干扰精脒/精胺N1乙酰基转移酶基因拮抗多胺类似物二乙基去甲精胺抗前列腺癌细胞增殖活性

Silence of spermidine/spermine N1-acetyltransferase expression by small interference RNA antagonizing anti-proliferation of N1,N11-diethylnorspermine on prostate cancer cells

目的探讨多胺类似物二乙基去甲精胺(DENSPM)对去势抵抗性前列腺癌PC3细胞的体外作用,以及干扰精脒/精胺N1乙酰基转移酶(SSAT)基因表达对DENSPM作用的影响。方法构建SSAT小干扰RNA质粒(siRNA),并应用脂质体转染试剂Lipofectamine 2000转染PC3细胞,转染24h后加入预先配置好的DENSPM。DENSPM处理48h后收集细胞进行细胞计数试剂盒(CCK-8)增殖活性实验、流式细胞周期实验,抽提RNA和总蛋白分别进行实时荧光定量PCR和蛋白质免疫印迹杂交,验证SSAT siRNA的干扰效果,检测DENSPM处理后SSAT mRNA和蛋白质的表达水平。采用高效液相色谱法(HPLC法)检测转染和药物处理后各组细胞内的多胺含量。结果空白对照组和DENSPM+siRNA组培养36、48、72h时的PC3细胞增殖活性均显著高于DENSPM组同时间点(P值均<0.05),空白对照组和DENSPM+siRNA组G0G1期细胞数均显著多于DENSPM组同期(P值均<0.05),G2M、S期细胞数均显著少于DENSPM组同期(P值均<0.05),空白对照组和DENSPM+siRNA组PC3细胞内精脒、腐胺、精胺水平均显著高于DENSPM组(P值均<0.05);空白对照组与DENSPM+siRNA组间培养各时间点的PC3细胞增殖活性,G0G1、G2M、S期的PC3细胞数,以及PC3细胞内精脒、腐胺、精胺水平的差异均无统计学意义(P值均>0.05)。DENSPM组的SSAT mRNA和蛋白表达量分别为15.10±0.12和1.56±0.06,显著高于空白对照组的1.00±0.00和0.60±0.01,以及DENSPM±siRNA组的1.07±0.03和0.62±0.02(P值均<0.01)。结论多胺类似物DENSPM可抑制去势抵抗性前列腺癌PC3细胞增殖,诱导S期阻滞,诱导SSAT表达上调,促进多胺降解代谢。干扰SSAT表达可拮抗DENSPM的抗癌作用。

Objective To explore the effects of silencing spermidine/spermine N1 acetyltransferase, （SSAT） mRNA on the anti-proliferation of N1, N11-diethylnorspermine （DENSPM） in castration resistant prostate cancer PC3 cells in vitro. Methods SSAT small interference RNA （siRNA） were constructed and transfected into PC3 cells by Lipofectamine 2000. DENSPM was added 24 h after transfection. After treated with DENSPM 48 h, cell proliferation was detected by cell counting Kit-8 （CCK-8）, and cell cycle was tested on flow cytometry. SSAT mRNA and protein were extracted and detected by quantitative-real-time-polymerase chain reaction （PCR） and Western blotting to verify silencing effect. The content of polyamines were determined by high performance liquid chromatography （HPLC） after transfection and DENSPM treatment. Results PC3 cell proliferation of control group and DENSPM-I-siRNA group were significantly higher than that in DENSPM group 36 h, 48 h and 72 h after culture （all P〈0.05）. PC3 cell counts of control group and DENSPM ＋ siRNA group in GOG1 phrase were significantly more than that in DENSPM group （both P〈0.05）, and the PC3 cell counts of control group and DENSPM＋ siRNA group in G2M and S phrases were significantly less than those in DENSPM group （all P〈0.05）. The contents of Spd, Put and Spm in control group and DENSPM ＋ siRNA group were significantly higher than those in DENSPM group （all P〈0.05）. There were no significant differences in terms of PC3 cell proliferation, PC3 cell counts in GOG1 ,G2M or S phrases, or the content of Spd, Put, Spm in PC3 cells between DENSPM＋ siRNA group and control group （all P〉0.05）. The expression of SSAT mRNA and protein in DENSPM group were 15.10±0.12 and 1.56 ± 0.06, which was significantly higher than those in control group （ 1.00 ±0.00 and 0.60± 0.01 ） and DENSPM＋siRNAgroup （1.07±0.03 and 0.62±0.02, all P〈0.01）. Conclusion DENSPM can suppress castration resistant prostate cancer PC3 cell proliferation by inducing S-phase block, up-regulating SSAT, promoting poiyamine degradation. But silence of SSAT will invert the effect.