Silencing Of GAP-43 Gene In Colorectal Cancer And Its Mechanism

Cancer has become a leading killer of human health,and new cases are increasing every year.China has a high incidence of cancer and mortality,with colorectal cancer at the forefront.Colorectal cancer(CRC)is a primary tumor that occurs mainly in the colon and rectum.It has a high incidence,mortality,and recurrence rate and is one of the common malignant tumors of the digestive tract.In recent years,the treatment of colorectal cancer has been considerably improved,but its recurrence and metastasis are still the main causes of postoperative death,and it has become a bottleneck limiting the survival of patients and improving prognosis.Growth associated protein 43(GAP-43)is a membrane phosphoprotein closely related to neural development,prominent formation and the like.At present,it has been reported that GAP-43 has an inhibitory effect on the proliferation of glioma cells and breast cancer cells,but the mechanism of inhibition is still unclear.In the early stage of the research group,through the analysis of genomic data of colorectal cancer in TCGA database,it was found that GAP-43 gene has significant expression silencing in colorectal cancer,and there is methylation modification in its gene promoter region,but its specific The mechanism of silence is not yet clear.In order to further confirm this finding,this paper first selected the clinical tissue samples of colorectal cancer patients for real-time quantitative PCR(qRT-PCR)experiments,and found that the expression level of GAP-43 mRNA in colorectal cancer tissues was significantly lower than normal.Paracancerous tissues;and by immunohistochemistry(IHC)experiments,it was found that the expression level of GAP-43 in colorectal cancer tissues was also lower than that in normal adjacent tissues,and we also found GAP-43 in highly differentiated tissues of colorectal cancer.The expression is higher than that of poorly differentiated organizations.This demonstrates that the GAP-43 gene is silently expressed in colorectal cancer.In order to explore the possible mechanism of GAP-43 gene expression silencing in colorectal cancer,this paper uses bisulfite sequencing to detect GAP-in normal tissue samples of colorectal cancer cells SW620,HCT116 and colorectal cancer clinical patients.The level at which the base of the GAP-43 gene promoter is methylated.We found that in two colorectal cancer cells,cytosine of multiple CpG islands in the promoter region of GAP-43 gene is methylated,whereas in normal tissues of colorectal cancer patients,cytosine of these CpG islands No methylation modification occurred;we subsequently treated colorectal cancer cells with DNA methyltransferase inhibitor 5-aza-dc,and found that GAP-43 mRNA of colorectal cancer cells was treated by qRT-PCR.The expression level is significantly higher than that of normal colorectal cancer cells.Therefore,we conclude that the expression of the GAP-43 gene in colorectal cancer is silenced,most likely due to methylation of the promoter region DNA.In order to study the effect of GAP-43 on the phenotype of colorectal cancer cells,we successfully constructed SW620/GAP43 and HCT116/GAP43 two overexpression stable cell lines,and carried out a series of phenotypic experiments.The MTT proliferation curve assay and colony formation assay showed that the up-regulation of GAP-43 inhibited the proliferation of colorectal cancer cells.Transwell chamber assay showed that the expression of GAP-43 was up-regulated and the migration and invasion ability of colorectal cancer cells.At the same time,in order to explore the possible signaling pathways of GAP43 in colorectal cancer cells,we sequenced the single-cell transcriptome of over-expressed colorectal cancer cells and analyzed the sequencing results using bioinformatics software,and found FILP1 L,KRT6A,S100A14,etc.Multiple differentially expressed genes,KEGG enrichment analysis showed that the differentially expressed genes with high expression were mainly enriched in ABC transporters,neuroactive ligand-receptor interactions,and the differentially expressed genes were mainly enriched in ribosomes.Related,antibiotic synthesis and other pathways.In recent years,the treatment of colorectal cancer has been considerably improved,but its recurrence and metastasis are still the main causes of postoperative death,and it has become a bottleneck limiting the survival of patients and improving prognosis.The above experimental results demonstrate that the expression of GAP-43 gene is silenced in colorectal cancer,and it is most likely caused by methylation modification of DNA in its promoter region;GAP-43 expression up-regulates significantly inhibits proliferation of colorectal cancer cells,migration and invasion ability;transcriptome sequencing analysis found differentially expressed genes such as FILP1 L,KRT6A,S100A14,and these differential genes were enriched in a variety of different signaling pathways.