Tumor Biology

, Volume 37, Issue 4, pp 5621–5632 | Cite as

Methylation-mediated repression of potential tumor suppressor miR-203a and miR-203b contributes to esophageal squamous cell carcinoma development

  • Yibing Liu
  • Zhiming Dong
  • Jia Liang
  • Yanli Guo
  • Xin Guo
  • Supeng Shen
  • Gang Kuang
  • Wei Guo
Original Article


MiRNAs regulate gene expression and play pivotal roles in biological processes. MiRNAs can be inactivated by epigenetic mechanisms, such as DNA hypermethylation of CpG sites within CpG islands. Here, we investigated the role and methylation status of miR-203a and miR-203b in esophageal cancer cell lines and primary esophageal squamous cell carcinoma (ESCC) tumors and further elucidate the role of both miRNAs in the prognosis of ESCC. The present study revealed a strong downregulation of miR-203a and miR-203b in esophageal cancer cell lines and primary ESCC samples. Treatment of esophageal cancer cells with demethylating agent 5-Aza-dC led to increased miR-203a and miR-203b expression, confirming the epigenetic regulation of both miRNAs. The inhibition of proliferation and invasiveness in esophageal cancer cells after treated with 5-Aza-dC or transfected with miR-203a or miR-203b mimics, suggesting the tumor suppressor role of both miRNAs in esophageal cancer. Furthermore, the critical CpG sites of miR-203a and miR-203b were found to be located in proximal promoter region, and the proximal promoter hypermethylation of both miRNAs was found to influence transcriptional activity. Downregulation and hypermethylation of miR-203a and miR-203b were associated with TNM stage, pathological differentiation, and lymph node metastasis. ESCC patients in stages III and IV, with reduced expression of miR-203a or hypermethylation of miR-203a or miR-203b, demonstrated poor patient survival. In summary, our results suggest that miR-203a and miR-203b may function as tumor-suppressive miRNAs that are inactivated through proximal promoter hypermethylation and miR-203a expression and methylation may be useful prognostic marker in ESCC patients.


miR-203a miR-203b Esophageal squamous cell carcinoma Expression Methylation 



We thank the patients for taking part in this study. This research was supported by the National Natural Science Foundation (no. 81472335).

Compliance with ethical standards

Conflicts of interest


Supplementary material

13277_2015_4432_MOESM1_ESM.docx (15 kb)
Supplementary Table 1 Clinicopathologic characteristics of esophageal squamous cell carcinoma cases (DOCX 15 kb)
13277_2015_4432_MOESM2_ESM.docx (17 kb)
Supplementary Table 2 Primer sequences and reaction conditions of miR-203a and miR-203b used in this study (DOCX 16 kb)


  1. 1.
    Mirnezami AH, Pickard K, Zhang L, Primrose JN, Packham G. MicroRNAs: key players in carcinogenesis and novel therapeutic targets. Eur J Surg Oncol. 2009;35:339–47.CrossRefPubMedGoogle Scholar
  2. 2.
    Oom AL, Humphries BA, Yang C. MicroRNAs: novel players in cancer diagnosis and therapies. Biomed Res Int. 2014;2014:959461.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Xue J, Niu J, Wu J, Wu ZH. MicroRNAs in cancer therapeutic response: friend and foe. World J Clin Oncol. 2014;5:730–43.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, Noch E, et al. Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A. 2002;99:15524–9.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Tian L, Li M, Ge J, Guo Y, Sun Y, Liu M, et al. MiR-203 is downregulated in laryngeal squamous cell carcinoma and can suppress proliferation and induce apoptosis of tumours. Tumour Biol. 2014;35:5953–63.CrossRefPubMedGoogle Scholar
  6. 6.
    Liu Y, Ren F, Rong M, Luo Y, Dang Y, Chen G. Association between underexpression of microrna-203 and clinicopathological significance in hepatocellular carcinoma tissues. Cancer Cell Int. 2015;15:62.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Lee SA, Kim JS, Park SY, Kim HJ, Yu SK, Kim CS, et al. miR-203 downregulates Yes-1 and suppresses oncogenic activity in human oral cancer cells. J Biosci Bioeng. 2015.Google Scholar
  8. 8.
    Xu M, Gu M, Zhang K, Zhou J, Wang Z, Da J. miR-203 inhibition of renal cancer cell proliferation, migration and invasion by targeting of FGF2. Diagn Pathol. 2015;10:24.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Liao H, Bai Y, Qiu S, Zheng L, Huang L, Liu T, et al. MiR-203 downregulation is responsible for chemoresistance in human glioblastoma by promoting epithelial-mesenchymal transition via SNAI2. Oncotarget. 2015;6:8914–28.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Mao L, Zhang Y, Mo W, Yu Y, Lu H. BANF1 is downregulated by IRF1-regulated microRNA-203 in cervical cancer. PLoS One. 2015;10, e0117035.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Qu Y, Li WC, Hellem MR, Rostad K, Popa M, McCormack E, et al. MiR-182 and miR-203 induce mesenchymal to epithelial transition and self-sufficiency of growth signals via repressing SNAI2 in prostate cells. Int J Cancer. 2013;133:544–55.CrossRefPubMedGoogle Scholar
  12. 12.
    Tang G, Wu J, Xiao G, Huo L. miR-203 sensitizes glioma cells to temozolomide and inhibits glioma cell invasion by targeting E2F3. Mol Med Rep. 2015;11:2838–44.PubMedGoogle Scholar
  13. 13.
    Chang X, Sun Y, Han S, Zhu W, Zhang H, Lian S. MiR-203 inhibits melanoma invasive and proliferative abilities by targeting the polycomb group gene BMI1. Biochem Biophys Res Commun. 2015;456:361–6.CrossRefPubMedGoogle Scholar
  14. 14.
    Zhou X, Xu G, Yin C, Jin W, Zhang G. Down-regulation of miR-203 induced by Helicobacter pylori infection promotes the proliferation and invasion of gastric cancer by targeting CASK. Oncotarget. 2014;5:11631–40.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Zhou M, Chen J, Zhou L, Chen W, Ding G, Cao L. Pancreatic cancer derived exosomes regulate the expression of TLR4 in dendritic cells via miR-203. Cell Immunol. 2014;292:65–9.CrossRefPubMedGoogle Scholar
  16. 16.
    Wang N, Liang H, Zhou Y, Wang C, Zhang S, Pan Y, et al. miR-203 suppresses the proliferation and migration and promotes the apoptosis of lung cancer cells by targeting SRC. PLoS One. 2014;9, e105570.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Chen Z, Li D, Cheng Q, Ma Z, Jiang B, Peng R, et al. MicroRNA-203 inhibits the proliferation and invasion of U251 glioblastoma cells by directly targeting PLD2. Mol Med Rep. 2014;9:503–8.PubMedGoogle Scholar
  18. 18.
    Ralfkiaer U, Hagedorn PH, Bangsgaard N, Løvendorf MB, Ahler CB, Svensson L, et al. Diagnostic microRNA profiling in cutaneous T-cell lymphoma (CTCL). Blood. 2011;118:5891–900.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Wang S, Zhao X, Wang J, Wen Y, Zhang L, Wang D, et al. Upregulation of microRNA-203 is associated with advanced tumor progression and poor prognosis in epithelial ovarian cancer. Med Oncol. 2013;30:681.CrossRefPubMedGoogle Scholar
  20. 20.
    Iorio MV, Visone R, Di Leva G, Donati V, Petrocca F, Casalini P, et al. MicroRNA signatures in human ovarian cancer. Cancer Res. 2007;67:8699–707.CrossRefPubMedGoogle Scholar
  21. 21.
    Li Z, Du L, Dong Z, Yang Y, Zhang X, Wang L, et al. MiR-203 suppresses ZNF217 upregulation in colorectal cancer and its oncogenicity. PLoS One. 2015;10, e0116170.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Yantiss RK, Goodarzi M, Zhou XK, Rennert H, Pirog EC, Banner BF, et al. Clinical, pathologic, and molecular features of early-onset colorectal carcinoma. Am J Surg Pathol. 2009;33:572–82.CrossRefPubMedGoogle Scholar
  23. 23.
    Taipaleenmäki H, Browne G, Akech J, Zustin J, van Wijnen AJ, Stein JL, et al. Targeting of Runx2 by miR-135 and miR-203 impairs progression of breast cancer and metastatic bone disease. Cancer Res. 2015;75:1433–44.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Wang C, Zheng X, Shen C, Shi Y. MicroRNA-203 suppresses cell proliferation and migration by targeting BIRC5 and LASP1 in human triple-negative breast cancer cells. J Exp Clin Cancer Res. 2012;31:58.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Zhang Z, Zhang B, Li W, Fu L, Fu L, Zhu Z, et al. Epigenetic silencing of miR-203 upregulates SNAI2 and contributes to the invasiveness of malignant breast cancer cells. Genes Cancer. 2011;2:782–91.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Saini S, Arora S, Majid S, Shahryari V, Chen Y, Deng G, et al. Curcumin modulates microRNA-203-mediated regulation of the Src-Akt axis in bladder cancer. Cancer Prev Res (Phila). 2011;4:1698–709.CrossRefPubMedCentralGoogle Scholar
  27. 27.
    Gottardo F, Liu CG, Ferracin M, Calin GA, Fassan M, Bassi P, et al. Micro-RNA profiling in kidney and bladder cancers. Urol Oncol. 2007;25:387–92.CrossRefPubMedGoogle Scholar
  28. 28.
    Joyce CE, Zhou X, Xia J, Ryan C, Thrash B, Menter A, et al. Deep sequencing of small RNAs from human skin reveals major alterations in the psoriasis miRNAome. Hum Mol Genet. 2011;20:4025–40.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Yan B, Guo JT, Zhu CD, Zhao LH, Zhao JL. miR-203b: a novel regulator of MyoD expression in tilapia skeletal muscle. J Exp Biol. 2013;216:447–51.CrossRefPubMedGoogle Scholar
  30. 30.
    Furuta M, Kozaki KI, Tanaka S, Arii S, Imoto I, Inazawa J. miR-124 and miR-203 are epigenetically silenced tumor-suppressive microRNAs in hepatocellular carcinoma. Carcinogenesis. 2010;31:766–76.CrossRefPubMedGoogle Scholar
  31. 31.
    Chim CS, Wan TS, Wong KY, Fung TK, Drexler HG, Wong KF. Methylation of miR-34a, miR-34b/c, miR-124-1 and miR-203 in Ph-negative myeloproliferative neoplasms. J Transl Med. 2011;9:197.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Huang YW, Kuo CT, Chen JH, Goodfellow PJ, Huang TH, Rader JS, et al. Hypermethylation of miR-203 in endometrial carcinomas. Gynecol Oncol. 2014;133:340–5.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Diao Y, Guo X, Jiang L, Wang G, Zhang C, Wan J, et al. miR-203, a tumor suppressor frequently down-regulated by promoter hypermethylation in rhabdomyosarcoma. J Biol Chem. 2014;289:529–39.CrossRefPubMedGoogle Scholar
  34. 34.
    Botezatu A, Goia-Rusanu CD, Iancu IV, Huica I, Plesa A, Socolov D, et al. Quantitative analysis of the relationship between microRNA-124a, −34b and −203 gene methylation and cervical oncogenesis. Mol Med Rep. 2011;4:121–8.PubMedGoogle Scholar
  35. 35.
    Chim CS, Wong KY, Leung CY, Chung LP, Hui PK, Chan SY, et al. Epigenetic inactivation of the hsa-miR-203 in haematological malignancies. J Cell Mol Med. 2011;15:2760–7.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Jamali Z, Asl Aminabadi N, Attaran R, Pournagiazar F, Ghertasi Oskouei S, Ahmadpour F. MicroRNAs as prognostic molecular signatures in human head and neck squamous cell carcinoma: a systematic review and meta-analysis. Oral Oncol. 2015;51:321–31.CrossRefPubMedGoogle Scholar
  37. 37.
    Nagaraja V, Eslick GD. Forthcoming prognostic markers for esophageal cancer: a systematic review and meta-analysis. J Gastrointest Oncol. 2014;5:67–76.PubMedPubMedCentralGoogle Scholar
  38. 38.
    Guohong Z, Min S, Duenmei W, Songnian H, Min L, Jinsong L, et al. Genetic heterogeneity of oesophageal cancer in high-incidence areas of southern and northern China. PLoS One. 2010;5, e9668.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Hezova R, Kovarikova A, Srovnal J, Zemanova M, Harustiak T, Ehrmann J, et al. Diagnostic and prognostic potential of miR-21, miR-29c, miR-148 and miR-203 in adenocarcinoma and squamous cell carcinoma of esophagus. Diagn Pathol. 2015;10:42.CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Chen X, Hu H, Guan X, Xiong G, Wang Y, Wang K, et al. CpG island methylation status of miRNAs in esophageal squamous cell carcinoma. Int J Cancer. 2012;130:1607–13.CrossRefPubMedGoogle Scholar
  41. 41.
    Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods. 2001;25:402–8.CrossRefPubMedGoogle Scholar
  42. 42.
    Sasaki M, Anast J, Bassett W, Kawakami T, Sakuragi N, Dahiya R. Bisulfite conversion-specific and methylation-specific PCR: a sensitive technique for accurate evaluation of CpG methylation. Biochem Biophys Res Commun. 2003;309:305–9.CrossRefPubMedGoogle Scholar
  43. 43.
    Yu L, Liu C, Vandeusen J, Becknell B, Dai Z, Wu YZ, et al. Global assessment of promoter methylation in a mouse model of cancer identifies ID4 as a putative tumor-suppressor gene in human leukemia. Nat Genet. 2005;37:265–74.CrossRefPubMedGoogle Scholar
  44. 44.
    Zhang K, Dai L, Zhang B, Xu X, Shi J, Fu L, et al. miR-203 is a direct transcriptional target of E2F1 and causes G1 arrest in esophageal cancer cells. J Cell Physiol. 2015;230:903–10.CrossRefPubMedGoogle Scholar
  45. 45.
    Okumura T, Shimada Y, Moriyama M, Takei Y, Omura T, Sekine S, et al. MicroRNA-203 inhibits the progression of esophageal squamous cell carcinoma with restored epithelial tissue architecture in vivo. Int J Oncol. 2014;44:1923–32.PubMedGoogle Scholar
  46. 46.
    Zhang F, Yang Z, Cao M, Xu Y, Li J, Chen X, et al. MiR-203 suppresses tumor growth and invasion and down-regulates MiR-21 expression through repressing Ran in esophageal cancer. Cancer Lett. 2014;342:121–9.CrossRefPubMedGoogle Scholar
  47. 47.
    Takeshita N, Mori M, Kano M, Hoshino I, Akutsu Y, Hanari N, et al. miR-203 inhibits the migration and invasion of esophageal squamous cell carcinoma by regulating LASP1. Int J Oncol. 2012;41:1653–61.PubMedGoogle Scholar
  48. 48.
    He J, Deng Y, Yang G, Xie W. MicroRNA-203 down-regulation is associated with unfavorable prognosis in human glioma. J Surg Oncol. 2013;108:121–5.CrossRefPubMedGoogle Scholar
  49. 49.
    Ikenaga N, Ohuchida K, Mizumoto K, Yu J, Kayashima T, Sakai H, et al. MicroRNA-203 expression as a new prognostic marker of pancreatic adenocarcinoma. Ann Surg Oncol. 2010;17:3120–8.CrossRefPubMedGoogle Scholar
  50. 50.
    Chen HY, Han ZB, Fan JW, Xia J, Wu JY, Qiu GQ, et al. miR-203 expression predicts outcome after liver transplantation for hepatocellular carcinoma in cirrhotic liver. Med Oncol. 2012;29:1859–65.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Yibing Liu
    • 1
  • Zhiming Dong
    • 2
  • Jia Liang
    • 2
  • Yanli Guo
    • 2
  • Xin Guo
    • 2
  • Supeng Shen
    • 2
  • Gang Kuang
    • 2
  • Wei Guo
    • 2
  1. 1.Department of Internal Medicine-Oncologythe Fourth Hospital of Hebei Medical UniversityShijiazhuangChina
  2. 2.Laboratory of Pathology, Hebei Cancer Institutethe Fourth Hospital of Hebei Medical UniversityShijiazhuangChina

Personalised recommendations