Sp1 and Sp3 transcription factors upregulate the proximal promoter of the human prostate-specific antigen gene in prostate cancer cells

The serum level of prostate-specific antigen (PSA) is useful as a clinical marker for diagnosis and assessment of the progression of prostate cancer, and in evaluating the effectiveness of treatment. We characterized four Sp1/Sp3 binding sites in the proximal promoter of the PSA gene. In a luciferase assay, these sites contributed to the basal promoter activity in prostate cancer cells. In an electrophoretic mobility shift assay and chromatin immunoprecipitation assay, we confirmed that Sp1 and Sp3 bind to these sites. Overexpression of wild-type Sp1 and Sp3 further upregulated the promoter activity, whereas overexpression of the Sp1 dominant-negative form or addition of mithramycin A significantly reduced the promoter activity and the endogenous mRNA level of PSA. Among the four binding sites, a GC box located at nucleotides -53 to -48 was especially critical for basal promoter activity. These results indicate that Sp1 and Sp3 are involved in the basal expression of PSA in prostate cancer cells.     

人LOXL2启动子的鉴定与初步分析

LOXL2（lysyl oxidase like 2）是赖氨酰氧化酶（LOX）家族的一个重要成员,不仅可促进细胞外基质中胶原蛋白和弹性蛋白的交联,而且在转录调控、细胞信号转导以及细胞粘附等生物学过程中也有重要作用。多篇研究表明,LOXL2在多种肿瘤中高表达,且与多种肿瘤细胞的增殖迁移等生物学行为密切相关。LOXL2的表达调控机制目前仍不清楚。为了进一步研究LOXL2的转录调控机制,本研究克隆鉴定了LOXL2的启动子。首先通过数据库对LOXL2基因结构及潜在启动子区域进行了分析,进而以人的基因组DNA为模板,通过PCR定向克隆策略,构建了5个长度不同并覆盖LOXL2基因转录起始位点附近约1.7 kb的LOXL2基因启动子荧光素酶报告基因重组体。启动子活性分析结果表明,与对照组相比,5个重组体均具有启动子活性(P<0.05),提示LOXL2基因核心启动子定位于转录起始位点附近约185 bp的区域内。转录因子结合位点分析结果表明,LOXL2基因启动子缺乏典型的TATA盒,但含有GC盒以及Sp1、NFkB等潜在的转录因子结合位点。外源转染Sp1表达质粒能显著增强LOXL2基因启动子的活性（P<0.05）,提示Sp1能直接激活LOXL2的转录。     

抑瘤基因NGX6启动子的克隆与功能鉴定

NGX6是一个结直肠癌候选抑瘤基因,其转录调控机制不明．采用生物信息学技术预测其启动子区,并构建NGX6启动子荧光素酶报告基因重组体pGL3/Enhancer/1126．荧光素酶活性检测结果表明该区域具有强启动子活性．应用PromoterInspector program,FistEF,CpGplot和MatInspector Professional软件分析发现,NGX6基因转录调控区为一个不含TATA盒,而含有CAAT盒的GC富集区．凝胶迁移阻滞实验确定NGX6基因启动子区域具有Sp1特异性结合位点,Sp1特异性阻断剂光神霉素(mithramycin A)能明显抑制NGX6启动子的活性和NGX6基因的表达;封闭内源性Sp1能下调NGX6基因mRNA表达水平．