Methylation and Expression Analysis of POU4F2, HOXA9, RBM46, and TSGA10 Genes in Bladder Cancer Using Methyl-Sensitive Restriction Enzyme PCR (MSRE-PCR)

Shadi Mahdipour,1 Mohammad Hossein Modarressi,2 Mehrdad Noruzinia1 1Department of Medical Genetics, Faculty of Medicine, Tarbiat Modares University, Tehran, Iran; 2Department of Medical Genetics, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, IranCorrespondence: Mohammad Hossein Modarressi, Email [email protected] Mehrdad Noruzinia, Email [email protected]: Bladder cancer is a prevalent malignancy, with high recurrence rates for non-muscle invasive cases and significant progression risks. Traditional diagnostic methods, such as cystoscopy and urine cytology, are limited by their invasiveness and low sensitivity for detecting low-grade tumors, respectively. Advances in non-invasive diagnostics focus on biomarkers such as cfDNA and DNA methylation, offering promising tools for early detection.Patients and Methods: This study investigated methylation and expression changes in POU4F2, HOXA9, RBM46, and TSGA10 genes in bladder cancer. A total of 22 patients and 30 controls were enrolled, with urine and plasma samples collected for analysis. cfDNA and total RNA were extracted using commercial kits. Methylation was assessed via MSRE-PCR, and gene expression was evaluated with Real-Time PCR. Statistical analysis was performed using GraphPad Prism software, with an unpaired t-test and ANOVA to compare the differences between the cancer and control groups. A p-value of less than 0.05 was considered statistically significant.Results: Urine cfDNA from bladder cancer patients showed significantly higher promoter methylation of POU4F2, HOXA9, RBM46, and TSGA10 compared with controls (approximately 2.2-, 9.0-, 3.6- and 6.1-fold increases; all p < 0.0001), with smaller but still significant differences in plasma (p = 0.0005– 0.0278). ROC analysis of urine methylation yielded AUCs of 0.97 for POU4F2, 0.95 for HOXA9, 0.88 for RBM46 and 0.98 for TSGA10 (sensitivities 95– 100%, specificities 80– 100%; all p < 0.0001). A PCA-derived composite methylation score further improved discrimination (AUC = 0.993, 95% CI 0.974– 1.000; sensitivity 100%, specificity 95%; p < 0.001). Consistently, gene expression analysis in urine showed significant downregulation of POU4F2 and HOXA9 (≈7- and 9-fold decreases; p = 0.0099 and p = 0.025) and upregulation of RBM46 and TSGA10 (≈2.5- and 2.3-fold increases; p = 0.0037 and p = 0.0114) in patients versus controls.Conclusion: The results indicate that these methylation and expression profiles can be used as non-invasive biomarkers for the early diagnosis of bladder cancer. The use of these methods provides both greater sensitivity and accuracy than traditional methods and can pave the way for the development of more effective screening tests.Keywords: bladder cancer, DNA methylation, non-invasive biomarkers, cell-free DNA, PCR-based methods