TMEPAI genome editing in triple negative breast cancer cells

Bantari W.K. Wardhani, Meidi U. Puteri, Yukihide Watanabe, Melva Louisa, Rianto Setiabudy, Mitsuyasu Kato



DOI: http://dx.doi.org/10.13181/mji.v26i1.1871

Abstract


Background: Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) is a powerful genome editing technique. It consists of RNA-guided DNA endonuclease Cas9 and single guide RNA (gRNA). By combining their expressions, high efficiency cleavage of the target gene can be achieved, leading to the formation of DNA double-strand break (DSB) at the genomic locus of interest which will be repaired via NHEJ (non-homologous end joining) or HDR (homology-directed repair) and mediate DNA alteration. We aimed to apply the CRISPR/Cas9 technique to knock-out the transmembrane prostate androgen-induced protein (TMEPAI) gene in the triple negative breast cancer cell line.

Methods: Designed gRNA which targets the TMEPAI gene was synthesized, annealed, and cloned into gRNA expression vector. It was co-transfected into the TNBC cell line using polyethylenimine (PEI) together with Cas9-GFP and puromycin resistant gene vector. At 24-hours post-transfection, cells were selected by puromycin for 3 days before they were cloned. Selected knock-out clones were subsequently checked on their protein levels by western blotting.

Results: CRISPR/Cas9, a genome engineering technique successfully knocked-out TMEPAI in the Hs578T TNBC cell line. Sequencing shows a frameshift mutation in TMEPAI. Western blot shows the absence of TMEPAI band on Hs578T KO cells.

Conclusion: TMEPAI gene was deleted in the TNBC cell line using the genomic editing technique CRISPR/Cas9. The deletion was confirmed by genome and protein analysis.


Keywords


CRISPR/Cas9; gene editing; knock-out cell lines

Full Text:

PDF

References


  1. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281–308. https://doi.org/10.1038/nprot.2013.143
  2. Tycko J, Myer VE, Hsu PD. Methods for optimizing CRISPR-Cas9 genome editing specificity. Mol Cell. 2016;63(3):356–70. https://doi.org/10.1016/j.molcel.2016.07.004
  3. Sánchez-Rivera FJ, Jacks T. Applications of CRISPR-Cas9 system in cancer biology. Nat Rev Cancer. 2015;15(7):387–95. https://doi.org/10.1038/nrc3950
  4. Weinberg RA. The biology of cancer. 2nd edition. New York: Garland Sence; 2013.
  5. Singha PK, Pandeswara S, Geng H, Lan R, Venkatachalam MA, Saikumar P. TGF-β induced TMEPAI/PMEPA1 inhibits canonical Smad signaling through R-Smad sequestration and promotes non-canonical PI3K/Akt signaling by reducing PTEN in triple negative breast cancer. Genes Cancer. 2014;5(9-10):320–36. DOI: 10.18632/genesandcancer.30
  6. Foulkes WD, Smith IE, Reis-Fielho JS. Triple negative breast cancer. N Eng J Med. 2010;363:1938–48. https://doi.org/10.1056/NEJMra1001389
  7. Watanabe Y, Itoh S, Goto T, Ohnishi E, Inamitsu M, Itoh F, et al. TMEPAI, a transmembrane TGF-β-inducible protein, sequesters Smad proteins from active participation in TGF-beta signaling. Mol Cell. 2010;37(1):123–34. https://doi.org/10.1016/j.molcel.2009.10.028
  8. Vo Nguyen TT, Watanabe Y, Shiba A, Noguchi M, Itoh S, Kato M. TMEPAI/PMEPA1 enhances tumorigenic activities in lung cancer cells. Cancer Sci. 2014;105(3):334–41. https://doi.org/10.1111/cas.12355
  9. CRISPRdirect-Rational design of CRISPR/Cas target [available from: https://crispr.dbcls.jp]
  10. Itoh S, Thorikay M, Kowanetz M, Moustakas A, Itoh F, Heldin CH, et al. Elucidation of Smad requirement in transforming growth factor-beta type I receptor-induced responses. J Biol Chem. 2003;278(6):3751–61. https://doi.org/10.1074/jbc.M208258200
  11. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339(6121):819–23. https://doi.org/10.1126/science.1231143
  12. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339(6121):823–6. https://doi.org/10.1126/science.1232033
  13. Falahi F, Sgro A, Blancafort P. Epigenome engeenering in cancer: fairytale or a realistic path to the clinics?. Front Oncol. 2015;5:1–11. https://doi.org/10.3389/fonc.2015.00022
  14. Vo Nguyen TT. Tumorigenic function of TMEPAI in cancer. Tulips University of Tsukuba Library. 2014. p.13
  15. N, Triaspolitica. "Kanker Payudara: Informasi, Penyebab, Gejala, Stadium Dan Pengobatan." Mau Nanya Dong Dok. N.p, 28 June 2017. Web. 30 June 2017. Retreived from: https://nanyadongdok.blogspot.com/2017/06/kanker-payudara-informasi-penyebab.html
  16. Giannini G, Ambrosini MI, Di Marcotullio L, Cerignoli F, Zani M, MacKay AR, et al. EGF- and cell-cycle-regulated STAG1/PMEPA1/ERG1.2 belongs to a conserved gene family and is overexpressed and amplified in breast and ovarian cancer. Mol Carcinog. 2003;38(4):188–200. https://doi.org/10.1002/mc.10162
  17. Fournier PG, Juárez P, Jiang G, Clines GA, Niewolna M, Kim HS, et al. The TGF-β signaling regulator PMEPA1 supresses prostate cancer metastases to bone. Cancer Cell. 2015;27(6):809–21. https://doi.org/10.1016/j.ccell.2015.04.009
  18. Brunschwig EB, Wilson K, Mack D, Dawson D, Lawrence E, Willson JK, et al. PMEPA1, a transforming growth factor-beta-induced marker of terminal colonocyte differentiation whose expression is maintained in primary and metastatic colon cancer. Cancer Res. 2003;63(7):1568–75.





Copyright (c) 2017 Bantari W.K. Wardhani, Meidi U. Puteri, Yukihide Watanabe, Melva Louisa, Rianto Setiabudy, Mitsuyasu Kato

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

All articles and issues in Medical Journal of Indonesia have unique DOI number registered in Crossref.
 
Romeo
 
http://mji.ui.ac.id/journal/index.php/mji/pages/view/stat
Unique Visitors