抑制核小体结合蛋白1基因对人前列腺癌细胞LNCaP增殖的作用  

Effects of inhibiting nucleosomal binding protein 1 on proliferation of human prostate cancer cells

作　　者：周利群[1] 宋刚[1] 何志嵩[1] 郝金瑞[1] 那彦群[1] 

机构地区：[1]北京大学第一医院泌尿外科 北京大学泌尿外科研究所,100034

出　　处：《中华医学杂志》2007年第6期404-408,共5页National Medical Journal of China

基　　金：国家自然科学基金资助项目(30371420、30672099).

摘　　要：目的利用 RNA 干扰(RNAi)技术,探讨核小体结合蛋白1(NSBP1)基因对人激素依赖前列腺癌细胞系 LNCaP 增殖的作用。方法设计并合成针对 NSBP1的4种小分子干扰 RNA(siRNA)(包含1种阴性对照),构建能抑制 NSBP1 mRNA 表达的重组 pSilencer 2.1-U6 neo 质粒,转染LNCaP 细胞。应用逆转录聚合酶链反应(RT-PCR)、Western 印迹实验检测不同质粒对 NSBP1表达的抑制效率,选取抑制效率最高的质粒转染 LNCaP 细胞,用四甲基偶氮唑盐(MTT)法测定细胞增殖活性,用流式细胞光度术检测细胞周期的变化。结果筛选出抑制效率最高的质粒 pSilencer-81(mRNA水平抑制80%,蛋白水平抑制85%),与阴性对照 pSilencer-Neg 质粒分别转染 LNCaP 细胞。转染60 h后,经过84 h、108 h,一直到132 h,LNCaP/81细胞 A 值(代表细胞活性)低于 LNCaP/Neg 细胞 A 值,差异均有统计学意义(t=4.501,4.282,5.229,4.759,均 P<0.05)。抑制率随时间延长而增加,在84h 有所降低,在108 h 达最大值30.2%。转染60 h 后,经过84 h,一直到108 h,LNCaP/81细胞的G_2M+S 期细胞百分率较 LNCaP/Neg 细胞的降低,差异均有统计学意义(t=3.705,3.887,8.220,均P<0.05)。结论针对 NSBP1的 siRNA 通过抑制前列腺癌 LNCaP 细胞中 NSBP1基因的表达,能明显抑制细胞的增殖,NSBP1可能参与前列腺癌细胞生长增殖过程。Objective To explore the role of nucleosomal binding protein 1 （ NSBP1 ） in the proliferation of human hormone-dependent prostate cancer cells by inhibiting its mRNA expression. Methods Four well-designed short-hairpin RNA （shRNA） targeting NSBP1, including a negative control shRNA, were synthesized and inserted into the pSilencer 2. 1-U6 neo plasmid. The pSilencer-81 plasmid was identified as the most efficient. The 4 recombinant plasmids, pSilencer-58, 81, 126, and Neg, were then transfected to human hormone-dependent prostate cancer cells of the line LNCaP, i. e. , LNCap/81, LNCap/ 58, LNCap/126, and LNCap/Neg cells. RT-PCR and Western blotting were used to detect the inhibitory efficiency of different plasmids. MTT method was used to detect cell viability and flow cytometry was used to observe cell cycle distribution. Results Western blotting showed that the protein expression of NSBP1 in the LNCap/81 cells was lower by 85% compared to that in the LNCap/Neg cells. Compared with the control cells, the A values of the LNCap/81 cells 60, 84, 108, and 132 hours after transfection were all significantly lower than those of the LNCap/Neg cells （ t = 4. 501, 4. 282, 5. 229, and 4. 759, all P 〈 0.05 ）, showing that the cell viability was reduced . The proportions of cells in the G2 M and S phases 60, 84, and 108 hours after transfection of the LNCap/81 cells were all significantly lower than those of the LNCap/Neg cells （ t = 3. 705, 3.887, and 8.220, all P 〈 0.05）. Conclusion The suppressed expression of NSBP1 in prostate cancer cells mediated by shRNA inhibits cell proliferation significantly, which indicates that NSBP1 may play an important role in the proliferation of prostate cancer cells.

关 键 词：前列腺肿瘤 载体蛋白质类 RNA 高速泳动族蛋白 

分 类 号：R737.25[医药卫生—肿瘤]