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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 investigate the in vitro effect of polyamine analogue DENSPM on castration-resistant prostate cancer PC3 cells and its effect on DENSPM induced by spermine / spermine N1 acetyltransferase (SSAT) gene expression influences. Methods Small interfering RNA (siRNA) plasmid was constructed and transfected into PC3 cells with Lipofectamine 2000. The transfected cells were transfected with pre-configured DENSPM. 48 h after DENSPM treatment, cells were harvested for cell counting kit (CCK-8) proliferation activity assay, flow cytometry, RNA extraction and total protein were detected by real-time fluorescence quantitative PCR and Western blotting to verify the interference effect of SSAT siRNA The expression of SSAT mRNA and protein was detected after DENSPM treatment. The contents of polyamines in each group of cells after transfection and drug treatment were detected by high performance liquid chromatography (HPLC). Results The proliferative activity of PC3 cells in DENSPM + siRNA group and DENSPM + siRNA group were significantly higher than those in DENSPM group (P <0.05). The number of G0G1 cells in blank control group and DENSPM + siRNA group (P <0.05), the number of cells in G2M, S phase was significantly less than that in DENSPM group (P <0.05), and the concentration of spermidine in PC3 cells of blank control group and DENSPM + siRNA group was significantly higher than that of DENSPM group The levels of PC3 cells in G0G1, G2M and S phases were significantly higher than those in DENSPM group (P <0.05) There was no significant difference in the levels of spermine, putrescine and spermine in PC3 cells (P> 0.05). The SSAT mRNA and protein expression levels in DENSPM group were 15.10 ± 0.12 and 1.56 ± 0.06, respectively, which were significantly higher than 1.00 ± 0.00 and 0.60 ± 0.01 in blank control group and 1.07 ± 0.03 and 0.62 ± 0.02 in DENSPM ± siRNA group (P value All <0.01). Conclusions DENSPM, a polyamine analogue, can inhibit the proliferation of PC3 cells in ovariectomized prostate cancer cells, induce the arrest of S phase, induce the up-regulation of SSAT expression and promote the degradation and metabolism of polyamines. Disruption of SSAT expression can antagonize the anticancer effect of DENSPM.