组蛋白伴侣ASF1B在前列腺癌细胞的表达及其对体外细胞活力的影响

Expression of histone chaperone ASF1B in prostate cancer cells and its effect on cell viability in vitro

目的:探讨组蛋白伴侣抗沉默功能蛋白1B(ASF1B)在前列腺癌细胞中的表达情况及其对体外细胞活力的影响。方法:以人前列腺癌PC-3细胞为研究对象,通过小干扰RNA(siRNA)来敲减ASF1B表达。细胞分为对照组、siRNA阴性对照载体(mock)组和siRNA-ASF1B组。采用MTT法测定ASF1B-siRNA处理PC-3细胞12、24和48 h的细胞活力。采用流式细胞术检测细胞凋亡和细胞周期分布情况。采用RT-qPCR检测细胞中凋亡相关因子的mRNA表达水平,Western blot检测细胞中MAPK/JNK/ERK信号通路相关蛋白的表达。结果:正常细胞(良性前列腺增生)中ASF1B的蛋白水平显著低于PC-3细胞(P<0.01)。与对照组和mock组相比,用siRNA-ASF1B质粒转染PC-3细胞后的ASF1B蛋白表达水平显著降低(P<0.01),并且PC-3细胞的活力显著降低(P<0.01)。流式细胞术结果显示,转染siRNA-ASF1B质粒的PC-3细胞凋亡率较对照组显著升高(P<0.01),且细胞周期被阻滞于G1期。RT-qPCR结果表明,与对照组和mock组相比,转染siRNA-ASF1B质粒的PC-3细胞中p53、caspase-3、Bax和PARP-1的mRNA水平上调(P<0.01)。此外,与mock组相比,siRNA-ASF1B组PC-3细胞中MAP2K4和p-JNK蛋白水平显著升高(P<0.01),而p-ERK蛋白水平显著降低(P<0.01)。结论:ASF1B沉默诱导PC-3细胞G1期阻滞,促进细胞凋亡。激活MAPK/JNK/ERK信号通路可能是siRNA-ASF1B抗前列腺癌作用的机制之一。

AIM:To investigate the expression of histone chaperone anti-silencing function 1B(ASF1B)in prostate cancer cells and its effect on cell viability in vitro.METHODS:Human prostate cancer PC-3 cells were used,and knockdown of ASF1B was conducted by small interfering RNA(siRNA)transfection into the cells.The cells were divided into control group,siRNA negative control vector(mock)group and siRNA-ASF1B group.The viability of the PC-3 cells treated with ASF1B-siRNA for 12,24 and 48 h was measured by MTT assay.Flow cytometry was used to analyze cell apoptosis and cell cycle distribution.The mRNA expression of apoptosis-related molecules was detected by RT-qPCR,and the expression levels of MAPK/JNK/ERK signaling pathway-related proteins were determined by Western blot.RESULTS:The protein level of ASF1B in the normal cells(benign prostatic hyperplasia)was significantly lower than that in the PC-3 cells(P<0.01).Compared with control group and mock group,the protein expression level of ASF1B in the PC-3 cells transfected with siRNA-ASF1B plasmid and the viability of the PC-3 cells were significantly decreased(P<0.01).The apoptotic rate of the PC-3 cells transfected with siRNA-ASF1B plasmid was significantly higher than that in control group(P<0.01),and the cell cycle was arrested at G1 phase.The mRNA levels of p53,caspase-3,Bax and PARP-1 in the PC-3 cells transfected with siRNA-ASF1B plasmid were up-regulated compared with those in control group and Mack group(P<0.01).In addition,the protein levels of MAP2K4 and p-JNK in the PC-3 cells in siRNA-ASF1B group were significantly higher than those in mock group(P<0.01),while the protein level of p-ERK was significantly lower than that in mock group(P<0.01).CONCLUSION:ASF1B silencing induces G1 arrest and promotes apoptosis of PC-3 cells.Activating MAPK/JNK/ERK signaling pathway may be a possible contributor to the anti-prostate cancer effect of siRNAASF1B.