LSD1 acetylation mediates TSA-induced apoptosis of ovarian cancer cells / 医学研究生学报

ABSTRACT

Objective Lysine-specific demethylase 1 (LSD1) is a flavin adenine dinucleotide-dependent oxidase, which participates in many biological processes , such as cell proliferation and differentiation and gene activation and repression .The aim of this study was to investigate LSD1 acetylation by histone deacetylase inhib -itor trichostatin A ( TSA) and its effect on TSA-induced apoptosis of ovarian cancer cells . Methods LSD1 shRNA was synthesized and implanted into the pLKO-Tet-On lentiviral vector , which was transfected into HO8910 and SKOV3 ovarian cancer cell lines , and then the transfected cells were screened with 1.5μg/mL puromycin for one week until stable clones were established .The cells were treated for 48 hours with methanol (2 mg/mL, control), TSA (200 nmol/L), TCP (100μmol/L), or TSA+TCP.And in the experiment of RNA interfering the LSD1 expression, the cells were also treated for 48 hours with methanol (2 mg/mL, control), TSA (200 nmol/L), Dox (100 ng/mL), or TSA +Dox.The levels of LSD1 acetylation and its substrate histone H3 lysine 4 dimethylation (H3K4me2) were de-tected by immunoprecipitation (IP) and Western blot.The apoptosis of the cells was determined by Annexin Ⅴ/PI staining and flow cytometry, the transcription levels of the Bax and p21 genes detected by real-time quantitative PCR, and the H3K4me2levels in the promoter regions of Bax and p21 measured by chromatin immunoprecipitation ( ChIP ) .Results In comparison with the methanol control, the TSA group showed significantly increased levels of LSD 1 acetylation in the HO8910(1.00±0.29 vs 5.83±0.46, P<0.01) and SKOV3 cells ( 1.00±0.24 vs 5.07±0.35, P<0.01) as well as that of H3K4me2 ( P<0.01);the total apoptosis rates of HO 8910 and SKOV3 cells were remarkably increased in the TSA, TCP, and TSA+TCP groups (P<0.05), even more significantly in the TSA+TCP than in the TSA and TCP groups ( P<0.05) .The mRNA expressions of Bax and p21 in the HO8910 cells were markedly upregulated in the TSA, Dox, and TSA+Dox groups (P<0.05), even more significantly in the latter than in the former two groups (P<0.05).The TSA group exhibited a higher level of H 3K4me2 than the methanol control in the promoters of Bax(2 .92±0.26 vs 0.68±0.19, P<0.01) and p21 (3.07±0.29 vs 0.93±0.17, P<0.01). Conclusion TSA induces the LSD1 acetylation, while suppression of LSD1 expres-sion and activity may enhance the antitumor activity of TSA .