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MicroRNA-223 functions as an oncogene in human gastric cancer by targeting FBXW7/hCdc4

  • Jinhai Li
  • Yuanyuan Guo
  • Xiaodi Liang
  • Ming Sun
  • Guoliang Wang
  • Wei De
  • Wenxi WuEmail author
Original Paper

Abstract

Aims

The aim of this study was (a) to determine the role of micro-223 (miR-223) in gastric cancer and (b) to elucidate its regulatory mechanism on the FBXW7/hCdc4 gene.

Materials and methods

Artificial miR-223 and control oligonucleotide was transfected into gastric cancer cell line SGC7901 by using Lipofectamine2000. Apoptosis of miR-223 group and control group cells was analyzed by flow cytometry, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and colony formation assays were performed to detect the cell viability, to survey migration of miR-223 group and control group cells; scratch wound-healing motility assays, Transwell Assay, and Western blot test were performed to measure the variance of hFBXW7. Luciferase Reporter Assay, which was done by pLUC-hFBXW7 WT-3′-UTR co-transfected with pLMP-hsa-miR-223 or pLMP plasmid (as control) into HEK293T cells, used to detect whether hFBXW7 is a direct target gene of miR-223. Gastric cancer cell line SGC7901 transfected with miR-223 or control oligonucleotide was resuspended in ECM gel and then was injected into the flank of nude mice, 4 weeks later, the nude mice were euthanized. The tumors were excised then were measured and weighted. SYBR-Green I-based real-time RT-PCR study was used to detect the level of miR-223 in 22 gastric cancer tissue and corresponding gastric mucosa tissues. Immunohistochemical method was applied to detect the protein of hFBXW7.

Results

Gastric cancer cell line SGC7901, transfected with miR-223, showed significant reduction in cellular apoptosis and increased proliferation and invasion in vitro. Similar results were found in tumorigenesis assays performed in nude mice. Moreover, 19 of 22 cancer tissue samples highly expressed miR-223, when compared with patient-matched normal gastric mucosa. Specifically, patients with lymph node metastasis or metastatic disease (M1) at an advanced pathological stage showed significantly higher expression of miR-223. FBXW7/hCdc4 protein (FBW7) levels in gastric cancer cases were inversely correlated with miR-223 expression. Overexpression of miR-223 in gastric cancer cell lines decreased FBW7 expression at the translational level and decreased FBXW7/hCdc4-driven luciferase-reporter activity.

Conclusion

In summary, the data indicated that miR-223 targets FBXW7/hCdc4 expression at the post-transcriptional level and appears to regulate cellular apoptosis, proliferation, and invasion in gastric cancer. MiR-223 may serve as a novel therapeutic target in gastric cancer.

Keywords

MicroRNAs MiR-223 Proliferation FBXW7/hCdc4 Gastric cancer 

Notes

Acknowledgments

We thank Dr. Bin Cao and Lizong Shen of Jiangsu province hospital for helpful. This work was supported by National Science Foundation of China (No. 81070381 and No. 30872532).

Conflict of interest

We declare that we have no conflict of interest.

Supplementary material

432_2012_1154_MOESM1_ESM.tif (6.6 mb)
Fig. 1 Sample QC report (A) Total SGCS RNA sample, the ratio of 28/18 s was 1.9. (B) The ratio for SGCS0 sample was 2.1. They were validated samples and used for additional experiments (TIFF 6783 kb)
432_2012_1154_MOESM2_ESM.tif (2.2 mb)
Fig. 2 RT-PCR products using SYBR-Green I for miR-223. Amplicons were analyzed by 8% PAGE electrophoresis. MiR-223 bands at 60 bp position and GAPDH at 180 bp position indicated that the PCR products were as desired (G0, SGCG0; S, SGCS) (TIFF 2271 kb)
432_2012_1154_MOESM3_ESM.docx (39 kb)
Supplementary material 3 (DOCX 39 kb)

References

  1. Akhoondi S, Sun D, von der Lehr N, Apostolidou S, Klotz K, Maljukova A, Cepeda D, Fiegl H, Dafou D, Marth C, Mueller-Holzner E, Corcoran M, Dagnell M, Nejad SZ, Nayer BN, Zali MR, Hansson J, Egyhazi S, Petersson F, Sangfelt P, Nordgren H, Grander D, Reed SI, Widschwendter M, Sangfelt O, Spruck C (2007) FBXW7/hCDC4 is a general tumor suppressor in human cancer. Cancer Res 67(19):9006–9012PubMedCrossRefGoogle Scholar
  2. Alvarez-Garcia I, Miska EA (2005) MicroRNA functions in animal development and human disease. Development 132(21):4653–4662PubMedCrossRefGoogle Scholar
  3. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116(2):281–297PubMedCrossRefGoogle Scholar
  4. Behm-Ansmant I, Rehwinkel J, Izaurralde E (2006) MicroRNAs silence gene expression by repressing protein expression and/or by promoting mRNA decay. Cold Spring Harb Symp Quant Biol 71:523–530PubMedCrossRefGoogle Scholar
  5. Calhoun ES, Jones JB, Ashfaq R, Adsay V, Baker SJ, Valentine V, Hempen PM, Hilgers W, Yeo CJ, Hruban RH, Kern SE (2003) BRAF and FBXW7 (CDC4, FBW7, AGO, SEL10) mutations in distinct subsets of pancreatic cancer: potential therapeutic targets. Am J Pathol 163(4):1255–1260PubMedCrossRefGoogle Scholar
  6. Croce CM, Calin GA (2005) miRNAs, cancer, and stem cell division. Cell 122(1):6–7PubMedCrossRefGoogle Scholar
  7. Earle JS, Luthra R, Romans A, Abraham R, Ensor J, Yao H, Hamilton SR (2010) Association of microRNA expression with microsatellite instability status in colorectal adenocarcinoma. J Mol Diagn 12(4):433–440PubMedCrossRefGoogle Scholar
  8. Gottardo F, Liu CG, Ferracin M, Calin GA, Fassan M, Bassi P, Sevignani C, Byrne D, Negrini M, Pagano F, Gomella LG, Croce CM, Baffa R (2007) Micro-RNA profiling in kidney and bladder cancers. Urol Oncol 25(5):387–392PubMedCrossRefGoogle Scholar
  9. Gregory RI, Shiekhattar R (2005) MicroRNA biogenesis and cancer. Cancer Res 65(9):3509–3512PubMedCrossRefGoogle Scholar
  10. Guo C, Sah JF, Beard L, Willson JK, Markowitz SD, Guda K (2008) The noncoding RNA, miR-126, suppresses the growth of neoplastic cells by targeting phosphatidylinositol 3-kinase signaling and is frequently lost in colon cancers. Genes Chromosom Cancer 47(11):939–946PubMedCrossRefGoogle Scholar
  11. Hartgrink HH, Jansen EP, van Grieken NC, van de Velde CJ (2009) Gastric cancer. Lancet 374(9688):477–490PubMedCrossRefGoogle Scholar
  12. Huang GL, Zhang XH, Guo GL, Huang KT, Yang KY, Shen X, You J, Hu XQ (2009) Clinical significance of miR-21 expression in breast cancer: SYBR-Green I-based real-time RT-PCR study of invasive ductal carcinoma. Oncol Rep 21(3):673–679PubMedGoogle Scholar
  13. Koepp DM, Schaefer LK, Ye X, Keyomarsi K, Chu C, Harper JW, Elledge SJ (2001) Phosphorylation-dependent ubiquitination of cyclin E by the SCFFbw7 ubiquitin ligase. Science 294(5540):173–177PubMedCrossRefGoogle Scholar
  14. Laios A, O’Toole S, Flavin R, Martin C, Kelly L, Ring M, Finn SP, Barrett C, Loda M, Gleeson N, D’Arcy T, McGuinness E, Sheils O, Sheppard B, OL J (2008) Potential role of miR-9 and miR-223 in recurrent ovarian cancer. Mol Cancer 7:35PubMedCrossRefGoogle Scholar
  15. Lee JW, Soung YH, Kim HJ, Park WS, Nam SW, Kim SH, Lee JY, Yoo NJ, Lee SH (2006) Mutational analysis of the hCDC4 gene in gastric carcinomas. Eur J Cancer 42(14):2369–2373PubMedCrossRefGoogle Scholar
  16. Li X, Zhang Y, Ding J, Wu K, Fan D (2010) Survival prediction of gastric cancer by a seven-microRNA signature. Gut 59(5):579–585PubMedCrossRefGoogle Scholar
  17. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4):402–408PubMedCrossRefGoogle Scholar
  18. Mathe EA, Nguyen GH, Bowman ED, Zhao Y, Budhu A, Schetter AJ, Braun R, Reimers M, Kumamoto K, Hughes D, Altorki NK, Casson AG, Liu CG, Wang XW, Yanaihara N, Hagiwara N, Dannenberg AJ, Miyashita M, Croce CM, Harris CC (2009) MicroRNA expression in squamous cell carcinoma and adenocarcinoma of the esophagus: associations with survival. Clin Cancer Res 15(19):6192–6200PubMedCrossRefGoogle Scholar
  19. McManus MT (2003) MicroRNAs and cancer. Semin Cancer Biol 13(4):253–258PubMedCrossRefGoogle Scholar
  20. Mendell JT (2005) MicroRNAs: critical regulators of development, cellular physiology and malignancy. Cell Cycle 4(9):1179–1184PubMedCrossRefGoogle Scholar
  21. Mi S, Lu J, Sun M, Li Z, Zhang H, Neilly MB, Wang Y, Qian Z, Jin J, Zhang Y, Bohlander SK, Le Beau MM, Larson RA, Golub TR, Rowley JD, Chen J (2007) MicroRNA expression signatures accurately discriminate acute lymphoblastic leukemia from acute myeloid leukemia. Proc Natl Acad Sci USA 104(50):19971–19976PubMedCrossRefGoogle Scholar
  22. Miko E, Czimmerer Z, Csanky E, Boros G, Buslig J, Dezso B, Scholtz B (2009) Differentially expressed microRNAs in small cell lung cancer. Exp Lung Res 35(8):646–664PubMedCrossRefGoogle Scholar
  23. Milne AN, Sitarz R, Carvalho R, Carneiro F, Offerhaus GJ (2007) Early onset gastric cancer: on the road to unraveling gastric carcinogenesis. Curr Mol Med 7(1):15–28PubMedCrossRefGoogle Scholar
  24. Milne AN, Carneiro F, O’Morain C, Offerhaus GJ (2009) Nature meets nurture: molecular genetics of gastric cancer. Hum Genet 126(5):615–628PubMedCrossRefGoogle Scholar
  25. Milne AN, Leguit R, Corver WE, Morsink FH, Polak M, de Leng WW, Carvalho R, Offerhaus GJ (2010) Loss of CDC4/FBXW7 in gastric carcinoma. Cell Oncol 32(5–6):347–359PubMedGoogle Scholar
  26. Nateri AS, Riera-Sans L, Da Costa C, Behrens A (2004) The ubiquitin ligase SCFFbw7 antagonizes apoptotic JNK signaling. Science 303(5662):1374–1378PubMedCrossRefGoogle Scholar
  27. O’Neil J, Grim J, Strack P, Rao S, Tibbitts D, Winter C, Hardwick J, Welcker M, Meijerink JP, Pieters R, Draetta G, Sears R, Clurman BE, Look AT (2007) FBW7 mutations in leukemic cells mediate NOTCH pathway activation and resistance to gamma-secretase inhibitors. J Exp Med 204(8):1813–1824PubMedCrossRefGoogle Scholar
  28. Petrocca F, Visone R, Onelli MR, Shah MH, Nicoloso MS, de Martino I, Iliopoulos D, Pilozzi E, Liu CG, Negrini M, Cavazzini L, Volinia S, Alder H, Ruco LP, Baldassarre G, Croce CM, Vecchione A (2008) E2F1-regulated microRNAs impair TGFbeta-dependent cell-cycle arrest and apoptosis in gastric cancer. Cancer Cell 13(3):272–286PubMedCrossRefGoogle Scholar
  29. Stamatopoulos B, Meuleman N, Haibe-Kains B, Saussoy P, Van Den Neste E, Michaux L, Heimann P, Martiat P, Bron D, Lagneaux L (2009) microRNA-29c and microRNA-223 down-regulation has in vivo significance in chronic lymphocytic leukemia and improves disease risk stratification. Blood 113(21):5237–5245PubMedCrossRefGoogle Scholar
  30. Takamizawa J, Konishi H, Yanagisawa K, Tomida S, Osada H, Endoh H, Harano T, Yatabe Y, Nagino M, Nimura Y, Mitsudomi T, Takahashi T (2004) Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res 64(11):3753–3756PubMedCrossRefGoogle Scholar
  31. Tang F, Hajkova P, Barton SC, Lao K, Surani MA (2006) MicroRNA expression profiling of single whole embryonic stem cells. Nucleic Acids Res 34(2):e9PubMedCrossRefGoogle Scholar
  32. van der Fits L, van Kester MS, Qin Y, Out-Luiting JJ, Smit F, Zoutman WH, Willemze R, Tensen CP, Vermeer MH (2011) MicroRNA-21 expression in CD4+ T cells is regulated by STAT3 and is pathologically involved in Sezary syndrome. J Invest Dermatol 131(3):762–768PubMedCrossRefGoogle Scholar
  33. Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, Visone R, Iorio M, Roldo C, Ferracin M, Prueitt RL, Yanaihara N, Lanza G, Scarpa A, Vecchione A, Negrini M, Harris CC, Croce CM (2006) A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci USA 103(7):2257–2261PubMedCrossRefGoogle Scholar
  34. Wong QW, Lung RW, Law PT, Lai PB, Chan KY, To KF, Wong N (2008) MicroRNA-223 is commonly repressed in hepatocellular carcinoma and potentiates expression of Stathmin1. Gastroenterology 135(1):257–269PubMedCrossRefGoogle Scholar
  35. Wu WK, Lee CW, Cho CH, Fan D, Wu K, Yu J, Sung JJ (2010) MicroRNA dysregulation in gastric cancer: a new player enters the game. Oncogene 29(43):5761–5771PubMedCrossRefGoogle Scholar
  36. Xu T, Zhu Y, Xiong Y, Ge YY, Yun JP, Zhuang SM (2009) MicroRNA-195 suppresses tumorigenicity and regulates G1/S transition of human hepatocellular carcinoma cells. Hepatology 50(1):113–121PubMedCrossRefGoogle Scholar
  37. Yamaoka Y, Kato M, Asaka M (2008) Geographic differences in gastric cancer incidence can be explained by differences between Helicobacter pylori strains. Intern Med 47(12):1077–1083PubMedCrossRefGoogle Scholar
  38. Yang F, Sarangarajan R, Le Poole IC, Medrano EE, Boissy RE (2000) The cytotoxicity and apoptosis induced by 4-tertiary butylphenol in human melanocytes are independent of tyrosinase activity. J Invest Dermatol 114(1):157–164PubMedCrossRefGoogle Scholar
  39. Yeh E, Cunningham M, Arnold H, Chasse D, Monteith T, Ivaldi G, Hahn WC, Stukenberg PT, Shenolikar S, Uchida T, Counter CM, Nevins JR, Means AR, Sears R (2004) A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells. Nat Cell Biol 6(4):308–318PubMedCrossRefGoogle Scholar
  40. Yokobori T, Mimori K, Iwatsuki M, Ishii H, Onoyama I, Fukagawa T, Kuwano H, Nakayama KI, Mori M (2009) p53-Altered FBXW7 expression determines poor prognosis in gastric cancer cases. Cancer Res 69(9):3788–3794PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Jinhai Li
    • 1
  • Yuanyuan Guo
    • 2
  • Xiaodi Liang
    • 2
  • Ming Sun
    • 2
  • Guoliang Wang
    • 1
  • Wei De
    • 2
  • Wenxi Wu
    • 1
    Email author
  1. 1.Department of Gastrointestinal SurgeryFirst Affiliated Hospital of Nanjing Medical UniversityNanjingPeople’s Republic of China
  2. 2.Department of Biochemistry and Molecular BiologyNanjing Medical UniversityNanjingPeople’s Republic of China

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