human breast cancer and 16 normal adjacent tissues (NATs) (P < 0.05). (b) MiR-34a expression is associated with lymph node metastasis of breast cancer (P < 0.05). (c) MiR-34a expression in different clinical stages of breast cancer patients. Statistical analyses were carried out using Student's t-test (a–c). (d) Inverse correlation between miR-34a and Notch1 mRNA expression in breast cancer tissues (n = 45) by Spearman's correlation analysis. (e) Representative immunohistochemical analyses of Notch1 in normal breast tissue and breast cancer tissue with different Notch1 scores (×100). (f) Immunohistochemistry scores of Notch1 in NATs and breast cancer tissues. (g) MiR-34a expression negatively correlated with Notch1 scores in breast cancer tissues. (Spearman's correlation analysis, r = −0.692; P < 0.05).

Figure 3

MicroRNA-34a (MiR-34a) regulates cell proliferation, migration, and invasion by targeting Notch1. (a) Inhibitory effects of miR-34a mimics and Notch1 siRNA on the proliferation of MCF-7 cells by Cell Counting Kit-8 assay. (b) Inhibitory effects of miR-34a mimics and Notch1 siRNA on migration and invasion of MCF-7 cells by Transwell assay (×100). *P < 0.05, compared to negative controls.

Figure 4

MicroRNA-34a (MiR-34a) suppresses BCSCs in MCF-7 cells partly by targeting Notch1. (a) MiR-34a expression in breast cancer stem cells (BCSCs) and non-BCSCs. U6 was used as control. (b) Notch1 mRNA expression in BCSCs and non-BCSCs. GAPDH was used as control. (c) Percentage of CD44⁺/CD24⁻ BCSCs was decreased by overexpression of miR-34a or knockdown of Notch1. (d) Aldehyde dehydrogenase 1 (ALDH1) expression was decreased by miR-34a overexpression or Notch1 knockdown. *P < 0.05, compared to negative controls.

Figure 5

Notch1 restoration counteracts the inhibitory effects of microRNA-34a (miR-34a) in MCF-7 cells. (a) Notch1 restoration significantly increased the cell migration suppressed by miR-34a. (b) Notch1 restoration partly reversed the miR-34a inhibition of cell proliferation. (c) Expression of Notch1, Hes-1, and aldehyde dehydrogenase 1 (ALDH1) were detected after miR-34a overexpression or Notch1 restoration. GAPDH was used for normalization. (d) Percentage of CD44⁺/CD24⁻ breast cancer stem cells BCSCs decreased by miR-34a was partly reversed by Notch1 restoration. *P < 0.05.

Figure 6

MicroRNA-34a (MiR-34a) increases chemosensitivity to paclitaxel (PTX) in MCF-7 cells through inhibition of breast cancer stemness. (a) Cell viability was quantitated by Cell Counting Kit-8 assay after treatment with miR-34a mimics or combined with PTX for indicated times. (b) MiR-34a mimic-transfection alone or combined with PTX resulted in decreased volume (left) (×400, scale bar = 100 μm) and number (right) of mammospheres. (c) Expression (exp) levels of aldehyde dehydrogenase 1 (ALDH1), Notch1, Hes-1, and ATP-binding cassette sub-family G member 2 (ABCG2) were tested after miR-34a overexpression or combined treatment with PTX (left). GAPDH was used for normalization. *P < 0.05, **P < 0.01, compared to control (miR-Ctrl).