Tumor Biology

, Volume 36, Issue 12, pp 9733–9738 | Cite as

The effect of recombinant lentiviral vector encoding miR-145 on human esophageal cancer cells

  • Tian-Yun Wang
  • Qing-qing Zhang
  • Xi Zhang
  • Qiu-Li Sun
  • Chun-Peng Zhao
  • Xiao-Yin Wang
Research Article

Abstract

miR-145, a newly identified microRNA molecule, is hypothesized to function as a tumor suppressor, but this activity has not been investigated in esophageal l carcinoma (EC). The aim of this study was to investigate the effect of miR-145 on the biological features of EC cells. miR-145 was obtained using PCR technology and cloned into the lentiviral vector, pLVX-IRES-ZsGreen1, to construct the resulting vector, pLVX-IZ-miR-145. The vector was packaged, the viral titer was tested, and ECA109 cells were infected with the optimal viral titer. Cells that were stably transfected with miR-145 were screened. Flow cytometry was used to analyze enhanced green fluorescence protein gene expression, and to measure cell apoptosis and cell cycle. miR-145 expression was detected by real-time fluorescent quantitative PCR. Furthermore, cell proliferation was assayed using CCK-8 assay. The pLVX-IZ-miR-145 vector was successfully constructed, and the viral titer achieved up to 5.0 × 108 TU/mL. The transfection efficiency was 90 %. Compared to the control group, the expression level of miR-145 in the transfected group was significantly higher (185-fold, P < 0.05). miR-145 overexpression significantly inhibited esophageal cancer cell proliferation (P < 0.05). Moreover, the number of cells at the G2/M stage, as well as the cell apoptotic rate, in the miR-145-transfected group was significantly increased (P < 0.05). Our study reveals that overexpression of miR-145 inhibits cell proliferation, increases apoptosis, and influences the cell cycle progression of EC cell.

Keywords

MicroRNA Metastasis Apoptosis Esophageal carcinoma Lentiviralvector 

Notes

Acknowledgments

This work was partly supported by grants from the National Natural Science Foundation of China (Nos. 31371332 and 31300702).

Conflicts of interest

None

References

  1. 1.
    Parthipun A, Diamantopoulos A, Shaw A, Dourado R, Sabharwal T. Self-expanding metal stents in palliative malignant oesophageal dysplasia. Ann Palliat Med. 2014;3:92–103.PubMedGoogle Scholar
  2. 2.
    Jemal A, Bray F, Center MM, Ferlay J, Ward R, Forman D. Global cancer statistics. CAC Cancer J Clin. 2011;61:69–90.CrossRefGoogle Scholar
  3. 3.
    Kim T, Grobmyer SR, Smith R, Ben-David K, Ang D, Vogen SB, et al. Esophageal cancer: the five year survivors. J Surg. 2011;103:179–83.Google Scholar
  4. 4.
    Tie J, Pan Y, Zhao L, Wu K, Liu J, Sun S, et al. MiR-218 inhibits invasion and metastasis of gastric cancer by targeting the Robo1 receptor. PLoS Genet. 2010;6, e1000879.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Ambros V. The functions of animal microRNAs. Nature. 2004;431:350–5.CrossRefPubMedGoogle Scholar
  6. 6.
    Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116:281–97.CrossRefPubMedGoogle Scholar
  7. 7.
    He L, Hannon GJ. MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet. 2004;5:522–31.CrossRefPubMedGoogle Scholar
  8. 8.
    Song JL, Nigam P, Tektas SS, Selva E. MicroRNA regulation of Wnt signaling pathways in development and disease. Cell Signal. 2015;27:1380–91.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Zheng W, Liu Z, Zhang W, Hu X. miR-31 functions as an oncogene in cervical cancer. Arch Gynecol Obstet. 2015 Apr 18. [Epub ahead of print].Google Scholar
  10. 10.
    Adams BD, Kasinski AL, Slack FJ. Aberrant regulation and function of microRNAs in cancer. Curr Biol. 2014;24:R762–76.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Ni Y, Meng L, Wang L, Dong W, Shen H, Wang G, et al. MicroRNA-143 functions as a tumor suppressor in human esophageal squamous cell carcinoma. Gene. 2013;517:197–204.CrossRefPubMedGoogle Scholar
  12. 12.
    Iorio MV, Ferracin M, Liu CG, Veronese A, Spizzo R, Sabbioni S, et al. MicroRNA gene expression deregulation in human breast cancer. Cancer Res. 2005;65:7065–70.CrossRefPubMedGoogle Scholar
  13. 13.
    Michael MZ, O' Connor SM, van Holst Pellekaan NG, Young GP, James RJ. Reduced accumulation of specific microRNAs in colorectal neoplasia. Mol Cancer Res. 2003;1:882–91.PubMedGoogle Scholar
  14. 14.
    Tabrizi M, Khalili M, Vasei M, Nouraei N, Mansour Samaei N, Khavanin A, et al. Evaluating the miR-302b and miR-145 expression in formalin-fixed paraffin-embedded samples of esophageal squamous cell carcinoma. Arch Iran Med. 2015;18:173–8.PubMedGoogle Scholar
  15. 15.
    Fu HL, de Wu P, Wang XF, Wang JG, Jiao F, Song LL, et al. Altered miRNA expression is associated with differentiation, invasion, and metastasis of esophageal squamous cell carcinoma (ESCC) in patients from Huaian. China Cell Biochem Biophys. 2013;67:657–68.CrossRefPubMedGoogle Scholar
  16. 16.
    Sachdeva M, Mo YY. MicroRNA-145 suppresses cell invasion and metastasis by directly targeting mucin 1. Cancer Res. 2010;70:378–87.CrossRefPubMedGoogle Scholar
  17. 17.
    Wang S, Bian C, Yang Z, Bo Y, Li J, Zeng L, et al. miR-145 inhibits breast cancer cell growth through RTKN. Int J Oncol. 2009;34:1461–6.PubMedGoogle Scholar
  18. 18.
    Guo CJ, Pan Q, Li DG, Sun H, Liu BW. miR-15b and miR-16 are implicated in activation of the rat hepatic stellate cell: an essential role for apoptosis. J Hepatol. 2009;50:766–78.CrossRefPubMedGoogle Scholar
  19. 19.
    Si ML, Zhu S, Wu H, Lu Z, Wu F, Mo YY. miR-21-mediated tumor growth. Oncogene. 2007;26:2799–803.CrossRefPubMedGoogle Scholar
  20. 20.
    Turato C, Vitale A, Fasolato S, Ruvoletto M, Terrin L, Quarta S, et al. SERPINB3 is associated with TGF-beta1 and cytoplasmic beta-catenin expression in hepatocellular carcinomas with poor prognosis. Br J Cancer. 2014;110:2708–15.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Jiang L, Wang Y, Rong Y, Xu L, Chu Y, Zhang Y, et al. miR-1179 promotes cell invasion through SLIT2/ROBO1 axis in esophageal squamous cell carcinoma. Int J Clin Exp Pathol. 2015;8:319–27.PubMedPubMedCentralGoogle Scholar
  22. 22.
    Pagliuca A, Valvo C, Fabrizi E, di Martino S, Biffoni M, Runci D, et al. Analysis of the combined action of miR-143 and miR-145 on oncogenic pathways in colorectal cancer cells reveals a coordinate program of gene repression. Oncogene. 2013;32:4806–13.CrossRefPubMedGoogle Scholar
  23. 23.
    Gotte M, Mohr C, Koo CY, Götte M, Mohr C, Koo CY, et al. miR-145-dependent targeting of junctional adhesion molecule A and modulation of fascin expression are associated with reduced breast cancer cell motility and invasiveness. Oncogene. 2010;29:6569–80.CrossRefPubMedGoogle Scholar
  24. 24.
    Derouet MF, Liu G, Darling GE. MiR-145 expression accelerates esophageal adenocarcinoma progression by enhancing cell invasion and anoikis resistance. PLoS One. 2014;9, e115589.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Wang F, Xia J, Wang N, Zong H. miR-145 inhibits proliferation and invasion of esophageal squamous cell carcinoma in part by targeting c-Myc. Onkologie. 2013;36:754–8.CrossRefPubMedGoogle Scholar
  26. 26.
    Cho WC, Chow AS, Au JS. MiR-145 inhibits cell proliferation of human lung adenocarcinoma by targeting EGFR and NUDT1. RNA Biol. 2011;8:125–31.CrossRefPubMedGoogle Scholar
  27. 27.
    Nam EJ, Yoon H, Kim SW, Kim H, Kim YT, Kim JH, et al. MicroRNA expression profiles in serous ovarian carcinoma. Clin Cancer Res. 2008;14:2690–5.CrossRefPubMedGoogle Scholar
  28. 28.
    Shen H, Shen J, Wang L, Shi Z, Wang M, Jiang BH, et al. Low miR-145 expression level is associated with poor pathological differentiation and poor prognosis in non-small cell lung cancer. Biomed Pharmacother. 2015;69:301–5.CrossRefPubMedGoogle Scholar
  29. 29.
    Zhang X, Dong Y, Ti H, Zhao J, Wang Y, Li T, et al. Down-regulation of miR-145 and miR-143 might be associated with DNA methyltransferase 3B overexpression and worse prognosis in endometrioid carcinomas. Hum Pathol. 2013;44:2571–80.CrossRefPubMedGoogle Scholar
  30. 30.
    Karakatsanis A, Papaconstantinou I, Gazouli M, Lyberopoulou A, Polymeneas G, Voros D. Expression of microRNAs, miR-21, miR-31, miR-122, miR-145, miR-146a, miR-200c, miR-221, miR-222, and miR-223 in patients with hepatocellular carcinoma or intrahepatic cholangiocarcinoma and its prognostic significance. Mol Carcinog. 2013;52:297–303.CrossRefPubMedGoogle Scholar
  31. 31.
    Dong R, Liu X, Zhang Q, Jiang Z, Li Y, Wei Y, et al. miR-145 inhibits tumor growth and metastasis by targeting metadherin in high-grade serous ovarian carcinoma. Oncotarget. 2014;5:10816–29.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Feng Y, Zhu J, Ou C, Deng Z, Chen M, Huang W, et al. MicroRNA-145 inhibits tumour growth and metastasis in colorectal cancer by targeting fascin-1. Br J Cancer. 2014;110:2300–9.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Zhang H, Pu J, Qi T, Qi M, Yang C, Li S, et al. MicroRNA-145 inhibits the growth, invasion, metastasis and angiogenesis of neuroblastoma cells through targeting hypoxia-inducible factor 2 alpha. Oncogene. 2014;33:387–97.CrossRefPubMedGoogle Scholar
  34. 34.
    Fan L, Wu Q, Xing X, Wei Y, Shao Z. MicroRNA-145 targets vascular endothelial growth factor and inhibits invasion and metastasis of osteosarcoma cells. Acta Biochim Biophys Sin (Shanghai). 2012;44:407–14.CrossRefGoogle Scholar
  35. 35.
    Peng W, Hu J, Zhu XD, Liu X, Wang CC, Li WH, et al. Overexpression of miR-145 increases the sensitivity of vemurafenib in drug-resistant colo205 cell line. Tumour Biol. 2014;35:2983–8.CrossRefPubMedGoogle Scholar
  36. 36.
    Shi M, Du L, Liu D, Qian L, Hu M, Yu M, et al. Glucocorticoid regulation of a novel HPV-E6-p53-miR-145 pathway modulates invasion and therapy resistance of cervical cancer cells. J Pathol. 2012;228:148–57.CrossRefPubMedGoogle Scholar
  37. 37.
    Spizzo R, Nicoloso MS, Lupini L, Lu Y, Fogarty J, Rossi S, et al. miR-145 participates withTP53 in a death-promoting regulatory loop and targets estrogen receptor-alpha in human breast cancer cells. Cell Death Differ. 2010;17:246–54.CrossRefPubMedGoogle Scholar
  38. 38.
    Liu R, Liao J, Yang M, Sheng J, Yang H, Wang Y, et al. The cluster of miR-143 and miR-145 affects the risk for esophageal squamous cell carcinoma through co-regulating fascin homolog 1. PLoS One. 2012;7, e33987.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Kano M, Seki N, Kikkawa N, Fujimura L, Hoshino I, Akutsu Y, et al. miR-145, miR-133a and miR-133b: tumor-suppressive miRNAs target FSCN1 in esophageal squamous cell carcinoma. Int J Cancer. 2010;127:2804–14.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Tian-Yun Wang
    • 1
  • Qing-qing Zhang
    • 2
  • Xi Zhang
    • 1
  • Qiu-Li Sun
    • 1
  • Chun-Peng Zhao
    • 1
  • Xiao-Yin Wang
    • 1
  1. 1.Department of Biochemistry and Molecular BiologyXinxiang Medical UniversityXinxiangChina
  2. 2.Department of Psychosomatic Medicinethe Second Affiliated Hospital of Xinxiang Medical UniversityXinxiangChina

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