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The knockdown of c-myc expression by RNAi inhibits cell proliferation in human colon cancer HT-29 cells in vitro and in vivo

Abstract

We investigated the effects of RNA interference-mediated silencing of the c-myc gene on celluar proliferation and apoptosis in human colon cancer HT-29 cells in vitro and in vivo. A small interfering RNA (siRNA) targeting c-myc was designed, the DNA template was synthesized, and the siRNA was obtained by in vitro transcription. After siRNA transfection into HT-29 and human neuroblastoma IMR-32 cells with Lipofectamine 2000™, the proliferation of the HT-29 and IMR-32 cells was assessed via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetry, and Hoechst 33258 staining was used to observe cell apoptosis. Following gene transfer to HT-29 cells, the expression of c-myc mRNA was examined via reverse transcription polymerase chain reaction, and the level of the protein via Western blot assay. Growth curves were constructed and in vivo experiments were performed on nude mice to assess the effects of c-myc silencing on tumor growth. The c-myc expression in the tumor tissue was measured by reverse transcription polymerase chain reaction and subsequently by immunohistochemistry. Our paper demonstrates that the delivery of siRNA directed against c-myc not only efficiently down-regulated the expression of c-myc, inhibited the proliferation of HT-29 cells and induced apoptosis in vitro, but also suppressed the growth of colon cancer cells in vivo.

Abbreviations

DMEM:

Dulbecco’s modified Eagle’s medium

FBS:

fetal bovine serum

GAPDH:

glyceraldehyde-3-phosphate dehydrogenase

MTT:

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide

PBS:

phosphate-buffered saline

RT-PCR:

reverse transcription polymerase chain reaction

SDS:

sodium dodecyl sulphate

siRNA:

short interfering RNA

References

  1. Ahmed, F.E. Molecular markers that predict response to colon cancer therapy. Expert Rev. Mol. Diagn. 5 (2005) 353–375.

    PubMed  Article  CAS  Google Scholar 

  2. Grady, W.M. and Carethers, J.M. Genomic and Epigenetic Instability in Colorectal Cancer Pathogenesis. Gastroenterology 135 (2008) 1079–1099.

    PubMed  Article  CAS  Google Scholar 

  3. Grandori, C., Cowley, S.M., James, L.P. and Eisenman, R.N. The Myc/Max/Mad network and the transcriptional control of cell behavior. Annu. Rev. Cell Dev. Biol. 16 (2000) 653–699.

    PubMed  Article  CAS  Google Scholar 

  4. Bondi, J., Bukholm, G., Nesland, J.M. and Bukholm, I.R. Expression of non-membranous beta-catenin and gamma-catenin, c-Myc and cyclin D1 in relation to patient outcome in human colon adenocarcinomas. APMIS 112 (2004) 49–56.

    PubMed  Article  CAS  Google Scholar 

  5. Csontos, Z., Nádasi, E., Csejtey, A., Illényi, L., Kassai, M., Lukács, L., Kelemen, D., Kvarda, A. Zólyomi, A., Horváth, O.P. and Ember, I. Oncogene and tumor suppressor gene expression changes in the peripheral blood leukocytes of patients with colorectal cancer. Tumori 94 (2008) 79–82.

    PubMed  Google Scholar 

  6. Dang, C.V. c-Myc target genes involved in cell growth, apoptosis, and metabolism. Mol. Cell. Biol. 19 (1999) 1–11.

    PubMed  CAS  Google Scholar 

  7. Hongxing, Z., Nancai, Y., Wen, S., Guofu, H., Yanxia, W., Hanju, H., Qian, L., Wei, M., Yandong, Y. and Hao, H. Depletion of c-Myc inhibits human colon cancer colo 320 cells’ growth. Cancer Biother. Radiopharm. 23 (2008) 229–237.

    PubMed  Article  Google Scholar 

  8. Schwab, M., Reynders, V., Loitsch, S., Shastri, Y.M., Steinhilber, D., Schröder, O. and Stein, J. PPARgamma is involved in mesalazine-mediated induction of apoptosis and inhibition of cell growth in colon cancer cells. Carcinogenesis 29 (2008) 1407–1414.

    PubMed  Article  CAS  Google Scholar 

  9. Wilkins, J.A. and Sansom, O.J. C-Myc is a critical mediator of the phenotypes of Apc loss in the intestine. Cancer Res. 68 (2008) 4963–4966.

    PubMed  Article  CAS  Google Scholar 

  10. Colnot, S., Niwa-Kawakita, M., Hamard, G., Godard, C., Le Plenier, S., Houbron, C., Romagnolo, B., Berrebi, D., Giovannini, M. and Perret, C. Colorectal cancers in a new mouse model of familial adenomatous polyposis: influence of genetic and environmental modifiers. Lab. Invest. 84 (2004) 1619–1630.

    PubMed  Article  CAS  Google Scholar 

  11. Al-Kuraya, K., Novotny, H., Bavi, P., Siraj, A.K., Uddin, S., Ezzat, A., Sanea, N.A., Al-Dayel, F., Al-Mana, H., Sheikh, S.S., Mirlacher, M., Tapia, C., Simon, R., Sauter, G., Terracciano, L. and Tornillo, L. HER2, TOP2A, CCND1, EGFR and C-MYC oncogene amplification in colorectal cancer. J. Clin. Pathol. 60 (2007) 768–772.

    PubMed  Article  CAS  Google Scholar 

  12. Stewart, J., Evan, G., Watson, J. and Sikora, K. Detection of the c-myc oncogene product in colonic polyps and carcinomas. Br. J. Cancer 53 (1986) 1–6.

    PubMed  CAS  Google Scholar 

  13. Hannon, G.J. RNA interference. Nature 418 (2002) 244–251.

    PubMed  Article  CAS  Google Scholar 

  14. Watson, P.H., Pon, R.T. and Shiu, R.P.C. Inhibition of c-myc expression by phosphorothioate antisense oligonucleotide identifies a critical role for c-myc in the growth of human breast cancer. Cancer Res. 51 (1991) 3996–4000.

    PubMed  CAS  Google Scholar 

  15. Hong, J., Zhao, Y. and Huang, W. Blocking c-myc and stat3 by E. coli expressed and enzyme digested siRNA in mouse melanoma. Biochem. Biophys. Res. Commun. 348 (2006) 600–605.

    PubMed  Article  CAS  Google Scholar 

  16. Liao, D.J. and Dickson, R.B. c-Myc in breast cancer. Endocr. Relat. Cancer 7 (2000) 143–164.

    PubMed  Article  CAS  Google Scholar 

  17. Kruszewski, W., Kowara, R., Rzepko, R., Warezak, C., Zieliński, J., Gryglewski, G., Kopacz, A., Jastrzebski, T. and Pawełczyk, T. K-RAS point mutation, and amplification of C-MYC and C-ERBB2 in colon adenocarcinoma. Folia Histochem. Cytobiol. 42 (2004) 173–179.

    PubMed  CAS  Google Scholar 

  18. Nagy, B., Szendroi, A. and Romics, I. Overexpression of CD24, c-myc and Phospholipase 2A in prostate cancer tissue samples obtained by needle biopsy. Pathol. Oncol. Res. 2008.

  19. Steiner, M.S., Anthony, C.T., Lu, Y. and Holt, J.T. Antisense c-myc retroviral vector suppresses established human prostate cancer. Hum. Gene Ther. 9 (1998) 747–755.

    PubMed  Article  CAS  Google Scholar 

  20. Bressin, C., Bourgarel-Rey, V., Carré, M., Pourroy, B., Arango, D., Braguer, D. and Barra, Y. Decrease in c-Myc activity enhances cancer cell sensitivity to vinblastine. Anticancer Drugs 17 (2006) 181–187.

    PubMed  Article  CAS  Google Scholar 

  21. Zhao, H.Y., Ooyama, A., Yamamoto, M., Ikeda, R., Haraguchi, M., Tabata, S., Furukawa, T., Che, X.F., Iwashita, K.I., Oka, T., Fukushima, M., Nakagawa, M., Ono, M., Kuwano, M. and Akiyama, S.I. Down regulation of c-Myc and induction of an angiogenesis inhibitor, thrombospondin-1, by 5-FU in human colon cancer KM12C cells. Cancer Lett. 270 (2008) 156–163.

    PubMed  Article  CAS  Google Scholar 

  22. Bertrand, J.R., Pottier, M., Vekris, A., Opolon, P., Maksimenko, A. and Malvy, C. Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo. Biochem. Biophys. Res. Commun. 296 (2002) 1000–1004.

    PubMed  Article  CAS  Google Scholar 

  23. Prendergast, G.C. Mechanisms of apoptosis by c-Myc. Oncogene 1 (1999) 2967–2987.

    Article  Google Scholar 

  24. Wang, Y.H., Liu, S., Zhang, G., Zhou, C.Q., Zhu, H.X., Zhou, X.B., Quan, L.P., Bai, J.F. and Xu, N.Z. Knockdown of c-Myc expression by RNAi inhibits MCF-7 breast tumor cells growth in vitro and in vivo. Breast Cancer Res. 7 (2005) 220–228.

    Article  Google Scholar 

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Correspondence to Yin-Lin Ge.

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Zhang, X., Ge, YL. & Tian, RH. The knockdown of c-myc expression by RNAi inhibits cell proliferation in human colon cancer HT-29 cells in vitro and in vivo . Cell Mol Biol Lett 14, 305–318 (2009). https://doi.org/10.2478/s11658-009-0001-9

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  • DOI: https://doi.org/10.2478/s11658-009-0001-9

Key words

  • c-myc
  • Cell proliferation
  • HT-29
  • siRNA