Despite numerous observations regarding the relationship between DNA methylation changes and cancer progression, only a few genes have been verified as diagnostic biomarkers of colorectal cancer (CRC). To more practically detect methylation changes, we performed targeted bisulfite sequencing. Through co-analysis of RNA-seq, we identified cohort-specific DNA methylation markers: CpG islands of the intragenic regions of PDX1, EN2, and MSX1. We validated that these genes have oncogenic features in CRC and that their expression levels are increased in correlation with the hypermethylation of intragenic regions. The reliable depth of the targeted bisulfite sequencing data enabled us to design highly optimized quantitative methylation-specific PCR primer sets that can successfully detect subtle changes in the methylation levels of candidate regions. Furthermore, these methylation levels can divide CRC patients into two groups denoting good and poor prognoses. In this study, we present a streamlined workflow for screening clinically significant differentially methylated regions. Our discovery of methylation markers in the PDX1, EN2, and MSX1 genes suggests their promising performance as prognostic markers and their clinical application in CRC patients.
Bibliographical noteFunding Information:
We thank Prof. Sungsoon Fang and Dr. Hwang-Phil Kim for providing the colorectal cancer cell lines, Prof. Kyung Hyun Yoo for providing the dCas9-tet1 construct, Ms. Hyeran Shim for technical advice, and all the other members of Prof. Lark Kyun Kim’s laboratory for technical help. This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science and ICT (NRF-2016M3A9B6026918) and by the Ministry of Education (NRF-2016R1D1A1B01015292). This work was supported by the Collaborative Genome Program for Fostering New Post-Genome Industry through the National Research Foundation of Korea (NRF), funded by the Ministry of Science and ICT (NRF-2016M3C9A4921712). This work was also supported by a faculty research grant from Yonsei University College of Medicine (6-2020-0100).
© 2022, The Author(s).
ASJC Scopus subject areas
- Molecular Medicine
- Molecular Biology
- Clinical Biochemistry