Preliminary Study On Change Law And Regulatory Pathway Of CpG Island Methylator Phenotype In Colorectal Cancer

Colorectal cancer(CRC)is one of the most prevalent malignant tumors around the world,whose incidence and mortality ranked the third and fifth in malignant tumors,respectively,and with an increasing trend year by year.Cp G island methylation is one of the important epigenetic events in the development of colorectal cancer,especially the functional inactivation by Cp G island methylation of key tumor suppressor genes and mismatch repair gene promoter regions is the key link in triggering tumors.Cp G island methylation phenotype,CIMP,refers a high proportion of genes in the tumor genome undergo DNA methylation in the promoter region,of which CIMPH is a characteristic molecular marker of some colorectal cancers;however,its molecular pathological mechanism of CIMP remains unclear.In this study,200 cases of colorectal surgical resection or biopsy samples were collected first,and the CIMP status of normal colorectal mucosal tissues,adenoma and adenocarcinoma tissues was analyzed with the Methylight method based on fluorescence quantitative PCR technique to explore the changes of CIMP during the development of CRC.Findings disclosed that there was a significant difference in the proportion of CIMPH,CIMP-L and CIMP-0 between normal colorectal mucosa,adenoma and adenocarcinoma(P<0.01).With the occurrence and development of tumors,the proportion of CIMP-H is increasing and the proportion of CIMP-0 is gradually decreasing,indicating that the dynamic changes of CIMP may play a significant role in the occurrence and development of tumors,indicating that the dynamic changes of CIMP may play an important role in the process;this study also analyzed the differences in the methylation of tumor tissues and adjacent tissues,and found that the changes trends of CIMP between adjacent tissues and tumor tissues were positively correlated(P<0.01).as well as exposed that when the CIMP level in adjacent tissues exceeded that in tumor tissues,the lymph node metastasis rate of tumor tissues would also be increased(P<0.05),indicating that the CIMP status of tumor tissues may be correlated with lymph node metastasis.To further explore the molecular pathological mechanism between CIMP and the occurrence and development of colorectal cancer,we analyzed the mutations of 62 tumor genes in 40 cases of colorectal adenocarcinoma tissues by NGS technique to elucidate the relationship between CIMP and tumor gene mutations,and found that the number of methylated genes in CIMP indicators was positively correlated with the number of tumor gene mutations(P<0.05).Meanwhile,we also found that: DNA methyltransferase 1(DNMT1)and DNA methyltransferase 3A(DNMT3A),as important catalytic enzymes in the process of methylation occurrence,had significantly changed in the expression level after lymph node metastasis of tumorigenesis: DNMT1 m RNA and protein expression increased,while DNMT3 A m RNA and decreased(P<0.05).To verify the relevance of DNA methyltransferase effects on CIMP and tumor development,we overexpressed DNMT3 A in colon adenocarcinoma LOVO cells by eukaryotic plasmid vector technology.It was found that after overexpression of DNMT3 A in LOVO cells,DNMT3 A m RNA expression was significantly increased,CIMP-related indicators of methylation in this cell were with 1 increased indicator,and CIMP phenotype was changed from CIMP-L to CIMP-H;MLH1 gene was methylated,and protein expression was decreased,reducing the apoptotic ability of the cells and improving the cell viability;we also transiently silenced DNMT1 expression in LOVO cells using si RNA technology and found that the CIMP level of LOVO cells did not change significantly after DNMT1 silencing,but increased the apoptotic ability of the cells and decreased the cell viability.These results suggested that DNMT1 and DNMT3 A seem to have a certain synergistic effect and be involved in the dynamic regulation of CIMP in colorectal cancer,and their real interactions need to be further studied;this study also found that epigallocatechin gallate(EGCG)can decrease the CIMP level of LOVO cells,the DNMT1 gene and protein expression,cell viability,but increase apoptosis;however,there are some differences in the effect of DNMT3 A at different concentrations,40 mg/L and 60 mg/L can decrease the DNMT3 A expression,and the DNMT3 A expression increases at 80 mg/L,which provides an important clue for the study of targeted drug regulation of CIMP status in colorectal tumors.This experiment studied the dynamic changes of CIMP during the development of CRC and preliminarily explored its molecular regulatory pathway,which laid the foundation for revealing the molecular pathological mechanism of CIMP subtypes in colorectal cancer,and it also discovered that CIMP of colorectal cancer is closely related to tumor gene mutation load and lymph node metastasis.EGCG can reduce the level of CIMP in tumor cells,which has not been reported at home and abroad.