Effect Of BRAF Gene Mutation On CpG Island Methylation,Cell Proliferation And Apoptosis In Colorectal Cancer

Globally,the incidence and mortality of colorectal cancer(CRC)continues to rise.The BRAF gene is located downstream of the RAS gene in the MAPK signaling pathway.The BRAF gene is located downstream of the RAS gene in the MAPK signaling pathway.More than 90% of its mutations are manifested as a mutation of codon 600 in exon 15(V600E),that is,BRAFV600 E mutation.The methylation of CpG islands is one of the important epigenetic events.The CpG island sequence in the promoter region of tumor suppressor genes in the genome is widely methylated,and the state that causes partial gene function inactivation is called the CpG island methylation phenotype(CIMP).In order to explore the correlation between BRAF mutations and CIMP,we used convenient sampling methods to collect 148 cases of colorectal cancer surgical resection or biopsy samples,used next-generation sequencing technology to detect BRAF mutations,and used the Methylight method based on fluorescence quantitative PCR to detect CIMP status of colorectal cancer tissue.The results was statistically analyzed by the χ2 test.Our study showed that the correlation between the BRAF mutation status of the sample and the CIMP phenotype was statistically significant(P <0.05).That suggested that the BRAF mutation status is related to the CIMP phenotype,and BRAF mutations cases tends to accompanied by CIMP-H in patients with colorectal cancer.In order to explore the molecular pathological mechanism related to BRAF gene mutation and CIMP,130 colorectal cancer samples were collected by convenient sampling method and typical sampling method,and the expression of BRAF,MEK,ERK,DNMT1 and DNMT3 A m RNA were detected by q PCR method.Regression analysis method was used for statistical analysis.The results showed that BRAF,MEK,and ERK were positively correlated with DNMT1 and DNMT3A(P<0.05).That suggested that the correlation between BRAF mutation and CIMP-H may be caused by the influence of MAPK pathway on DNMT1 and DNMT3 A.In order to explore the relationship between BRAF mutation and CIMP phenotype,and to explore the relationship between BRAF mutation and CIMP-H.BRAF mutation/CIMP-H group and BRAF wild/CIMP-H group were selected,and q PCR was used to detect the expression of BRAF,MEK,ERK,DNMT1 and DNMT3 A m RNA,and immunohistochemistry method was used to detect BRAF,MEK,ERK,DNMT1 and DNMT3 A protein expression.We observed the expression difference between the two groups.The results were statistically analyzed by the t test method.The results showed that at the molecular level,the BRAF mutation/CIMP-H group showed lower expression of BRAF,MEK,DNMT1 and DNMT3A(P<0.05);at the protein level,the BRAF mutation/CIMP-H group BRAFV600 E mutant protein was positive related,BRAFV600 E mutant protein in BRAF wild/CIMP-H group was negative related,consistent with the molecular level comparison;BRAF mutant/CIMP-H group showed lower protein expression of BRAF,MEK,DNMT1 and DNMT3A(P<0.05).That suggested that BRAF mutation and CIMP-H have a synergistic effect in the occurrence and development of colorectal cancer,and may be the main driving factor for the carcinogenesis of serrated adenoma.In order to explore the mechanism of BRAF gene mutation in the occurrence of colorectal cancer,the BRAF wild-type colorectal cancer cell line SW480 was transfected with BRAFV600 E mutant plasmid and empty plasmid by liposome transfection technology.The experimental group was BRAFV600 E that the mutant plasmid group,and the control group were the empty plasmid negative control group and the SW480 blank control group.Then the BRAF gene of BRAFV600 E mutant colorectal cancer cell line HT29 was transiently silenced by si RNA technology.Methylight method was used to detect 8 methylation indicators of CIMP in each group of cells;q PCR was used to detect the expression of BRAF,MEK,ERK,DNMT1 and DNMT3 A m RNA;immunocytochemistry was used to detect the expression of BRAFV600 E mutant protein and MEK,ERK,DNMT1 and DNMT3A protein;CCK8 test to detect changes in cell proliferation;TUNEL test to detect cell apoptosis;Transwell test to detect changes in cell migration ability.The results were statistically analyzed by the t test method.The results showed that compared with the two control groups,the experimental group overexpressing the BRAFV600 E mutant plasmid in SW480 cells had significantly higher methylation levels of CACNA1 G,NEUROG1 and CRABP1 genes(P<0.05).Comparing the experimental group with BRAF gene silenced by HT29 cells and the two control groups,the methylation levels of CACNA1 G,NEUROG1,CRABP1,MLH1 and SOCS1 genes were significantly reduced(P<0.05);the expression of DNMT1 increased(P<0.05),the expression of DNMT3 A decreased(P<0.05);cell proliferation ability was weakened(P<0.05);apoptosis ability was enhanced(P<0.05).That suggested that BRAF gene mutation can increase the methylation level of CIMP-related gene CpG island of colorectal cancer cells,enhance cell proliferation ability and weaken apoptosis ability.In summary,this study found that BRAF mutations are mostly associated with CIMP-H in colorectal cancer samples.Based on fluorescence quantification and immunohistochemistry platforms,it could speculated from the m RNA and protein levels that the relationship between BRAF mutations and CIMP-H may be influenced by MEK,ERK,DNMT1 and DNMT3A.At the same time,it was believed that BRAF gene mutation and CIMP-H play a role in inducing tumor formation.The relationship between BRAF gene expression and CIMP and MAPK pathway-related genes showed that BRAF gene mutations can affect the methylation level of CIMP-related genes by regulating the expression of DNMT1 and DNMT3 A genes and proteins.BRAF mutation could increase the proliferation ability of cells and decrease the apoptotic ability of cells.BRAF mutation had no effect on the overall state of CIMP.