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目的:研究9-顺维甲酸对前列腺癌细胞系 LNCaP 中同源盒基因 NKX3.1表达的调节作用。方法:采用流式细胞术、反转录 PCR 和 Western Blot 技术检测9-顺维甲酸对 LNCaP 细胞周期及 NKX3.1表达的影响;构建和转染 NKX3.1启动子-报告基因质粒及其缺失突变体,通过报告基因活性测定,鉴定 NKX3.1启动子中受9顺-维甲酸调控的区域。结果:通过转染及报告基因检测,发现9-顺维甲酸在 LNCaP 细胞中可明显提高 NKX3.1启动子的活性:RT-PCR 和 Western Blot 结果显示,9-顺维甲酸可提高 NKX3.1mRNA 和蛋白的表达,并呈剂量依赖性;通过启动子缺失突变分析,发现 NKX3.1基因上游-936至-921区明显受9-顺维甲酸诱导调节;流式细胞术分析细胞周期的结果显示,9-顺维甲酸可阻止 LNCaP 细胞于 G_1期,减少 G_2/M 期细胞。结论:9-顺维甲酸作为诱导分化剂可阻止 LNCaP 细胞于 G_1期,减少细胞有丝分裂,并明显上调前列腺特异的抑癌基因 NKX3.1的表达。
OBJECTIVE: To investigate the regulatory effect of 9-cis-retinoic acid on the expression of NKX3.1 gene in prostate cancer cell line LNCaP. Methods: The effects of 9-cis-retinoic acid on the cell cycle and the expression of NKX3.1 in LNCaP cells were detected by flow cytometry, reverse transcription PCR and Western Blot. The NKX3.1 promoter-reporter plasmids and their deletion were constructed and transfected Mutants, identified by reporter gene activity, identified the cis-retinoic acid-regulated region of the NKX3.1 promoter. Results: 9-cis-retinoic acid could significantly enhance the activity of NKX3.1 promoter in LNCaP cells by transfection and reporter gene assay. The results of RT-PCR and Western Blot showed that 9-cis-retinoic acid increased NKX3.1 mRNA And protein expression in a dose-dependent manner. By the promoter deletion mutation analysis, it was found that the -936 to -921 region of NKX3.1 gene was significantly regulated by 9-cis-retinoic acid. The results of flow cytometry analysis of cell cycle showed 9-cis-retinoic acid can prevent LNCaP cells from G_1 phase and reduce G_2 / M phase cells. CONCLUSION: 9-cis-retinoic acid can prevent LNCaP cells from G 1 phase, decrease cell mitosis and upregulate the expression of prostate specific anti-oncogene NKX3.1 as an inducer of differentiation.