Advertisement

Identification of Cancer Stem Cell-Related MicroRNAs in Hepatocellular Carcinoma

  • Junfang Ji
  • Xin Wei WangEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 826)

Abstract

Cancer Stem cells (CSCs) are the source of many solid tumor types including hepatocellular carcinoma. MicroRNAs (miRNAs) are small noncoding RNAs and have been showed to be associated with hepatic CSCs. Here, we described methods to screen hepatic CSC-related miRNAs, and to validate and examine their expressions and functions in vitro and in vivo, which contribute to the maintenance of stemness and differentiation of hepatic CSCs.

Key words

Hepatic cancer stem cells MicroRNAs Hepatocellular carcinoma EpCAM Fluorescence-activated cell sorting MicroRNA microarray Quantitative reverse transcription-polymerase chain reaction Transfection Spheroid assay Tumorigenicity assay 

Notes

Acknowledgment

This work was supported by the Intramural Research Program of the Center for Cancer Research, the National Cancer Institute (Z01 BC 010313 and Z01 BC 010876).

References

  1. 1.
    Gupta PB, Chaffer CL, and Weinberg RA (2009) Cancer stem cells:mirage or reality? Nat. Med 15:10101012PubMedCrossRefGoogle Scholar
  2. 2.
    Al Hajj M, Wicha MS, Benito-Hernandez A et al (2003) Prospective identification of tumorigenic breast cancer cells. Proc. Natl. Acad. Sci USA 100:39833988PubMedCrossRefGoogle Scholar
  3. 3.
    Singh SK, Hawkins C, Clarke ID et al (2004) Identification of human brain tumour initiating cells. Nature 432:396401PubMedCrossRefGoogle Scholar
  4. 4.
    Bonnet D. and Dick JE (1997) Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat. Med. 3:730737PubMedCrossRefGoogle Scholar
  5. 5.
    Ricci-Vitiani L, Lombardi DG, Pilozzi E et al (2007) Identification and expansion of human colon-cancer-initiating cells. Nature 445:111115PubMedCrossRefGoogle Scholar
  6. 6.
    O’Brien CA, Pollett A, Gallinger S et al (2007) A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 445:106110PubMedCrossRefGoogle Scholar
  7. 7.
    Yamashita T, Ji J, Budhu A et al (2009) EpCAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features. Gastroenterology 136:10121024PubMedCrossRefGoogle Scholar
  8. 8.
    Parkin DM, Bray F, Ferlay J et al (2005) Global cancer statistics, 2002. CA Cancer J. Clin 55:74108Google Scholar
  9. 9.
    Yang ZF, Ho DW, Ng MN et al (2008) Significance of CD90(+) Cancer Stem Cells in Human Liver Cancer. Cancer Cell 13:153166PubMedCrossRefGoogle Scholar
  10. 10.
    Ma S, Chan KW, Hu L et al (2007) Identification and characterization of tumorigenic liver cancer stem/progenitor cells. Gastroenterology 132:25422556PubMedCrossRefGoogle Scholar
  11. 11.
    Yamashita T, Forgues M, Wang W et al (2008) EpCAM and alpha-fetoprotein expression defines novel prognostic subtypes of hepatocellular carcinoma. Cancer Res. 68:14511461PubMedCrossRefGoogle Scholar
  12. 12.
    Lagos-Quintana M, Rauhut R, Lendeckel W et al (2001) Identification of novel genes coding for small expressed RNAs. Science 294:853858PubMedCrossRefGoogle Scholar
  13. 13.
    Lau NC, Lim LP, Weinstein EG et al (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294:858862PubMedCrossRefGoogle Scholar
  14. 14.
    Ji J, Yamashita T, Budhu A et al (2009) Identification of microRNA-181 by genome-wide screening as a critical player in EpCAM-positive hepatic cancer stem cells. Hepatology 50:472480PubMedCrossRefGoogle Scholar
  15. 15.
    Ji J, Shi J, Budhu A et al (2009) MicroRNA expression, survival, and response to interferon in liver cancer. N. Engl. J Med 361:14371447PubMedCrossRefGoogle Scholar
  16. 16.
    Ji J. and Wang X.W (2009) New kids on the block:Diagnostic and prognostic microRNAs in hepatocellular carcinoma. Cancer Biol Ther. 8:1686–1693PubMedGoogle Scholar
  17. 17.
    Budhu A, Jia HL, Forgues M et al (2008) Identification of metastasis-related microRNAs in hepatocellular carcinoma. Hepatology 47:897907PubMedCrossRefGoogle Scholar
  18. 18.
    Lu J, Getz G, Miska EA et al (2005) MicroRNA expression profiles classify human cancers. Nature 435:834838PubMedCrossRefGoogle Scholar
  19. 19.
    Croce CM. and Calin GA (2005) miRNAs, cancer, and stem cell division. Cell 122:67PubMedCrossRefGoogle Scholar
  20. 20.
    Rogler CE, Levoci L, Ader T et al (2009) MicroRNA-23b cluster microRNAs regulate transforming growth factor-beta/bone morphogenetic protein signaling and liver stem cell differentiation by targeting Smads. Hepatology 50:575584PubMedCrossRefGoogle Scholar
  21. 21.
    Hatfield SD, Shcherbata HR, Fischer KA et al (2005) Stem cell division is regulated by the microRNA pathway. Nature 435:974978PubMedCrossRefGoogle Scholar
  22. 22.
    Bernstein E, Kim SY, Carmell MA et al (2003) Dicer is essential for mouse development. Nat. Genet. 35:215217PubMedCrossRefGoogle Scholar
  23. 23.
    Strauss WM, Chen C, Lee CT et al (2006) Nonrestrictive developmental regulation of microRNA gene expression. Mamm. Genome 17:833840PubMedCrossRefGoogle Scholar
  24. 24.
    Suh MR, Lee Y, Kim JY et al (2004) Human embryonic stem cells express a unique set of microRNAs. Dev. Biol. 270:488498PubMedCrossRefGoogle Scholar
  25. 25.
    Chen CZ, Li L, Lodish HF et al (2004) MicroRNAs modulate hematopoietic lineage differentiation. Science 303:8386PubMedCrossRefGoogle Scholar
  26. 26.
    Schratt GM, Tuebing F, Nigh EA et al (2006) A brain-specific microRNA regulates dendritic spine development. Nature 439:283289PubMedCrossRefGoogle Scholar
  27. 27.
    Yi R, Poy MN, Stoffel M et al (2008) A skin microRNA promotes differentiation by repressing ‘stemness’. Nature 452:225229PubMedCrossRefGoogle Scholar
  28. 28.
    Kota J, Chivukula RR, O’donnell KA et al (2009) Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model. Cell 137:10051017PubMedCrossRefGoogle Scholar
  29. 29.
    Liu CG, Calin GA, Meloon B et al (2004) An oligonucleotide microchip for genome-wide microRNA profiling in human and mouse tissues. Proc Natl Acad Sci USA 101:97409744PubMedCrossRefGoogle Scholar
  30. 30.
    Voorhoeve PM, le Sage C, Schrier M et al (2006) A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors. Cell 124:11691181PubMedCrossRefGoogle Scholar
  31. 31.
    Krutzfeldt J, Rajewsky N, Braich R et al (2005) Silencing of microRNAs in vivo with ‘antagomirs’. Nature 438:685689PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Laboratory of Human CarcinogenesisBethesdaUSA

Personalised recommendations