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目的 :研究赖氨酸特异性组蛋白去甲基化酶(1lysine-specific demethylase 1,LSD1)表达下调及其酶活性下降对人卵巢癌HO8910细胞增殖和凋亡的影响。方法:采用慢病毒感染系统将携带有特异性针对LSD1基因的LSD1-shRNA重组慢病毒载体(pLKO-Tet-On-LSD1-shRNA)转入HO8910细胞,并用嘌呤霉素(puromycin)筛选LSD1-shRNA稳定转入的HO8910细胞(HO8910-LSD1-shRNA细胞);采用不同质量浓度的多西环素(doxycycline,Dox)(1、10和100 ng/m L)处理HO8910-LSD1-shRNA细胞,并用实时荧光定量PCR和蛋白质印迹法分别检测细胞中LSD1 mRNA和蛋白以及组蛋白(histones)H3第4位赖氨酸的二甲基化(H3K4me2)蛋白表达的变化。分别用LSD1特异性抑制剂苯环丙胺(tranylcypromine,TCP)和Dox处理HO8910细胞和HO8910-LSD1-shRNA细胞后,用MTT法和EdU染色法检测细胞的增殖情况;FCM法检测抑制LSD1活性或沉默LSD1基因表达对HO8910细胞周期及凋亡的影响;最后,采用蛋白质印迹法检测抑制LSD1活性或沉默LSD1基因表达对HO8910细胞中p21、Bax、Bcl-2和survivin蛋白表达的影响。结果:建立了稳定沉默LSD1表达的卵巢癌HO8910细胞株;LSD1表达沉默后,H3K4me2蛋白的表达水平随着Dox浓度的增加而上调;干扰LSD1基因表达或抑制LSD1的活性,均能够显著抑制HO8910细胞的增殖,并促进细胞的凋亡(P值均<0.05)。此外,LSD1活性的抑制或LSD1表达的下调均可使细胞周期阻滞在G_1期,并下调Bcl-2和survivin蛋白的表达,以及上调p21和Bax蛋白的表达。结论 :沉默人卵巢癌细胞HO8910中LSD1基因的表达能有效抑制细胞的增殖,使细胞阻滞在G_1期,促进细胞的凋亡,提示LSD1可能是卵巢癌治疗的一个新靶点。
AIM: To investigate the effects of down-regulation of lysine-specific histone demethylase 1 (LSD1) and the decrease of its enzymatic activity on the proliferation and apoptosis of human ovarian cancer HO8910 cells. Methods: LSD1-shRNA recombinant lentiviral vector carrying LSD1 gene (pLKO-Tet-On-LSD1-shRNA) was transfected into HO8910 cells by lentivirus infection system and purifed with LSD1-shRNA HO8910-LSD1-shRNA cells were stably transfected; HO8910-LSD1-shRNA cells were treated with doxycycline (Dox) Fluorescence quantitative PCR and Western blotting were used to detect the expression of LSD1 mRNA and protein in cells and the lysine methylation of H3K4me2 in histone H3. Cell proliferation was detected by MTT assay and EdU staining after treatment of HO8910 cells and HO8910-LSD1-shRNA cells with LSD1 specific inhibitor of tranylcypromine (TCP) and Doxox, respectively. The inhibition of LSD1 activity or silencing by FCM LSD1 gene expression on HO8910 cell cycle and apoptosis. Finally, the effect of inhibiting LSD1 activity or silencing LSD1 gene expression on p21, Bax, Bcl-2 and survivin protein expression in HO8910 cells was detected by Western blot. Results: The expression of H3K4me2 protein was up-regulated in HO8910 ovarian cancer cell line with stable expression of LSD1. After silencing LSD1, the expression of H3K4me2 protein was up-regulated with the increase of Dox concentration. Interfering LSD1 gene expression or inhibiting the activity of LSD1 significantly inhibited HO8910 cells Proliferation and promote cell apoptosis (all P <0.05). In addition, the inhibition of LSD1 activity or the down-regulation of LSD1 expression could block the cell cycle in G_1 phase, down-regulate the expression of Bcl-2 and survivin protein, and up-regulate the expression of p21 and Bax protein. Conclusion: The expression of LSD1 gene in human ovarian cancer cell line HO8910 can effectively inhibit the cell proliferation, arrest the cells in G 1 phase and promote the apoptosis of cells, suggesting that LSD1 may be a new therapeutic target for ovarian cancer.