Gene Expression, DNA Methylation and Prognostic Significance of DNA Repair Genes in Human Bladder Cancer

Abstract

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. Methods: 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. Results: 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). Conclusion: 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.

Prognostic Implication of Urothelial Stem Cell Markers Differs According to Primary Tumour Location in Non-Muscle-Invasive Bladder Cancer

“…Total RNA from bladder cancer and paired normal bladder tissues was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions [21]. The extracted RNA was pretreated with RNase-free DNase, and 2μg RNA from each sample was used for cDNA synthesis with random hexamers.…”

The polymorphic variant rs1800734 influences methylation acquisition and allele-specific TFAP4 binding in the MLH1 promoter leading to differential mRNA expression

“…While the functional role of rs1800734 in the pathways of CRC development is becoming clearer, it is also interesting to note how readily the accumulation of MLH1 promoter methylation could be reversed resulting in re-expression of MLH1. The importance of MLH1 promoter methylation in other cancer types is less well understood however it is frequently found in endometrial 22 , and gastric 23 ) cancers, as well as lung 24 , bladder 25 , and some haematological malignancies 26 . Drugs such as Azacitidine that inhibit DNA methylation are already approved for the treatment of some cancers.…”

Epigenetic Silencing of the MLH1 Promoter in Relation to the Development of Gastric Cancer and its use as a Biomarker for Patients with Microsatellite Instability: a Systematic Analysis

“…The absence or downregulation of MLH1 gene expression via promoter methylation was reported in GC [15, 32-34, 36, 37, 39, 42, 44-46, 50, 56, 59, 60, 62, 64, 72, 76, 77]. MLH1 promoter methylation is detected in some cancers, such as bladder cancer [86], colorectal cancer [87] and GC [18]. However, some studies reported that MLH1 promoter methylation exhibited a low frequency in GC [18, 34, 43, 44, 57, 65, 67, 70, 75, 78, 81].…”