Ogishima Tatsuya, Shiina Hiroaki, Breault Julia E, Terashima Masaharu, Honda Satoshi, Enokida Hideki, Urakami Shinji, Tokizane Takashi, Kawakami Toshifumi, Ribeiro-Filho Leopoldo A, Fujime Makoto, Kane Christopher J, Carroll Peter R, Igawa Mikio, Dahiya Rajvir
Department of Urology, Veterans Affairs Medical Center, San Francisco and University of California, San Francisco, 4150 Clement Street, CA 94121, USA.
Oncogene. 2005 Oct 13;24(45):6765-72. doi: 10.1038/sj.onc.1208811.
Heparanase plays a critical role in the degradation of extracellular matrix and cell membrane and is frequently upregulated in malignant tumors. Transcription factor, early growth response 1 (EGR1), is closely associated with inducible transcription of the heparanase gene. We hypothesized that promoter CpG hypomethylation with increased EGR1 expression could determine heparanase expression during the pathogenesis of bladder cancer. Bladder cancer cell lines (J82, T24 and transitional cell carcinoma) significantly restored heparanase expression after 5-Aza-dC treatment. Transfection of EGR1 siRNA with T24 bladder cancer cell line significantly downregulated heparanase expression compared to the control siRNA transfection. In 54 bladder cancer and paired normal bladder samples, heparanase expression was significantly higher in bladder cancer than in normal bladder (P<0.01). We performed methylation-specific PCR targeting the CpG sites within the core-binding consensus motifs of EGR1 (GGCG) and Sp1 (GGGCGG). Methylation prevalence was significantly higher in normal bladder than in bladder cancer (P<0.05) and inversely correlated with heparanase expression (P=0.055). In the total series of bladder cancer and normal bladder samples, the combination of promoter CpG methylation and EGR1 expression regulated heparanase expression in a stepwise manner, where heparanase expression was the lowest in methylation-positive and EGR1-negative samples and the highest in methylation-negative and EGR1-positive samples. To our knowledge, this is the first study demonstrating that increased heparanase expression during the pathogenesis of bladder cancer is due to promoter hypomethylation and transcription factor EGR1.
乙酰肝素酶在细胞外基质和细胞膜的降解中起关键作用，且在恶性肿瘤中常上调表达。转录因子早期生长反应因子1（EGR1）与乙酰肝素酶基因的诱导转录密切相关。我们推测，启动子CpG低甲基化伴EGR1表达增加可能在膀胱癌发病机制中决定乙酰肝素酶的表达。膀胱癌细胞系（J82、T24和移行细胞癌）经5-氮杂-2'-脱氧胞苷（5-Aza-dC）处理后，乙酰肝素酶表达显著恢复。与对照小干扰RNA（siRNA）转染相比，用EGR1 siRNA转染T24膀胱癌细胞系可显著下调乙酰肝素酶表达。在54例膀胱癌及配对的正常膀胱组织样本中，膀胱癌中乙酰肝素酶的表达显著高于正常膀胱组织（P<0.01）。我们针对EGR1（GGCG）和Sp1（GGGCGG）核心结合共有基序内的CpG位点进行了甲基化特异性PCR。正常膀胱组织中的甲基化发生率显著高于膀胱癌（P<0.05），且与乙酰肝素酶表达呈负相关（P=0.055）。在整个膀胱癌和正常膀胱组织样本系列中，启动子CpG甲基化和EGR1表达的组合以逐步方式调节乙酰肝素酶表达，其中甲基化阳性和EGR1阴性样本中乙酰肝素酶表达最低，甲基化阴性和EGR1阳性样本中乙酰肝素酶表达最高。据我们所知，这是第一项证明膀胱癌发病机制中乙酰肝素酶表达增加是由于启动子低甲基化和转录因子EGR1所致的研究。
