Regional Hypermethylation Of Ascl2 Promoter Regulates The Stemness Of CRC Progenitor Cell And Regulation Of SMOC2 Expression By Ascl2

Background and Objectives:Colorectal cancer is the third most common cancer in the world.With the development of economy and the change of diet,the incidence of colorectal cancer is gradually increasing worldwide.With the deepening of research on colorectal cancer,it has been found that there is a class of "Cancer stem cells" in colorectal cancer,which have been found to have continuous self-renewal,chemotherapy resistance and greater ability to invade.Ascl2,a basic helix-loop-helix transcription factor(TF),is a downstream target of Wnt signaling pathway and may control the fate of intestinal Lgr5+ crypt stem cells and CRC progenitor cells.Our previous studies have found that Ascl2 can affect the epithelial mesenchymal transformation of colorectal cancer by regulating the mi R-200 family,and blocking of Ascl2 expression in CRC cells will result in tumor growth arrest in vivo and in vitro.Although Ascl2 plays an important role in the development and progression of colorectal cancer,the regulatory mechanism of Ascl2 expression in colorectal cancer cells is still unclear.Abnormal methylation is one of the characteristics of tumors,in which there is usually global hypomethylation and regional hypermethylation.The TET family plays a key role in the demethylation of DNA by catalyzing the hydroxylation of 5-methylcytosine(5m C)to5-hydroxymethylcytosine(5hm C).TET2 is frequently mutated in hematologic malignancies,and patients with TET2 mutations often show significant reductions in overall 5hm C levels,and TET2 mutation is thought to be associated with low genomic 5hm C levels.Loss of 5hm C has also been observed in many solid tumors,in which the role of TET2 needs further investigation.SMOC2 is an extracellular matrix protein involved in many biological processes such as embryonic development,cell cycle progression,cell attachment and migration,and angiogenesis.Overexpression of SMOC2 can lead to enhanced invasion and metastatic ability of CRC cells,but the role of SMOC2 in the occurrence and development of colorectal cancer still requires further study.This study mainly explores the role of regional hypermethylation of Ascl2-dependent colorectal cancer progenitor cell stemness maintenance,as well as the regulatory mechanism of Ascl2 on SMOC2 transcription and its impact on the prognosis of colorectal cancer patients.Methods:1.DNA methylation data were downloaded from MethHC database,and the methylation levels of different regions of Ascl2 locus were compared between colon cancer and normal intestinal mucosa tissues.2.Comparing the expression levels of Ascl2 and TET2 genes between CRC tissues and normal colon mucosa tissues in the TCGA database,analyzing the correlation between Ascl2 and TET2 mRNA expression in CRC tissues,and verifying the results in CRC cells by using RT-PCR and Western blot..3.Stable HCT116 and SW480 cell lines knockdowning TET2 expression using sh RNA interference were constructed,and SW620 cell lines stably overexpressing TET2 catalytic domain and mutated TET2 catalytic domain were constructed.The effect of TET2knock-down or TET2 catalytic domain overexpression on Ascl2 expression was detected by RT-PCR and Western blot.4.The effect of TET2 knockdown or TET2 catalytic domain overexpression on Ascl2 and its downstream stem genes expression in CRC cells was detected by RT-PCR and Western blot.5.The effect of TET2 knockdown or TET2 catalytic domain overexpression on the stemness phenotypes of CRC cells was studied by tumorsphere formation assay and mouse xenografts assay.6.By analyzing the GEO database,comparing the mRNA expression differences of Ascl2 and SMOC2 between CRC tissues and normal colon mucosal tissues,as well as the correlation between Ascl2 and SMOC2 mRNA expression in CRC tissues.7.Downloading the data from GEO database,we obtained the mRNA expression levels of each gene after Ascl2 interference,and drew volcano map and heat map to show the results.8.We detected SMOC2 expression levels in CRC cells with Ascl2 interference by RT-PCR and Western blot.9.Performing a dual luciferase assay and a chromatin immunoprecipitation experiment to determine whether Ascl2 directly interacts with the SMOC2 promoter.10.The clinical data and gene expression data were downloaded from the TCGA database.Kaplan-Meier and Cox regression were used to analyze the prognostic significance of Ascl2 and SMOC2 expression in CRC tissues.Results:1.The methylation level of Ascl2 promoter TSS1500 region in CRC tissues was higher than that in normal tissues(P<0.0001).The methylation level of TSS1500 region was higher than that of other regions of Ascl2 locus.2.There was a negative correlation between TET2 and Ascl2 mRNA levels in CRC tissues and CRC cells(P<0.0001).3.TET2 catalytic domain overexpression inhibited the expression of Ascl2 and its downstream stem genes,and weakened the stemness phenotypes of CRC cells;TET2knockdown enhanced the expression of Ascl2 and downstream stem genes,and enhanced the stemness phenotypes of CRC cells.4.The mRNA expression levels of Ascl2 and SMOC2 were significantly higher in colorectal cancer tissues than in normal colon mucosal tissues,and the mRNA expression levels of Ascl2 and SMOC2 were positively correlated.5.SMOC2 mRNA expression levels were decreased after Ascl2 knockdown in CRC cells.6.The dual luciferase assay and chromatin immunoprecipitation experiment confirmed that Ascl2 can directly bind to the proximal promoter of SMOC2 and promote its expression..7.The progression-free survival and overall survival in high SMOC2 expression group were significantly worse than low SMOC2 expression group,and the progression-free survival and overall survival in colorectal cancer patients with combined abnormal Ascl2 and SMOC2 expression were significantly worse than its control groups.SMOC2 expression level is an independent risk factor for progression-free survival and overall survival of colorectal cancer patients.Conclusion:1.TET2 regulates Ascl2 expression and affects the stemness phenotypes of CRC cells.2.Ascl2 enhances SMOC2 gene expression by acting on SMOC2 proximal promoter in colon cancer cells,which results in the worse prognosis of colorectal cancer patients.