Role Of Lysine-Specific Demethylase 1 In Proliferation And Apoptosis Of Ovarian Cancer Cells

Background and Objective:Histone lysine-specific demethylase 1(LSD1) was discovered and reported in 2004. It is classified as a member of amine oxidase superfamily, with the nature of flavin adenine dinucleotide(FAD) dependence. LSD1 acts as a histone methylation eraser, which specifically removes mono-or-dimethylated histone H3 lysine 4(H3K4) and H3 lysine 9(H3K9) through formaldehyde-generating oxidation. Related researchs demonstrate that LSD1 regulates activation and inhibition of gene transcription in the nucleus, playing important biological functions. Recent studies have shown that LSD1 plays a key function in the occurrence, development, proliferation, migration and invasion of tumors. More has been reported that LSD1 is over-expressed in ovarian cancer tissues and cell lines, while the relationship between LSD1 and proliferation of ovarian cancer cell is not clear. This research aims to discover the effects of targeting LSD1 on cell proliferation and apoptosis in HO8910 ovarian cancer cells.Methods: 1.To generate ovarian cancer HO8910 cell lines with stable interference of LSD1 through lentiviral vector, the specific LSD1-shRNA sequences were synthesized and inserted into pLKO-Tet-On vector to construct the recombinant plasmid pLKO-Tet-On-shLSD1. The pLKO-Tet-On-sh LSD1, pHR’-CMV-8.2ΔVPR, and pHR’-CMV-VSVG were then cotransfected into 293 T cells to package lentiviral particles using the Lipofectamine 2000 reagent. Infection was performed by adding the lentiviral particles to HO8910 cells. The cells were selected with puromycin until stable clones of LSD1-shRNA in HO8910 were established.2. To generate rTet-repressor expressing(rtTA) cell line, 293 T cells were transfected with p LVX-Tet-On, pHR’-CMV-8.2ΔVPR and pHR’-CMV-VSVG by using Lipofectamine 2000 reagent. After transfection, the viral supernatant was collected and used to infect HO8910 cells. HO8910-rtTA cells were selected with G418. Cells survived were stable HO8910-rtTA cells. HO8910-rtTA cells were then infected with lentiviral particles packaged with pLVX-tight-puro-LSD1. After maintainning with puromycin, the cells survived were considered as rTet-repressor expressing(rtTA) cell line.3. The expression levels of LSD1 mRNA and protein in the stable clones were detected by real-time PCR and Western blot analysis, respectively. The expression level of H3K4me2 was also observed by Western blot.4. The inducer doxycycline(Dox) was added to the stable clones, and the LSD1 inhibitor tranylcypromine(TCP) was added to HO8910 cells. Cell proliferation rate was detected by MTT and EdU assays.5. Apoptosis was detected by AnnexinV/PI, while cell cycle analysis was detected by flow cytometry.6. In addition, the protein levels of P21, Bax, Bcl-2, and Survivin were examined by Western blot.Results: Stable LSD1 knockdown and over-expressed HO8910 cell line were successfully generated. LSD1 knockdown or inhibition of LSD1 activity significantly inhibited cell proliferation and promoted cell apoptosis in HO8910 ovarian cancer cells(P﹤0.05). LSD1 overexpression promoted cell proliferation and inhibited cell apoptosis. In addition, LSD1 knockdown or inhibition of LSD1 activity caused cell-cycle arrest at G1 phase, and up-regulation of LSD1 induced cell-cycle differentiation at G1/S phase. Mechanistic analyses showed that LSD1 control P21, Bax, Bcl-2 and Survivin.Conclusion: LSD1 knockdown or inhibition of LSD1 activity can inhibit the proliferation and promote the apoptosis of HO8910 cells,which can prevent the progression in the G1 phase. However, LSD1 overexpression has an opposite effect. It is suggested that targeting LSD1 could represent a potential approach for preventing the progression of ovarian cancer.