miR-200a Inhibits Tumor Proliferation by Targeting AP-2γ in Neuroblastoma Cells

  • Gao, Shun-Li (Department of pediatrics, The First Affiliated Hospital, University of South China) ;
  • Wang, Li-Zhong (Department of pediatrics, The First Affiliated Hospital, University of South China) ;
  • Liu, Hai-Ying (Department of pediatrics, The First Affiliated Hospital, University of South China) ;
  • Liu, Dan-Li (Department of pediatrics, The First Affiliated Hospital, University of South China) ;
  • Xie, Li-Ming (Cancer Research Institute, University of South China, Key Laboratory of Cancer Cellular and Molecular Pathology of Hunan Provincial University) ;
  • Zhang, Zhi-Wei (Cancer Research Institute, University of South China, Key Laboratory of Cancer Cellular and Molecular Pathology of Hunan Provincial University)
  • Published : 2014.06.15


Background: MicroRNA-200a (miR-200a) has been reported to regulate tumour progression in several tumours but little is known about its role in neuroblastoma. Our aim was to investigate the potential role and mechanism of miR-200a in neuroblastomas. Materials and Methods: Expression levels of miR-200a in tissues were determined using RT-PCR. The effect of miR-200a and shAP-$2{\gamma}$ on cell viability was evaluated using MTS assays, and target protein expression was determined using Western blotting and RT-PCR. Luciferase reporter plasmids were constructed to confirm direct targeting. Results were reported as mean${\pm}$S.E.M and differences were tested for significance using the 2-tailed Students t-test. Results: We determined that miR-200a expression was significantly lower in neuroblastoma tumors than the adjacent non-cancer tissue. Over-expression of miR-200 are reduced cell viability in neuroblastoma cells and inhibited tumor growth in mouse xenografts. We identified AP-$2{\gamma}$ as a novel target for miR-200a in neuroblastoma cells. Thus miR-200a targets the 3'UTR of AP-$2{\gamma}$ and inhibits its mRNA and protein expression. Furthermore, our result showed that shRNA knockdown of AP-$2{\gamma}$ in neuroblastoma cells results in significant inhibit of cell proliferation and tumor growth in vitro, supporting an oncogenic role of AP-$2{\gamma}$ in neuroblastoma. Conclusions: Our study revealed that miR-200a is a candidate tumor suppressor in neuroblastoma, through direct targeting of AP-$2{\gamma}$. These findings re-enforce the proposal of AP-$2{\gamma}$ as a therapeutic target in neuroblastoma.


Supported by : National Natural Science Foundation of China


  1. Adam L, Zhong M, Choi W, et al (2009). miR-200 expression regulates epithelial-to-mesenchymal transition in bladder cancer cells and reverses resistance to epidermal growth factor receptor therapy. Clin Cancer Res, 15, 5060-72.
  2. Bai JX, Yan B, Zhao ZN, et al (2013). Tamoxifen represses miR-200 microRNAs and promotes epithelial-to-mesenchymal transition by up-regulating c-Myc in endometrial carcinoma cell lines. Endocrinology, 154, 635-45.
  3. Bracken CP, Gregory PA, Kolesnikoff N, et al (2008). A double-negative feedback loop between ZEB1-SIP1 and the microRNA-200 family regulates epithelial-mesenchymal transition. Cancer Res, 68, 7846-54.
  4. Cochrane DR, Howe EN, Spoelstra NS, Richer JK (2010). Loss of miR-200c: a marker of aggressiveness and chemoresistance in female reproductive cancers. J Oncol, 56, 8217-27.
  5. Bushati N, Cohen SM (2007). microRNA functions. Annu Rev Cell Dev Biol, 23, 175-205.
  6. Chakrabarti M, Banik NL, Ray SK (2013). miR-138 overexpression is more powerful than hTERT knockdown to potentiate apigenin for apoptosis inneuroblastoma in vitro and in vivo. Exp Cell Res, 19, 1575-85.
  7. Chen X, Pan M, Han L, et al (2013). miR-338-3p suppresses neuroblastoma proliferation, invasion and migration through targeting PREX2a. FEBS Lett, 587, 3729-37.
  8. Davis BN, Hata A (2010). microRNA in Cancer The involvement of aberrant microRNA biogenesis regulatory pathways. Genes Cancer, 1, 1100-14.
  9. Du Y, Xu Y, Ding L, et al (2009). Down-regulation of miR-141 in gastric cancer and its involvement in cell growth. J Gastroenterol, 44, 556-61.
  10. Dykxhoorn DM, Wu Y, Xie H, et al (2009). miR-200 enhances mouse breast cancer cell colonization to form distant metastases. PLoS One, 4, 7181.
  11. Eckert D, Buhl S, Weber S, Jager R, Schorle H (2005). The AP-2 family of transcription factors. Genome Biol, 6, 213-46.
  12. Gabriely G, Yi M, Narayan RS, et al (2011). Human glioma growth is controlled by microRNA-10b. Cancer Res, 71, 3563-72.
  13. Gee JM, Eloranta JJ, Ibbitt JC, et al (2009). Overexpression of TFAP2C in invasive breast cancer correlates with a poorer response to anti-hormone therapy and reduced patient survival. J PathoL, 217, 32-41.
  14. Gregory PA, Bert AG, Paterson EL, et al (2008). The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol, 10, 593-601.
  15. Kong D, Li Y, Wang Z, et al (2009). miR-200 regulates PDGF-D-mediated epithelial-mesenchymal transition, adhesion, and invasion of prostate cancer cells. Stem Cells, 27, 1712-21.
  16. Guo JX, Tao QS, Lou PR, et al (2012). miR-181b as a potential molecular target for anticancer therapy of gastric neoplasms. Asian Pac J Cancer Prev, 13, 2263-7.
  17. Hasleton MD, Ibbitt JC, Hurst HC (2003). Characterization of the human activator protein-2gamma (AP-2gamma) gene: control of expression by Sp1/Sp3 inbreast tumour cells.Biochem J, 373, 925-32.
  18. Huang F, Lin C, Shi YH, Kuerban G. (2013). MicroRNA-101 inhibits cell proliferation, invasion, and promotes apoptosis by regulating cyclooxygenase-2 in hela cervical carcinoma cells. Asian Pac J Cancer Prev, 14, 5915-20.
  19. Korpal M, Kang Y (2008). The emerging role of miR-200 family of microRNAs in epithelial-mesenchymal transition and cancer metastasis. RNA Biol, 5, 115-9.
  20. Lee JW, Park YA, Choi JJ, et al (2011). The expression of the miRNA-200 family in endometrial endometrioid carcinoma.Gynecol Oncol, 120, 56-62.
  21. Li Y, VandenBoom TG, Kong D, et al (2009). Up-regulation of miR-200 and let-7 by natural agents leads to the reversal of epithelial-to-mesenchymal transition in gemcitabine-resistant pancreatic cancer cells. Cancer Res, 69, 6704-12.
  22. Liu Y, Liu Q, Jia W, et al (2013). MicroRNA-200a regulates Grb2 and suppresses differentiation of mouse embryonic stem cells into endoderm and mesoderm. PLoS One, 8, 68990.
  23. Lynch J, Fay J, Meehan M, et al (2012). MiRNA-335 suppresses neuroblastoma cell invasiveness by direct targeting of multiple genes from the non-canonical TGF-$\beta$ signalling pathway. Carcinogenesis, 33, 976-85.
  24. Paterson EL, Kazenwadel J, Bert AG, et al (2013). Down-regulation of the miRNA-200 family at the invasive front of colorectal cancers with degraded basement membrane indicates EMT is involved in cancer progression. Neoplasia, 15, 180-91.
  25. McPherson JD, Apostol B, Wagner-McPherson CB, et al (1997). Radiation hybrid map of human chromosome 5 with integration of cytogenetic, genetic, and transcript maps.Genome Res, 7, 897-909.
  26. Osella-Abate S, Novelli M, Quaglino P, et al (2012). Expression of AP-2$\alpha$, AP-2$\gamma$ and ESDN in primary melanomas: correlation with histopathological features and potential prognostic value. J Dermatol Sci, 68, 202-4.
  27. Park SM, Gaur AB, Lengyel E, Peter ME (2008). The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. Genes Dev, 22, 894-907.
  28. Pellikainen JM, Kosma VM (2007). Activator protein-2 in carcinogenesis with a special reference to breast cancer--a mini review. Int J Cancer, 120, 2061-67.
  29. Perissi V, Menini N, Cottone E, et al (2000). AP-2 transcription factors in the regulation of ERBB2 gene transcription by oestrogen. Oncogene, 19, 280-8.
  30. Qiao J, Lee S, Paul P, et al (2013). miR-335 and miR-363 regulation of neuroblastoma tumorigenesis and metastasis.Surgery, 154, 226-33.
  31. Saydam O, Shen Y, Wurdinger T, et al (2009). Downregulated microRNA-200a in meningiomas promotes tumor growth by reducing E-cadherin and activating the Wnt/beta-catenin signaling pathway. Mol Cell Biol, 29, 5923-40.
  32. Soubani O, Ali AS, Logna F, et al (2012). Re-expression of miR-200 by novel approaches regulates the expression of PTEN and MT1-MMP in pancreatic cancer. Carcinogenesis, 33, 1563-71.
  33. Uhlmann S, Zhang JD, Schwager A, et al (2010). miR-200bc/429 cluster targets PLCgamma1 and differentially regulates proliferation and EGF-driven invasion thanmiR-200a/141 in breast cancer. Oncogene, 29, 4297-306.
  34. Stratmann J, Wang CJ, Gnosa S, et al (2011). Dicer and miRNA in relation to clinicopathological variables in colorectal cancer patients. BMC Cancer, 11, 345-54.
  35. Su J, Zhang A, Shi Z, et al (2012). MicroRNA-200a suppresses the Wnt/$\beta$-catenin signaling pathway by interacting with $\beta$-catenin. Int J Oncol, 40, 1162-70.
  36. Uhlmann S, Zhang JD, Schwager A, et al (2010). miR-200bc/429 cluster targets PLCgamma1 and differentially regulates proliferation and EGF-driven invasion than miR-200a/141 in breast cancer. Oncogene, 29, 4297-306.
  37. Williams CM, Scibetta AG, Friedrich JK, et al (2009). AP-2gamma promotes proliferation in breast tumour cells by direct repression of the CDKN1A gene. EMBO J, 28, 3591-601.
  38. Woodfield GW, Horan AD, Chen Y, Weigel RJ (2007). TFAP2C controls hormone response in breast cancer cells through multiple pathways of estrogen signaling. Cancer Res, 67, 8439-43.
  39. Yu Y, Wu J, Guan L, et al (2013). Kindlin 2 promotes breast cancer invasion via epigenetic silencing of the microRNA200 gene family. Int J Cancer, 133, 1368-79.

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