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. 2012 Jan 2;287(1):465-473.
doi: 10.1074/jbc.M111.280768. Epub 2011 Nov 10.

MicroRNA 34c gene down-regulation via DNA methylation promotes self-renewal and epithelial-mesenchymal transition in breast tumor-initiating cells

Fengyan Yu  1 , Yu Jiao  2 , Yinghua Zhu  2 , Ying Wang  1 , Jingde Zhu  3 , Xiuying Cui  4 , Yujie Liu  1 , Yinghua He  3 , Eun-Young Park  5 , Hongyu Zhang  3 , Xiaobin Lv  4 , Kelong Ma  3 , Fengxi Su  1 , Jong Hoon Park  6 , Erwei Song  7
Affiliations

MicroRNA 34c gene down-regulation via DNA methylation promotes self-renewal and epithelial-mesenchymal transition in breast tumor-initiating cells

Fengyan Yu et al. J Biol Chem. .

Abstract

Tumor-initiating cells (T-ICs), a subpopulation of cancer cells with stem cell-like properties, are related to tumor relapse and metastasis. Our previous studies identified a distinct profile of microRNA (miRNA) expression in breast T-ICs (BT-ICs), and the dysregulated miRNAs contribute to the self-renewal and tumorigenesis of these cells. However, the underlying mechanisms for miRNA dysregulation in BT-ICs remain obscure. In the present study, we demonstrated that the expression and function of miR-34c were reduced in the BT-ICs of MCF-7 and SK-3rd cells, a breast cancer cell line enriched for BT-ICs. Ectopic expression of miR-34c reduced the self-renewal of BT-ICs, inhibited epithelial-mesenchymal transition, and suppressed migration of the tumor cells via silencing target gene Notch4. Furthermore, we identified a single hypermethylated CpG site in the promoter region of miR-34c gene that contributed to transcriptional repression of miR-34c in BT-ICs by reducing DNA binding activities of Sp1. Therefore, miR-34c reduction in BT-ICs induced by a single hypermethylated CpG site in the promoter region promotes self-renewal and epithelial-mesenchymal transition of BT-ICs.

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Figures

FIGURE 1.
FIGURE 1.
miR-34c expression was reduced in BT-ICs. A, expression of primary miR-34b/c was reduced in BT-ICs (Sus.) compared with differentiated breast cancer cells (Ad.) using qRT-PCR analysis. B and C, expression of mature miR-34c and miR-34b was reduced in BT-ICs (Sus.) compared with differentiated breast cancer cells (Ad.) using qRT-PCR analysis. D, miR-34c function, as assessed using luciferase assays, was reduced in BT-ICs (Sus.) compared with differentiated breast cancer cells (Ad.). Data are shown as relative mRNA or microRNA levels normalized to β-actin or U6. *, p < 0.05; #, p < 0.01; error bars correspond to mean ± S.D.
FIGURE 2.
FIGURE 2.
Notch4 was negatively regulated by miR-34c in BT-ICs. A, Notch4 protein levels were reduced after transfection with miR-34c mimics in BT-ICs compared with controls. In addition, Notch4 protein levels were also lower in differentiated breast cancer cells compared with BT-ICs, as assayed using Western blotting. Histogram shows the gray scale quantitative analysis for Western blotting using Gel-pro software. B, miR-34c mimics significantly inhibited the luciferase activity of the 3′-UTR of the Notch4 gene and no longer inhibited activity when the binding site was mutated, as assayed using a dual luciferase reporter assay. #, p < 0.01; error bars correspond to mean ± S.D.
FIGURE 3.
FIGURE 3.
Ectopic expression of miR-34c in BT-ICs reduced self-renewal, migration, and EMT. A, mammosphere formation was reduced after transfection of BT-ICs with miR-34c mimics and Notch4 siRNA. B and C, proportion of ALDH+ and CD44+CD24−/low cells was reduced after transfection with miR-34c mimics and Notch4 siRNA, as analyzed using FACS. D, E-cadherin was up-regulated, and fibronectin and vimentin were down-regulated in BT-ICs after transfection with miR-34c mimics and Notch4 siRNA, as analyzed using Western blotting. Histogram shows the gray scale quantitative analysis for Western blotting using Gel-pro software. E, zeb1, slug, and snail were down-regulated in BT-ICs after transfection with miR-34c mimics and Notch4 siRNA, as analyzed using Western blotting. Histogram shows the grayscale quantitative analysis for Western blotting using Gel-pro software. F and G, cell migration was significantly reduced in BT-ICs after transfection with miR-34c mimics and Notch4 siRNA, as assessed using wound healing assays and Transwell assays using Boyden chambers. *, p < 0.05; #, p < 0.01, compared with untransfected cells; error bars correspond to mean ± S.D.
FIGURE 4.
FIGURE 4.
Reduction of miR-34c in BT-ICs was associated with the hypermethylation status of its neighboring CpG island. A, miR-34c-neighboring CpG islands were hypermethylated to a greater extent in the BT-ICs (Sus.) compared with differentiated breast cancer cells (Ad.), as assessed using MeDIP-qPCR. B–D, expression levels of primary miR-34b/c, mature miR-34c, and mature miR-34b in BT-ICs were up-regulated upon DAC treatment in a dose-dependent manner. Data are shown as relative mRNA or miRNA levels normalized to β-actin or U6. *, p < 0.05; #, p < 0.01; error bars correspond to mean ± S.D.
FIGURE 5.
FIGURE 5.
CpG1 and CpG2 in the miR-34c-neighboring CpG islands were hypermethylated in BT-ICs. A, in the SK-3rd cell line, CpG1 and CpG2 in the miR-34c-neighboring CpG islands were fully methylated in all of the BT-ICs clones, whereas only 25 and 50% of CpG1 and CpG2, respectively, were methylated in the differentiated breast cancer cells. B, in the MCF-7 cell line, CpG1 and CpG2 were 50 and 90% methylated in the BT-ICs compared with 10 and 60% methylation, respectively, in the differentiated breast cancer cells, as assessed using bisulfate sequencing PCR in the SK-3rd and MCF-7 cell lines. Each line represents the methylation status of a single clone, and the open and filled circles represent the unmethylated and methylated CpG sites, respectively.
FIGURE 6.
FIGURE 6.
CpG1 was located in a crucial regulatory element of the miR-34c promoter, and methylated CpG1 decreased binding activity of Sp1 to this site. A, promoter activities of different fragments, based on the position of CpG1 and CpG2 sites in the reported miR-34c promoter region, were assayed using a dual luciferase reporter assay. All of the constructs containing CpG1 exhibited a significant level of promoter activity, and the luciferase activity increased significantly from fragment miR-34c-178-Luc to fragment miR-34c-266-Luc. #, p < 0.01, compared with the pGL3-basic vector; error bars correspond to mean ± S.D. B, changes in the putative promoter activities after mutation of CpG1 and CpG2 in fragments miR-34c-496-Luc and miR-34c-573-Luc were assayed using a dual luciferase reporter assay. When CpG1 was mutated, the promoter activity was significantly reduced, whereas there was no significant reduction when CpG2 was mutated. #, p < 0.01, compared with unmutated miR-34c-496-Luc and miR-34c-573-Luc; error bars correspond to mean ± S.D. C, DNA sequence of miR-34 promoter was demonstrated. The CG in bold represents CpG1 and CpG2. The predicted Sp1 binding site was also indicated. D, co-transfection of Sp1 expression vector (p-EZ-MO2-Sp1) with miR-34c-573-Luc or miR-34c-573-CpG1mu-Luc into BT-ICs showed that the promoter activity of miR-34c-573-Luc was significantly up-regulated, whereas that of miR-34c-573-CpG1mu1-Luc was not affected. E, Sp1 expression was up-regulated after transfection of the Sp1 expression vector, as analyzed using Western blotting. F, methylated CpG1 decreased the binding activity of Sp1, as assessed using EMSAs.
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