The Study Of Epigenetically Regulated Genes In The Heterogeneous Progression Of Colorectal Cancer

Objective:Epigenetic changes,such as abnormal DNA methylation and micro RNA(miRNA),play a pivotal role in regulating gene expression and promoting heterogeneous cancer behaviour.In this study,we investigated whether epigenetic regulation by DNA methylation and miRNA exerts a synergistic effect in colorectal cancer(CRC)progression,and if so,whether this effect contributes to classify CRC.Next,we identifid the key epigenetically regulated gene in the heterogeneous progression of CRC and performed in vitro and in vivo experiments.Methods:1.The TCGA database was searched for multi-omics data of CRC patients with clinical prognostic information.In TCGA dataset,we used the pearson’s correlation analysis to identify DNA methylation-correlated(METcor)and miRNA-correlated(MIRcor)genes.Next,integrative clustering analysis for the expression profiles of METcor or MIRcor genes was applied to identify the CRC subtypes,and then survival analysis was performed for the CRC subtypes.2.Through linear models in R ‘limma’ package,we identify the signature genes of the individual subtype of TCGA dataset.The GEO database was searched for available CRC datasets with both gene expression and clinical prognostic information.Using the signature genes of the TCGA dataset,the nearest template prediction(NTP)algorithm was conducted to validate the CRC subtypes in GEO dataset.3.We analyzed correlations of the CRC subtypes with DNA methylation,miRNA expression,copy number variations(CNV),mutations and neoantigens in TCGA dataset.Using ss GSEA,NTP and ESTIMATE algorithms,we analyzed correlations of the CRC subtypes with biological processes and immune infiltration.The drug sensitivity of chemotherapy drugs,targeted drugs and immune checkpoint inhibitors were also analysed using Sub Map analysis in the four CRC subtypes.4.Based on the epigenetically regulated CRC classification,we identified DNA methylation regulated N-acetyltransferase 2(NAT2)in the heterogeneous progression of CRC.CRC cells(HCT116 and DLD1)were treated with 5-aza-2’-deoxycytidine,and then NAT2 expression and its promoter DNA methylation were detected to explore their regulation relationship.5.Using overexpression lentiviruses for NAT2 and control lentivirus,NAT2 overexpressed cells and its control cells were established in HCT116 and DLD1 cells.Then,CCK-8 and clonogenic survival assays were used to detect the effect of NAT2 overexpression on cell proliferation.Flow cytometry analysis was used to analyze cell cycle and cell apoptosis.Wound healing assay and transwell assay were used to detect the effect of NAT2 overexpression on cell migration and invasion.In addition,nude mouse subcutaneous and lung metastasis xenograft models were used to detect the effect of NAT2 overexpression on CRC growth and metastasis in vivo.6.In TCGA and GEO datasets,GSEA analysis was performed to investigate the effect of NAT2 expression on tumor related pathways.Based on GSEA results,microplate reader and western blot were used to detect the effect of NAT2 overexpression on cellular acetyl coenzyme A concentration and epithelial-mesenchymal transition(EMT)related proteins,respectively.Results:1.In TCGA database,363 CRC patients with matched data of DNA methylation,miRNA expression,mRNA expression,CNV and gene mutation were selected for subsequent analyses.we identified METcor(n=1397)and MIRcor(n=410)genes.Further analysis revealed that the frequencies of aberrant METcor and MIRcor genes were obviously positively correlated in CRC.Based on integrative clustering analysis for the expression profiles of METcor and MIRcor genes,four CRC molecular subtypes(S-I,S-II,S-III and S-IV)with distinct prognoses were identified and then validated in four independent datasets(n=1087).2.Compared with normal samples,the S-I subtype only showed upregulated miRNA expression and the remaining three subtypes exhibited a significant increase in the levels of promoter methylation and miRNA expression.In particular,S-III and S-IV subtypes showed the highest levels of DNA methylation and miRNA expression,respectively.In addition,CNV did not significantly differ among the CRC subtypes.The S-III subtype showed significantly higher tumor mutation burden and neoantigens,and lower aneuploidy score than other subtypes.3.By investigating important biological processes,we found that glucose and lipid metabolism-related pathways were upregulated in the S-I subtype.Both S-II and S-III subtypes were characterized by elevated glucose metabolism-related pathways and high levels of proliferation-related pathways.In addition,the S-III subtype exhibited moderate levels of metastasis-related pathways.As expected,S-IV subtype was distinguished by more aggressive characteristics,such as‘stem/serrated/mesenchymal–like’ phenotype.4.ESTIMATE analysis indicated that S-III and S-IV subtypes had higher immune and stromal scores and lower tumour purity than that of S-I and S-II subtypes.Based on the results of ss GSEA and NTP analyses,S-III was defined as immune-inflamed subtype,whereas S-IV was defined as immune-excluded subtype.5.For chemotherapy,S-I patients were more likely to benefit from 5-fluorouracil(5-FU)-based chemotherapy,whereas S-III patients did not respond to 5-FU or irinotecan therapies.For targeted therapy,S-III patients tended to benefit from cetuximab(EGFR inhibitor)and the trametinib(MEK inhibitor),but did not respond to dabrafenib(BRAF inhibitor).For immunotherapy,S-III patients might respond to anti-PD-1 therapy.6.The promoter methylation level of NAT2 was significantly lower in S-I and S-II subtypes than S-III and S-IV subtypes.On the contrary,NAT2 expression was significantly higher in S-I and S-II subtypes than S-III and S-IV subtypes.In addition,NAT2 expression was associated with good prognosis,whereas its promoter methylation was associated with poor prognosis.After 5-aza-2’-deoxycytidine treatment for HCT116 and DLD1 cells,the promoter methylation level of NAT2 was reduced and NAT2 m RNA and protein were increased,indicating that promoter methylation could silence NAT2 expression.7.NAT2 overexpression could inhibit HCT116 and DLD1 cell proliferation,migration and invasion in vitro.Meanwhile,in HCT116 and DLD1 cells,NAT2 overexpression triggered G0-G1 cell cycle arrest and reduce S phase,but showed no significant induction of apoptosis.Furthermore,NAT2 overexpression suppressed HCT116 and DLD1 cell xenograft growth and lung metastasis in nude mice.8.GSEA analysis showed that high NAT2 expression was closely related to acetyl coenzyme A metabolism,lipid and fatty acid catabolism,whereas low NAT2 expression was related to EMT,angiogenesis,tumor invasion,extracellular matrix remodeling,hypoxia,and stemness.In vitro experiments indicated that overexpression of NAT2 overexpression could reduce cellular acetyl coenzyme A,and inhibit EMT process in HCT116 and DLD1 cells.Conclusion:This study reveals that DNA methylation and miRNA have a synergistic regulatory effect on gene expression in CRC.Next,based on the expression profiles of METcor and MIRcor genes,we developed a epigenetically regulated CRC classification containing four molecular subtypes with distinct clinical and molecular features.Based this classification,we identified promoter methylation regulated NAT2 gene in the heterogeneous progression of CRC.In vivo and in vitro experiments have shown that NAT2 overexpression can inhibit the growth and metastasis of CRC.NAT2 overexpression might suppress EMT process by downregulating cellular acetyl coenzyme A in CRC cells,thereby inhibiting CRC progression.