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. 2015 Apr 30;6(12):10432-44.
doi: 10.18632/onco_target.3394.

Dysregulation of the miR-34a-SIRT1 axis inhibits breast cancer stemness

Wei Ma  1   2 , Gary Guishan Xiao  3   4 , Jun Mao  1   5 , Ying Lu  1 , Bo Song  1 , Lihui Wang  1 , Shujun Fan  5 , Panhong Fan  1 , Zhenhuan Hou  1 , Jiazhi Li  1 , Xiaotang Yu  1 , Bo Wang  1 , Huan Wang  5 , Honghai Wang  1 , Fei Xu  2 , Yan Li  2 , Qiang Liu  6 , Lianhong Li  1   5
Affiliations

Dysregulation of the miR-34a-SIRT1 axis inhibits breast cancer stemness

Wei Ma et al. Onco_target. .

Abstract

Enforced expression of miR-34a eliminates cancer stem cells in some malignant tumors. Sirtuin-1 (SIRT1) is a direct _target of miR-34a. Here we found low levels of miR-34a and high levels of SIRT1 in CD44+/CD24- breast cancer stem cells (BCSCs). MiR-34a overexpression and knockdown of SIRT1 decreased proportion of BSCSs and mammosphere formation. Expression of CSC markers, ALDH1, BMI1 and Nanog was decreased. In nude mice xenografts, stable expression of miR-34a and silencing of SIRT1 reduced tumor burden. Taken together, our results demonstrated that miR-34a inhibits proliferative potential of BCSCs in vitro and in vivo, at least partially by downregulating SIRT1. The miR-34a-SIRT1 axis may play role in self-renewal of BCSCs.

Keywords: CD44+/CD24− BCSCs; SIRT1; miR-34α; stemness.

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Conflict of interest statement

DECLARATION OF CONFLICTING INTERESTS

The authors declared no potential conflicts of interest.

Figures

Figure 1
Figure 1. The endogenous expression levels of miR-34a and SIRT1 in CD44+/CD24−BCSCs
A. Relative qRT-PCR quantification of miR-34a in MCF-7 cells, non-BCSCs, and CD44+/CD24− BCSCs. The relative expression levels (mean ± S.D) were normalized to RNU6B, and the expression of miR-34a in MCF-7cells set at one. Compared to MCF-7 cells and non-BCSCs, miR-34a in BCSCs showed significantly lower expression. *p < 0.05. B. Representative mRNA expression of SIRT1 in MCF-7(unseparated) cells, non-BCSCs, and BCSCs. The realtive expression levels are presented by setting mRNA levels in MCF-7 cells as one. GAPDH was used for normalization. Expression of SIRT1 mRNA in BCSCs showed > 2 fold higher compared to MCF-7 cells or non-BCSCs. **p < 0.01. C. The protein expression of SIRT1 in MCF-7 (unseparated) cells, non-BCSCs, and BCSCs was determined by Western blotting (left). The quantitative results were analyzed by Gel-Pro Analyzer 4.0 software (right), and GAPDH was used as an endogenous control. **p < 0.01. D. Immunofluorescence staining of SIRT1 in MCF-7 cells. a, d, PE-labeled anti-SIRT1(red). b, e, labeled with DAPI (a nuclear marker) (blue). c, f, represent overlay image of a and b, d and e, respectively. SIRT1 were highly expressed in BCSCs than non-BCSCs. scale bar = 20 μm. MCF-7 represents the cells before sorting, non-BCSCs represents non-CD44+/CD24− breast cancer cells, and BCSCs represents CD44+/CD24− breast cancer stem cells.
Figure 2
Figure 2. Down-regulation of SIRT1 and over-expression of miR-34a inhibit cell growth and colony formation abilities
A. Comparison of shRNA-1, shRNA-2, and shRNA-3 in silencing SIRT1 expression at protein levels. MCF-7 cells were transfected with three shRNAs (shRNA-SIRT1–1, shRNA-SIRT1–2, and shRNA-SIRT1–3) respectively. Scrambling nucleotide sequence of SIRT1 (shRNA-NC) was used as negative control. After 48 h, cells were cultured under antibiotic pressure for 21 days and collected for Western blotting analysis (left). The quantitative results (right) were analyzed by Gel-Pro Analyzer 4.0 software, GAPDH was used as an endogenous control. **p < 0.01. B. MiR-34a mimics up-regulates miR-34a expression in MCF-7 cells. MCF-7 cells were transfected with miR-34a mimics (miR-34a) or non-specific control (miR-NC) for 48 h, and then collected for qRT-PCR analysis. C. Over-expression of miR-34a down regulates SIRT1 expression. Upper: Western blotting analysis the expression of SIRT1 in miR-34a or miR-NC transfected cells. Lower: The quantitative results were analyzed by Gel-Pro Analyzer 4.0 software, GAPDH was used as an endogenous control. **p < 0.01. D. CCK8 assay showed either ShRNA-SIRT1 or over-expression miR-34a inhibits the proliferation rate of MCF-7 cells. E. Either ShRNA-SIRT1 or over-expression miR-34a reduces the number and average size of colony in MCF-7 cells. Each condition was repeated 3 times and error bars represent SEM.
Figure 3
Figure 3. Dysregulation of miR-34a-SIRT1 axis decrease BCSCs
A. The percentages of CD44+/CD24− BCSCs were reduced either by over-expression of miR-34a or silencing SIRT1. The phenotype of CD44+/CD24− cells was measured by flow cytometry. Similar results were obtained in three independent experiments, **p < 0.01. B. Either over-expression of miR-34a or down-regulation of SIRT1 decreased volume (left) and number (right) of mammospheres. After transfection with miR-34a mimics or shRNA-SIRT1, cells were cultured in ultra-low attachment plates with serum-free medium for 7 d. Mammospheres were collected and evaluated. C. SIRT1, ALDH1, Nanog, and BMI1 were down-regulated as over-expression of miR-34a or silencing SIRT1 (left). The quantitative results were analyzed by Gel-Pro Analyzer 4.0 software (right), and GAPDH was used as an endogenous control. *p < 0.05, **p < 0.01. D. Immunofluorescent staining confirmed lower Nanog protein expressions in miR-34a over-expressed cells and silenced SIRT1 cells. a, d, g, j, labeled with DAPI (a nuclear marker) (blue), b, e, h, k, FITC-labeled anti-Nanog (green), and c, f, i, l, represent overlay image of a and b, d and e, g and h, j and k, respectively. Scale bar = 50 μm.
Figure 4
Figure 4. Modulation of miR-34a- SIRT1 axis enhanced MCF-7 cell apoptosis
A. Over-expression of miR-34a and silenced SIRT1 increased the percentage of early period apoptotic cells labeled with Annexin V-FITC/PI. Representative scatter grams from flow cytometry profile represent Annexin V-FITC staining in x axis and PI in y axis. B. The apoptotic cells labeled with Hoechst 33342 increased either in miR-34a over-expression cells or silenced SIRT1 cells. The apoptotic cells emit strong blue fluorescence.
Figure 5
Figure 5. MiR-34a over-expression or silenced SIRT1 inhibit tumor growth in vivo
A. Over-expression of miR-34a or silenced SIRT1 remarkably reduced the tumor volume compared to the control groups. *p < 0.05, **p < 0.01. B. Subcutaneous tumor regeneration from MCF-7 cells infected with lentivirus-miR-NC (miR-NC) or lentivirus-miR-34a (miR-34a), or transfected with shRNA-NC(shRNA-NC) or shRNA-SIRT1(shRNA-SIRT1). C. HE and IHC staining with SIRT1 and ALDH1 antibodies. a-d: HE staining; c-l: IHC staining for SIRT1 (brown color in nuclei) and ALDH1 (brown color in cytoplasm), scale bar = 50 μm. D. qRT-PCR analyses of miR-34a expression in each group of xenograft tissues. Compared to each control group, miR-34a or shRNA-SIRT1 group showed a significantly high miR-34a expression level. *p < 0.05. E. Protein levels of SIRT1, and ALDH1 were evaluated by Western blotting analysis in each group of mice tumors (upper). As an internal control, GAPDH was used for normalization. The quantitative results were analyzed by Gel-Pro Analyzer 4.0 software (lower). Data are presented as mean ± SEM, n = 5, *p < 0.05.
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