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

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60h后,经过84h、108h,一直到132h,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108h达最大值30．2％。转染60h后,经过84h,一直到108h,LNCaP／81细胞的G2M＋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。

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

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 G(2)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.