Wojtczyk-Miaskowska Anita, Presler Malgorzata, Michajlowski Jerzy, Matuszewski Marcin, Schlichtholz Beata
Department of Biochemistry, Medical University of Gdansk, Gdansk, Poland.
Department of Urology, Medical University of Gdansk, Gdansk, Poland.
Cell Physiol Biochem. 2017;42(6):2404-2417. doi: 10.1159/000480182. Epub 2017 Aug 21.
BACKGROUND/AIMS: This study investigated the gene expression and DNA methylation of selected DNA repair genes (MBD4, TDG, MLH1, MLH3) and DNMT1 in human bladder cancer in the context of pathophysiological and prognostic significance.
To determine the relationship between the gene expression pattern, global methylation and promoter methylation status, we performed real-time PCR to quantify the mRNA of selected genes in 50 samples of bladder cancer and adjacent non-cancerous tissue. The methylation status was analyzed by methylation-specific polymerase chain reaction (MSP) or digestion of genomic DNA with a methylation-sensitive restriction enzyme and PCR with gene-specific primers (MSRE-PCR). The global DNA methylation level was measured using the antibody-based 5-mC detection method.
The relative levels of mRNA for MBD4, MLH3, and MLH1 were decreased in 28% (14/50), 34% (17/50) and 36% (18/50) of tumor samples, respectively. The MBD4 mRNA expression was decreased in 46% of non-muscle invasive tumors (Ta/T1) compared with 11% found in muscle invasive tumors (T2-T4) (P<0.003). Analysis of mRNA expression for TDG did not show any significant differences between Ta/T1 and T2-T4 tumors. The frequency of increased DNMT1 mRNA expression was higher in T2-T4 (52%) comparing to Ta/T1 (16%). The overall methylation rates in tumor tissue were 18% for MBD4, 25% for MLH1 and there was no evidence of MLH3 promoter methylation. High grade tumors had significantly lower levels of global DNA methylation (P=0.04). There was a significant association between shorter survival and increased expression of DNMT1 mRNA (P=0.002), decreased expression of MLH1 mRNA (P=0.032) and the presence of MLH1 promoter methylation (P=0.006).
This study highlights the importance of DNA repair pathways and provides the first evidence of the role of MBD4 and MLH3 in bladder cancer. In addition, our findings suggest that DNMT1 mRNA and MLH1 mRNA expression, as well as the status of MLH1 promoter methylation, are attractive prognostic markers in this pathology.
背景/目的：本研究在病理生理和预后意义的背景下，调查了人膀胱癌中选定的DNA修复基因（MBD4、TDG、MLH1、MLH3）和DNMT1的基因表达及DNA甲基化情况。
为了确定基因表达模式、整体甲基化和启动子甲基化状态之间的关系，我们进行了实时PCR，以量化50份膀胱癌样本及相邻非癌组织中选定基因的mRNA。甲基化状态通过甲基化特异性聚合酶链反应（MSP）或用甲基化敏感限制性内切酶消化基因组DNA并使用基因特异性引物进行PCR（MSRE-PCR）进行分析。使用基于抗体的5-mC检测方法测量整体DNA甲基化水平。
肿瘤样本中MBD4、MLH3和MLH1的mRNA相对水平分别在28%（14/50）、34%（17/50）和36%（18/50）的样本中降低。与肌肉浸润性肿瘤（T2-T4）中11%的样本相比，46%的非肌肉浸润性肿瘤（Ta/T1）中MBD4 mRNA表达降低（P<0.003）。TDG mRNA表达分析未显示Ta/T1和T2-T4肿瘤之间有任何显著差异。与Ta/T1（16%）相比，T2-T4中DNMT1 mRNA表达增加的频率更高（52%）。肿瘤组织中MBD4的总体甲基化率为18%，MLH1为25%，且没有证据表明MLH3启动子甲基化。高级别肿瘤的整体DNA甲基化水平显著较低（P=0.04）。DNMT1 mRNA表达增加、MLH1 mRNA表达降低以及MLH1启动子甲基化的存在与较短生存期之间存在显著关联（P=0.002、P=0.032和P=0.006）。
本研究突出了DNA修复途径的重要性，并首次提供了MBD4和MLH3在膀胱癌中作用的证据。此外，我们的研究结果表明，DNMT1 mRNA和MLH1 mRNA表达以及MLH1启动子甲基化状态是这种病理情况下有吸引力的预后标志物。
