下调垂体肿瘤转化基因1表达对前列腺癌LNCaP-AI细胞衰老的影响

Down-regulated PTTG1 expression promotes the senescence of human prostate cancer LNCaP-AI cells

目的:探讨下调垂体肿瘤转化基因1(PTTG1)的表达对人去势抵抗前列腺癌LNCaP-AI细胞衰老的影响。方法:采用体外诱导的人去势抵抗前列腺癌LNCaP-AI细胞模型, LNCaP-AI细胞转染靶向PTTG1基因的siRNA为siRNA-PTTG1组,只添加转染试剂为Mock组,转染阴性序列为NC组。于含雄激素培养液(FBS)/去除雄激素培养液(CSS)中培养各组细胞,细胞计数实验比较各组细胞数量变化。采用细胞衰老-β-半乳糖苷酶(SA-β-Gal)染色试剂盒检测衰老细胞数目;Western印迹检测β-半乳糖苷酶蛋白1(Glb1)、细胞周期相关蛋白(p-21^(CIP1)、p-27^(Kip1))、异染色质蛋白1γ(HP1γ)的表达情况。结果:siRNA有效抑制了LNCaP-AI细胞中PTTG1的表达。siRNA-PTTG1组在FBS中培养,细胞数量增加,而在CSS中培养,细胞数量呈减少趋势;Mock组及NC组在上述不同培养液中细胞数量均呈增长趋势,siRNA-PTTG1组与Mock组和NC组比较差异有统计学意义(P<0.05)。SA-β-Gal染色检测衰老细胞数量,Mock组、NC组、siRNA-PTTG1组细胞染色阳性率分别为(11.3±1.24)%、(12.4±1.15)%、(63.5±2.35)%,siRNA-PTTG1组显著高于Mock组和NC组(P<0.05)。Western印迹检测转染效率显示Mock组、NC组及siRNA-PTTG1组中PTTG1相对表达量分别为0.56±0.02、0.61±0.02、0.21±0.01,p-21^(CIP1)相对表达量分别为0.20±0.02、0.21±0.03、0.32±0.03,p-27^(Kip1)相对表达量分别为0.20±0.03、0.22±0.01、0.38±0.02,GlB1相对表达量为0.13±0.01、0.15±0.01、0.24±0.01,HP1γ相对表达量分别为0.26±0.01、0.27±0.02、0.41±0.01;siRNA-PTTG1组与Mock组和NC组比较差异均有统计学意义(P<0.05)。结论 :下调PTTG1基因表达可促进人去势抵抗前列腺癌细胞株LNCaP-AI细胞衰老。

Objective: To investigate the effect of the down-regulated expression of pituitary tumor-transforming gene 1(PTTG1) on the senescence of human castration-resistant prostate cancer LNCaP-AI cells. Methods: Human castration-resistant prostate cancer LNCaP-AI cells were induced in vitro and transfected with siRNA targeting PTTG1(the siRNA-PTTG1 group), the reagent lip3000 only(the mock group) or siRNA negative control vector(the NC group). All the cells were cultured in fetal bovine serum(FBS) or charcoal-stripped bovine serum(CSS) and counted with the cell counting chamber. The senescence characteristics of the transfected LNCaP-AI cells were examined by senescence-associated β-galactosidase(SA-β-Gal) staining, and the expressions of the senescence-related β-galactosidase-1-like proteins(Glb1), the cyclin-dependent kinase inhibitors p-21^CIP1 and p-27^Kip1, and the chromatin-regulating heterochromatin protein 1γ(HP1γ) were detected by Western blot. Results: The expression of PTTG1 in the human prostate cancer LNCaP-AI cells was significantly reduced in the siRNA-PTTG1 group compared with those in the mock and NC groups(0.21 ± 0.01 vs 0.56 ± 0.02 and 0.61 ± 0.02, P < 0.05). Culture with FBS markedly increased while that with CSS decreased the number of LNCaP-AI cells transfected with siRNA, but both FBS and CSS enhanced the proliferation of the LNCaP-AI cells in the mock and NC groups. SA-β-Gal staining revealed that reducing the expression of PTTG1 induced a remarkably higher positive rate of the LNCaP-AI cells in the siRNA-PTTG1 than in the mock and NC groups([63.5 ± 2.35]% vs [11.3 ± 1.24]% and [12.4 ± 1.15]%, P < 0.05). The siRNA-PTTG1 group, in comparison with the mock and NC groups, showed a significantly down-regulated expression of PTTG1(0.21 ± 0.01 vs 0.56 ± 0.02 and 0.61 ± 0.02, P < 0.05), but up-regulated expressions of p-21^CIP1(0.32 ± 0.03 vs 0.20 ± 0.02 and 0.21 ± 0.03, P < 0.05), p-27^Kip1(0.38 ± 0.02 vs 0.20 ± 0.03 and 0.22 ± 0.01, P < 0.05), Glb1(0.24 ± 0.01 vs 0.13 ± 0.01 and 0.15 ± 0.01, P < 0.05), and HP1γ(0.41 ± 0.01 vs 0.26 ± 0.01 and 0.27 ± 0.02, P < 0.05) in the LNCaP-AI cells. Conclusion: Down-regulated expression of PTTG1 induces senescence of human castration-resistant prostate cancer LNCaP-AI cells.