摘要
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。
Objective： To investigate the effects of lysine-specific demethylase 1 （LSD1） gene-silencing and LSD1 activity-decrease on cell proliferation and apoptosis of ovarian cancer HO8910 cells.Methods： The recombinant lentivirus carrying LSD1-shRNA （named as pLKO-Tet-On-LSD1- shRNA） was infected into HO8910 cells by lentivirus infection system. The HO8910 cells stably infected with LSDI-shRNA （named as HO8910-LSDl-shRNA cells） were screened by puromycin. After HO8910-LSD1-shRNA cells were treated with different concentrations of doxycycline （Dox） （1, 10 and 100 ng/mL）, the expression levels of LSD1 mRNA, LSD1 protein and histone H3 lysine-4 dimethylation （H3K4me2） protein were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. Furthermore, HO8910 cells were treated with LSDl-inhibitor tranylcypromine （TCP）, while HO8910-LSD1-shRNA cells were treated with Dox. Then the cell proliferation was detected by MTT and EdU stain assays, the cell cycle distribution and apoptotic rate were detected by FCM, and the protein levels of p21, Bax, Bcl-2 and survivin were examined by Western blotting. Results： The stable ISD1 gene-silenced HO8910 cell line was successfully established. The expression level of H3K4me2 protein in HO8910-LSD1-shRNA cells was increased with the increase of Dox concentration. ISD1 gene-silencing or LSD1 activity-inhibiting could significantly inhibit the proliferation and promote the apoptosis of HO8910 cells （both P 〈 0.05）. In addition, both the down-regulation of LSD1 expression and the inhibition of LSD1 activity could induce cell cycle arrest in G1 phase, down-regulate the expressions of Bcl-2 and survivin, and up-regulate the expressions of p21 and Bax. Conclusion： LSD1 gene-silencing can inhibit the proliferation and promote the apoptosis of human ovarian cancer HO8910 cells, as well as arrest cell cycle progression in the G1 phase. It is suggested that LSD1 may be a new target for the therapy of ovarian cancer.
出处
《肿瘤》
CAS
CSCD
北大核心
2016年第5期485-495,共11页
Tumor
基金
国家自然科学基金资助项目(编号:81170573)~~
