鉴定膀胱癌中肝细胞黏附分子基因启动子区的高甲基化。
Identification of hypermethylation in hepatocyte cell adhesion molecule gene promoter region in bladder carcinoma.
机构信息
1. Department of Laboratory Diagnosis, Chongqing Medical University, Chongqing, 400016 China;
出版信息
Int J Med Sci. 2013 Nov 11;10(13):1860-7. doi: 10.7150/ijms.6460. eCollection 2013.
BACKGROUND
Epigenetic regulation such as aberrant hypermethylation of CpG islands in promoter plays a key role in tumorigenesis. 5-Aza-2'-deoxycytidine (5-aza-CdR) which is a potent inhibitor of DNA methylation can reverse the abnormal hypermethylation of the silenced tumor suppressor genes (TSGs). It has been reported that hepatocyte cell adhesion molecule (hepaCAM) acts as a tumor suppressor gene and expression of its mRNA and protein were down-regulated in bladder cancer. Over-expression of hepaCAM can inhibit cancer growth and arrest renal cancer cells at G0/G1 phase. In this study, we investigated the methylation status of hepaCAM gene, as well as the influence of 5-aza-CdR on expression of hepaCAM gene in bladder cancer cells.
METHODS
CpG islands in hepaCAM promoter and methprimers were predicted and designed using bioinformatics program. Methylation status of hepaCAM promoter was evaluated in bladder cancer tissues and two cell lines (T24 and BIU-87) by Methylation-specific PCR; Western blot and Immunofluorescence were used to detect expression of hepaCAM protein after 5-aza-CdR treatment; Flow cytometry assay was performed to determine effectiveness of 5-aza-CdR on cell cycle profile.
RESULTS
CpG island in promoter of hepaCAM gene was hyper-methylated both in bladder carcinoma tissues and cell lines (T24 and BIU-87). Otherwise, aberrant methylation of its promoter was associated with its decreased expression. Hypermethylation of hepaCAM gene was reversed and expression of its mRNA and protein were re-activated in two cell lines by DNA methyltransferases inhibitor 5-aza-CdR. Flow cytometry assay demonstrated that 5-aza-CdR can inhibit growth of cancer cells by arresting cancer cells at G0/G1 phase.
CONCLUSION
Abnormal hypermethylation in CpG island of hepaCAM promoter is involved in absence of hepaCAM gene expression when bladder cancer occurs. Re-activation of hepaCAM gene by 5-aza-CdR can inhibit growth of cancer cells and arrest cells at G0/G1 phase.
背景
表观遗传调控，如启动子中 CpG 岛的异常高甲基化，在肿瘤发生中起着关键作用。5-氮杂-2'-脱氧胞苷（5-aza-CdR）是一种有效的 DNA 甲基化抑制剂，可逆转沉默的肿瘤抑制基因（TSGs）的异常高甲基化。据报道，肝细胞黏附分子（hepaCAM）作为一种肿瘤抑制基因，其 mRNA 和蛋白的表达在膀胱癌中下调。hepaCAM 的过表达可以抑制肿瘤生长，并使肾癌细胞停滞在 G0/G1 期。在这项研究中，我们研究了 hepaCAM 基因的甲基化状态，以及 5-aza-CdR 对膀胱癌细胞中 hepaCAM 基因表达的影响。
方法
使用生物信息学程序预测和设计了 hepaCAM 启动子中的 CpG 岛和 MethPrimers。通过甲基化特异性 PCR 评估膀胱癌组织和两个细胞系（T24 和 BIU-87）中 hepaCAM 启动子的甲基化状态；Western blot 和免疫荧光检测 5-aza-CdR 处理后 hepaCAM 蛋白的表达；流式细胞术检测 5-aza-CdR 对细胞周期谱的作用。
结果
hepaCAM 基因启动子中的 CpG 岛在膀胱癌组织和细胞系（T24 和 BIU-87）中均呈高甲基化状态。此外，其启动子的异常甲基化与其表达降低有关。DNA 甲基转移酶抑制剂 5-aza-CdR 可逆转 hepaCAM 基因的高甲基化，并使两个细胞系中其 mRNA 和蛋白的表达重新激活。流式细胞术分析表明，5-aza-CdR 可通过将癌细胞阻滞在 G0/G1 期来抑制癌细胞的生长。
结论
膀胱癌发生时，hepaCAM 启动子 CpG 岛的异常高甲基化导致 hepaCAM 基因表达缺失。5-aza-CdR 对 hepaCAM 基因的重新激活可抑制癌细胞的生长并将细胞阻滞在 G0/G1 期。
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