Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Dec 5:5:5671.
doi: 10.1038/ncomms6671.

miR-205 acts as a tumour radiosensitizer by targeting ZEB1 and Ubc13

Peijing Zhang  1 , Li Wang  1 , Cristian Rodriguez-Aguayo  2 , Yuan Yuan  3 , Bisrat G Debeb  4 , Dahu Chen  1 , Yutong Sun  5 , M James You  6 , Yongqing Liu  7 , Douglas C Dean  7 , Wendy A Woodward  4 , Han Liang  3 , Xianbin Yang  8 , Gabriel Lopez-Berestein  2 , Anil K Sood  9 , Ye Hu  10 , K Kian Ang  4 , Junjie Chen  11 , Li Ma  11
Affiliations

miR-205 acts as a tumour radiosensitizer by targeting ZEB1 and Ubc13

Peijing Zhang et al. Nat Commun. .

Abstract

Tumour cells associated with therapy resistance (radioresistance and drug resistance) are likely to give rise to local recurrence and distant metastatic relapse. Recent studies revealed microRNA (miRNA)-mediated regulation of metastasis and epithelial-mesenchymal transition; however, whether specific miRNAs regulate tumour radioresistance and can be exploited as radiosensitizing agents remains unclear. Here we find that miR-205 promotes radiosensitivity and is downregulated in radioresistant subpopulations of breast cancer cells, and that loss of miR-205 is highly associated with poor distant relapse-free survival in breast cancer patients. Notably, therapeutic delivery of miR-205 mimics via nanoliposomes can sensitize the tumour to radiation in a xenograft model. Mechanistically, radiation suppresses miR-205 expression through ataxia telangiectasia mutated (ATM) and zinc finger E-box binding homeobox 1 (ZEB1). Moreover, miR-205 inhibits DNA damage repair by targeting ZEB1 and the ubiquitin-conjugating enzyme Ubc13. These findings identify miR-205 as a radiosensitizing miRNA and reveal a new therapeutic strategy for radioresistant tumours.

PubMed Disclaimer

Figures

Figure 1
Figure 1. miR-205 increases radiosensitivity and is downregulated in radioresistant breast cancer cells
(a) Top: schematic representation of the generation of a radioresistant subline (SUM159-P2) from the parental SUM159 cells (SUM159-P0). Bottom: miRNA expression profiling of SUM159-P2 cells relative to SUM159-P0 cells using a qPCR-based miRNA array. The expression values are shown in Supplementary Table 1. (b) TaqMan qPCR analysis of miR-205 in SUM159-P0 and SUM159-P2 cells. n = 3 samples per group. (c) qPCR of miR-205 (top) and clonogenic survival assays (bottom) of mesenchymal-like and epithelial-like breast cancer cell lines. n = 3 samples per group. (d) Clonogenic survival assays of SUM159-P2 cells transduced with miR-205. n = 3 wells per group. (e) Clonogenic survival assays of SUM159-P0 cells transfected with the miR-205 inhibitor. n = 3 wells per group. Inset in d and e: immunoblotting of ZEB1 and GAPDH. Data in b, c, d and e are the mean of biological replicates from a representative experiment, and error bars indicate s.e.m. Statistical significance was determined by a two-tailed, unpaired Student's t-test. The experiments were repeated 3 times.
Figure 2
Figure 2. Therapeutic effect of miR-205 mimics in mice
(a) Kaplan-Meier curves showing the distant relapse-free survival of patients with high (n = 43) or low (n = 38) expression of miR-205 in their breast tumors. The P value was determined by a log-rank test. (b) Clonogenic survival assays of SUM159-P2 cells incubated with 100 nM DOPC-encapsulated miR-205 mimics. n = 3 wells per group. (c) Top: overview of the experimental scheme. Bottom: tumor size of mice bearing SUM159-P2 xenografts. Mice were treated with the vehicle or DOPC-encapsulated miR-205 mimics or scramble mimics. Tumors were locally irradiated with a 15-Gy single dose (RT). n = 5 mice per group. General linear model multivariate analysis was performed to determine statistical significance. (d) qPCR of miR-205 in tumor tissues from the mice described in c. n = 5 mice per group. (e) Immunoblotting of ZEB1 and GAPDH in tumor lysates. Data in b, c and d are the mean of biological replicates from a representative experiment, and error bars indicate s.e.m. Statistical significance in b and d was determined by a two-tailed, unpaired Student's t-test. The experiments were repeated 3 times.
Figure 3
Figure 3. miR-205 regulates DNA damage repair and radiosensitivity through ZEB1 and Ubc13
(a) γH2AX and DAPI staining of SUM159-P2 cells transfected with miR-205 or scramble control oligonucleotides, 24 hours after 6-Gy IR. Scale bar: 20 μm. (b) HR repair assays of U2OS_DR-GFP cells transfected with miR-205 alone or in combination with ZEB1. n = 3 wells per group. (c) Immunoblotting of ZEB1, Ubc13, BRCA1 and GAPDH in SUM159-P0 cells transfected with miR-205 mimics or the miR-205 inhibitor. (d) Luciferase activity of the wild-type (UTR-WT) or mutant (UTR-mut.) Ubc13 3′ UTR reporter gene in 293T cells transfected with the miR-205-expressing or empty vector. n = 3 wells per group. (e) Immunoblotting of K63-linked polyubiquitin chains, Ubc13 and GAPDH in SUM159-P0 and SUM159-P2 cells. (f) Clonogenic survival assays of SUM159-P2 cells transfected with Ubc13 siRNA. n = 3 wells per group. (g) Top: clonogenic survival assays of miR-205-transduced SUM159-P2 cells with or without ectopic expression of ZEB1 and Ubc13. Bottom: immunoblotting of ZEB1, Ubc13 and GAPDH. n = 3 wells per group. (h) HR repair assays of U2OS_DR-GFP cells transfected with miR-205 alone or in combination with ZEB1 or Ubc13, or both. n = 3 wells per group. Data in b, d, f, g and h are the mean of biological replicates from a representative experiment, and error bars indicate s.e.m. Statistical significance was determined by a two-tailed, unpaired Student's t-test. The experiments were repeated 3 times.
Figure 4
Figure 4. Radiation downregulates miR-205 expression through ZEB1 and ATM
(a) Immunoblotting of ZEB1 and GAPDH (top) and qPCR of miR-205 (bottom) in SUM159-P2 cells transduced with ZEB1 shRNA. n = 3 samples per group. (b) Immunoblotting of Zeb1 and Gapdh (top) and qPCR of miR-205 (bottom) in Zeb1+/+, Zeb1+/− and Zeb1−/- MEFs. n = 3 samples per group. (c) Human mir-205 genomic locus. The three short blue lines represent PCR amplicons specific for three E-box elements. (d) Top: 293T cells were transfected with SFB-GFP or SFB-ZEB1, immunoprecipitated with the FLAG antibody and immunoblotted with the FLAG antibody. Bottom: ChIP assays of 293T cells transfected with SFB-GFP or SFB-ZEB1. PCR was performed with primers specific for E-box elements 1, 2 and 3. (e) Activity of a luciferase reporter fused to the putative human mir-205 promoter in 293T cells transfected with SFB-GFP or SFB-ZEB1. n = 3 wells per group. (f) qPCR of miR-205 and miR-200c in irradiated SUM159-P0 cells with or without pretreatment with the ATM inhibitor Ku55933 (10 μM, 1 hour). n = 3 samples per group. (g) qPCR of miR-205 in SUM159-P0 cells transfected with ZEB1 siRNA or scramble control oligonucleotides. Cells were treated with 6-Gy IR. n = 3 samples per group. Data in a, b, e, f and g are the mean of biological replicates from a representative experiment, and error bars indicate s.e.m. Statistical significance was determined by a two-tailed, unpaired Student's t-test. The experiments were repeated 3 times.
Figure 5
Figure 5
The working model of regulation of radiosensitivity and DNA damage repair by miR-205.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Jameel JK, Rao VS, Cawkwell L, Drew PJ. Radioresistance in carcinoma of the breast. Breast. 2004;13:452–460. - PubMed
    1. Horsman MR, et al. Tumor radiosensitizers--current status of development of various approaches: report of an International Atomic Energy Agency meeting. Int J Radiat Oncol Biol Phys. 2006;64:551–561. - PubMed
    1. Calin GA, et al. Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proceedings of the National Academy of Sciences of the United States of America. 2002;99:15524–15529. - PMC - PubMed
    1. He L, et al. A microRNA polycistron as a potential human oncogene. Nature. 2005;435:828–833. - PMC - PubMed
    1. Ma L, Teruya-Feldstein J, Weinberg RA. Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature. 2007;449:682–688. - PubMed

Publication types

MeSH terms

Associated data