Study of SCNN۱A and SCNN۱G DNA Methylation at Blood Leukocytes as Potential Epigenetic Biomarkers for Bladder Urotherial Carcinoma (BLCA)Diagnosis

Introduction Bladder urothelial carcinoma (BLCA), the fourth most prevalent malignancy in males and the biggest proportion of women, is a heterogeneous cancer that can be characterized as aggressive or invasive, with a high condition-specific mortality rate. Aggressive bladder cancer tends to recur, necessitating long-term invasive surveillance. Individual BLCA diagnoses vary greatly, resulting in large differences in outcomes even among people with similar disease severity. Unlike traditional tissue biopsies, liquid biopsies allow for the non-invasive, real-time assessment of entire cancer profiles. The epithelial sodium channel (ENaC), which is responsible for Na+ reabsorption in the epithelium, is made up of two subunits: SCNN۱A and SCNN۱G. These subunit-encoding genes have been associated to the progression of a variety of malignancies, including BLCA. Recent discoveries in cancer epigenetics have found biomarkers that can aid in early disease detection and surveillance.Methylation of SCNN۱A and SCNN۱G promoters is a promising biomarker. In this investigation, we hypothesized that methylation levels of the SCNN۱A and SCNN۱G DNA promoter CpG sites in peripheral blood leukocytes might be used as PCa biomarkers. Materials and Methods DNA was extracted from blood samples of ۳۳۰ individuals, including ۲۰۰ patients with BLCA and ۱۳۰ healthy controls. We utilized high-throughput sequencing DNA methylation data from TCGA and GEO, focusing on two datasets, GSE۳۷۸۱۷ platform GPL۶۱۰۲ and GSE۳۷۸۱۷, for analyzing human bladder tumors and normal tissues. This study investigated the relationship between SCNN۱A and SCNN۱G DNA promoter (chr۱۲:۶۳۶۴۱۰۱–۶۳۶۴۱۹۲ and chr۱۲:۲۳۱۸۳۳۹۰-۲۳۱۸۳۴۸۱) CpG site methylation levels in blood leukocytes of BLCA patients and healthy controls to evaluate potential BLCA risk diagnosis. MSRE-PCR and Real-time-MSRE-qPCR were employed to assess the methylation levels of the SCNN۱A and SCNN۱G promoter CpG sites in the promoter region. The TIMER database was used to examine the correlation between these genes and immune cell infiltration. Results Methylation levels at the SCNN۱A CpG site were found to be ۸۳.۱۷ ± ۱۱.۵۸% in BLCA samples compared to ۰.۵ ± ۲.۴% in healthy control samples. The SCNN۱G CpG site displayed similar patterns, with methylation levels of ۷۱.۷ ± ۱۲.۷۷% in BLCA samples and ۰.۳ ± ۱.۴% in healthy control samples. These disparities between BLCA and healthy samples were statistically significant (P <۰.۰۰۰۱). ROC curve analysis indicated that SCNN۱A CpG methylation levels in patients with BLCA, when compared to healthy samples, exhibited ۱۰۰% sensitivity, ۹۸.۳% specificity, and ۹۵.۶% sensitivity at a threshold of <۴۳%. For the SCNN۱G, the analysis revealed ۸۸% sensitivity, ۷۷% specificity, and ۶۸.۸% sensitivity at a threshold of <۳۶%. An assessment of the relationship between SCNN۱A and SCNN۱G and the established clinical criteria for BLCA diagnosis, tumor grade, and stage showed positive correlations for both genes (p < ۰.۰۰۰۱) , with SCNN۱A demonstrating a stronger correlation than SCNN۱G. Furthermore, SCNN۱A showed a notable correlation with B cell, macrophage, and dendritic cell infiltration, while correlations for SCNN۱G were less prominent. Conclusion The hypermethylation of CpG islands in the promoter regions of SCNN۱A and SCNN۱G shows promise as a blood-based biomarker for the early diagnosis and screening of bladder urothelial carcinoma (BLCA), providing a non-invasive diagnostic method.SCNN۱A has a significant role in immune-cells-infiltration.

کلیدواژه ها:

Bladder Urotherial Carcinoma (BLCA) ، Methylation ، SCNN۱A ، SCNN۱G ، Blood Biomarker