We analyzed the relationship between CALN1 methylation percentage and clinicopathological data of bladder cancer patients. We found that there exists a significant association between a lower CALN1 methylation percentage and advanced tumor stage and histologically grade and a significantly increased risk of intravesical recurrence. To the best of our knowledge, this is the first study to show that CALN1 methylation percentage is associated with the clinicopathological features and prognosis of bladder cancer.
DNA methylation is considered to be an epigenetic modification, and its association with many biological phenomena, including carcinogenesis, has been established [13, 14]. Methylation analyses have also found some information (e.g., drug susceptibility [15], prognosis prediction, and risk [16]) that cannot be obtained with conventional test data.
Cao et al. used microarray analysis to show that calcium signal transduction was associated with the development of bladder cancer via the mitogen-activated protein kinase pathway [17]. We hypothesized that the regulation of calcium signal transduction through methylation of CALN1 was involved in the development and progression of bladder cancer. In addition, intron 2 of CALN1 is a DNase I hypersensitive site that is strongly associated with transcriptional activity [18]. Therefore, we suspected that CALN1 methylation was involved in the action of a DNase I hypersensitive site and, as a result, may affect the expression of CALN1. Regarding the relationship between bladder cancer and methylation, various analytical reports have centered on CpG sites [19, 20], and testing systems such as Bladder EpiCheck [21] have been established. Although various trials have been conducted regarding the diagnosis and treatment of bladder cancer, methylation analysis of CALN1 and its association with bladder cancer has not been probed before.
Bisulfite sequencing is widely used for methylation analyses. In this study, we performed methylation analysis using methylation-sensitive restriction enzyme (MSRE)-qPCR. This technique enables the analysis of a small amount of sample obtained by TURBT without bisulfite treatment. Bisulfite treatment involves mixing the corrosive chemical bisulfite salt with DNA and heating it to 50–70 ℃. It is known that DNA is cleaved in this process, and the yield is extremely low. Recently, high-yield methods have been developed, but fragmentation has not been completely avoided [22]. In addition, because bisulfite sequencing requires a large number of cells, it is not feasible when a small amount of DNA is available from clinical specimens, such as cell-free DNA and circulating tumor cells. In contrast, one of the advantages of MSRE-qPCR is the side-by-side comparison between control and experimental samples, even for very low amounts of DNA. In addition, MSRE-qPCR can be completed in less time than other methods with the same level of accuracy [23]. Comprehensive analysis using next-generation sequencing is also useful but less practical owing to high costs. MSRE-qPCR is useful for targeted analysis owing to its simple workflow. Further investigation exploring this diagnostic method with high sensitivity and specificity in combination with other diagnostic markers is necessary and will contribute to the development of new diagnostic systems for bladder cancer.
The current study has some limitations. First, there were no criteria for intravesical BCG immunotherapy, though there was no difference in BCG therapy between the low- and high-methylation groups. Second, the sample size was small and the follow-up period was short. Therefore, the findings of this study need to be validated in a larger study.