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Watanabe, Yukihide
Department of Experimental Pathology, Faculty of Medicine, University of Tsukuba, Ibaraki
Medicine & Pharmacology

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Knock-out transmembrane prostate androgen-induced protein gene suppressed triple-negative breast cancer cell proliferation Wardhani, Bantari W.K.; Puteri, Meidi U.; Watanabe, Yukihide; Louisa, Melva; Setiabudy, Rianto; Kato, Mitsuyasu
Medical Journal of Indonesia Vol 26, No 3 (2017): September
Publisher : Faculty of Medicine Universitas Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (646.527 KB) | DOI: 10.13181/mji.v26i3.1823

Abstract

Background: Triple negative breast cancer (TNBC) tends to grow more rapidly and has poorer prognosis compared to others. High expression of transmembrane prostate androgen-induced protein (TMEPAI) correlates with poor prognosis in TNBC patients. However, the mechanistic role of TMEPAI in tumorigenic remains unknown. This study aimed to knock-out TMEPAI in TNBC cell line to determine its function further in cells proliferation.Methods: CRISPR-Cas9 has been used previously to knock-out TMEPAI in Hs857T TNBC cell line. Hs587T TNBC parental cell line (wild-type/WT) and TMEPAI knock out Hs 586T cell lines were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and amphotericin B. Both cell lines were seeded in 24-well plates and counted every two days, then proliferation rates were plotted. Afterwards, total RNA were isolated from the cells and Ki-67, and TGF-β mRNA expression levels as proliferation markers were determined.Results: Cell proliferation rates as displayed in growth curve plots showed that WT-TMEPAI cell line grew more rapidly than KO-TMEPAI. In accordance, mRNA expression levels of  Ki-67 and TGF-β  were significantly decreased KO-TMEPAI as compare to TMEPAI-WT.Conclusion: Knock-out of TMEPAI attenuates cell proliferation in TNBC.
TMEPAI genome editing in triple negative breast cancer cells Wardhani, Bantari W.K.; Puteri, Meidi U.; Watanabe, Yukihide; Louisa, Melva; Setiabudy, Rianto; Kato, Mitsuyasu
Medical Journal of Indonesia Vol 26, No 1 (2017): March
Publisher : Faculty of Medicine Universitas Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (607.158 KB) | DOI: 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.
Co-Authors Kato, Mitsuyasu Melva Louisa Puteri, Meidi U. Rianto Setiabudy Wardhani, Bantari W.K.