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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 investigate the effect of nucleosome binding protein-1 (NSBP1) gene on the proliferation of human hormone-dependent prostate cancer cell line LNCaP by using RNA interference (RNAi) technique. Methods Four small interfering RNAs (siRNAs) targeting NSBP1 (including one negative control) were designed and synthesized. Recombinant pSilencer 2.1-U6 neo plasmid was constructed and transfected into LNCaP cells. The inhibitory efficiency of different plasmids on the expression of NSBP1 was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. LNCaP cells were transfected with plasmids with the highest inhibitory efficiency and then assayed by MTT assay Cell proliferation activity was measured by flow cytometry cell cycle changes. Results The plasmid pSilencer-81 (80% inhibition and 85% inhibition) of the plasmid pSilencer-Neg was selected and transfected into LNCaP cells with negative control pSilencer-Neg. After 60 h of transfection, the A value (representing cell viability) of LNCaP / 81 cells was lower than that of LNCaP / Neg cells after 84 h, 108 h and 132 h (t = 4.501, 4.82 , 5.229,4.759, all P <0.05). The inhibition rate increased with time and decreased at 84h, reaching a maximum of 30.2% at 108h. After 60 h of transfection, the percentage of cells in G 2 M + S phase of LNCaP / 81 cells was significantly lower than that of LNCaP / Neg cells after 84 h and 108 h (t = 3.705, 3.887 and 8.220, both P <0.05). Conclusion NSBP1 siRNA can inhibit the proliferation of prostate cancer cell line LNCaP by inhibiting the expression of NSBP1 gene. NSBP1 may be involved in the growth and proliferation of prostate cancer cells.