Molecular Role Of DNA Methylation-mediated Epigenetic Silencing Of The MAD1L1 Gene In Colorectal Carcinogenesis

Background and objectivesColorectal cancer is one of the most common malignant tumors in the world,ranking second in cancer incidence and mortality.Currently,the screening methods for early colorectal cancer are limited,and the sensitivity and specificity for the diagnosis of early colorectal cancer are still insufficient.During the occurrence and development of colorectal cancer,DNA methylation,as an epigenetic modification,is closely related to the occurrence and development of cancer,but its related mechanism has not been fully clarified,which has broad research potential and value.This study intends to screen markers through DNA methylation sequencing combined with bioinformatics analysis,and conduct in vitro cell verification and ATAC-seq combined with transcriptomics and bioinformatics analysis to further study the mechanism involved in the regulation of the development of colorectal cancer.Methods1.This study utilized methylation data from the Cancer Genome Atlas(TCGA)database to analyze colorectal cancer and paracancerous tissues.Additionally,15 pairs of Chinese population precancerous lesions,colorectal cancer,and paracancerous tissue samples were used to detect DNA methylation and perform differential methylation analysis.This process involved screening for differential sites and designing probes.2.In this study,DNA methylation was detected in 39 pairs of precancerous lesions,colorectal cancer,and paracancerous tissues using the appeal probe as the target.Differential analysis combined with bioinformatics analysis was used to screen out differential sites.The expression of candidate genes was detected in colorectal cancer cells treated with methylase inhibitors,and DNA methylation candidate genes were further screened.Using assay for Transposase Accessible Chromatin with high-throughput sequencing(ATAC-seq)combined with transcriptome sequencing,prediction of transcription factor binding sites,methylsensitive transcription factors were indentified.3.This study aims to create colorectal cancer cell lines HCT116 and HT29 that stably overexpress/interfere MAD1L1,and evaluate the effects of MAD1L1 overexpression/interference on the proliferation and migration of colorectal cancer cell lines using MTT assay,clone formation assay,Transwell assay,and scratch assay to evaluate their behavioral impact.4.In this study,we conducted cluster analysis of the biological functions of MAD1L1 protein and signaling pathways regulated by MAD1L1.Additionally,we explored the molecular mechanism of MAD1L1 in regulating the occurrence and development of colorectal cancer through in vitro experiments.Results1.Based on TCGA-derived colorectal cancer methylation data and methylsequencing results of tissue samples,2731 different methylation sites were examined,and there was a correlation between methylation level and functional genes between cancer and paracancerous tissues.Probes were specifically designed to target differential sites,and the methylation sequencing results of 39 pairs of precancerous lesions,colorectal cancer,and para-cancerous tissue were analyzed.As a result,11 genes(including 69 differential sites)were identified.Following treatment for methyltransferase inhibitors,the relative mRNA expression levels of MAD1L1,ANGPT2,and SEPTIN9 significantly increased(p<0.05).The results of ATAC combined transcriptome sequencing revealed that the MAD1L1-regulated potential transcription factors mainly include Sp1(Zf),Elk4(ETS),and Fli1(ETS).2.The findings from various experiments including MTT,clony formation,transwell,and scratch experiments indicate that MAD1L1 overexpression in colorectal cancer cell lines can effectively restrain their proliferation,migration,and other biological activities.Conversely,interfering with MAD1L1 expression led to the opposite outcome.3.The analysis of gene ontology(GO)showed that MAD1L1 regulates proteins primarily located in the cytoplasm and involved in regulating cell biological processes.Moreover,the KEGG pathway analysis revealed that MAD1L1 is mainly enriched in the cell cycle pathway.The flow cytometry results indicated that G2/M phase was arrested on cell cycle by the overexpression of MAD1L1 to inhibiting carcinomatosis.ConclusionAccording to DNA methylation sequencing,there is a significant difference in the methylation level of the MAD1L1gene in colorectal cancer tissues.The MAD1L1 gene is regulated by DNA methylation and overexpression of MAD1L1 can inhibit biological behaviors such as proliferation and migration of colorectal cancer cells.Furthermore,methyl-sensitive transcription factors and regulatory pathways have been identified.In summary,the study explored DNA methylationmediated epigenetic silencing of the MAD1L1 gene can promote colorectal carcinogenesis,indicating that MAD1L1,as a potential biomarker of colorectal cancer,play an important role in colorectal carcinogenesis.