Screening Of Super-enhancer-associated Genes In Colorectal Cancer And The Anticancer Effect Of THZ2

Background and purpose:Colorectal cancer is a very common gastrointestinal malignancy with high morbidity and mortality worldwide.The most effective strategy to treat colorectal cancer is surgical resection,but many patients are already in the middle and late stages of tumor at the time of diagnosis,causing them to lose the best opportunity for surgical treatment.Although some patients can be treated with surgery,postoperative recurrence and chemotherapy resistance will still lead to treatment failure.Therefore,it is extremely important to understand the pathogenesis of colorectal cancer.Super-enhancer is a cis-regulatory element bound by many transcription factors and cofactors,enriched in a high density of mediator complexes,BRD4,CDK7,RNA polymerase II and the histone acetylation modification of H3K27ac,and thus has the strong transcriptional activation ability and interference sensitivity.In recent years,super-enhancers have been reported to activate the expression of key oncogenes in various tumors and promote tumor progression.Since tumor-associated variants are significantly enriched in super-enhancers,the identification of super-enhancers and associated genes may help to understand the mechanism of tumorigenesis and discover key oncogenic targets.This study aimed to identify super-enhancer-associated genes in colorectal cancer and to investigate the effect of the super-enhancer inhibitor THZ2 on the phenotype of colorectal cancer cells.Methods:（1）In this study,super-enhancer-associated genes of colorectal cancer were downloaded from the database of Core transcriptional Regulatory Circuitries.The enrichment analysis of super-enhancer-associated genes was performed by Gene Ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）,and the genes with statistically significant expression were further screened by The Cancer Genome Atlas（TCGA）data.Finally,the expression of super-enhancer-associated genes in normal colon epithelial cells（HCo Epi C）and cancer cells（SW480,HCT116,SW620）was verified by RT-q PCR and Western blotting experiments.（2）Firstly,the effect of THZ2 on the cell toxicity of HCo Epi C,SW480,HCT116,and SW620 and their respective 50%inhibitory concentration(IC₅₀)were detected by CCK8 assay;Then SW480,HCT116,and SW620 were treated with appropriate concentration of THZ2,and the expression of THZ2 target CDK7 and super-enhancer-associated gene ETV4 were detected by RT-q PCR and western blotting assay;The effects of THZ2 on cell proliferation,migration and invasion were detected by clone formation assay,wound healing assay and transwell assay,respectively;The effects of cisplatin alone and in combination with THZ2 on cell viability were detected by CCK8 assay,and the combination index was calculated to determine whether the two had a synergistic effect;Finally,the effect of THZ2treatment on the expression of cancer stem cell-associated genes was detected by RT-q PCR and Western blotting experiments.Results:（1）By overlapping super-enhancer-associated genes in colorectal cancer cells and colorectal tissues,and excluding super-enhancer-associated genes in normal tissues,a total of 44super-enhancer-associated genes were obtained;GO and KEGG analysis found that these 44 genes were involved in the regulation of transcriptional processes,and 15 genes among them were related to tumor pathways;Further expression analysis revealed that the expressions of 6genes were statistically significant,including ETV4,MYC,SHH,BMP4,TRIB3,and GTF2IRD1,which had significantly higher expression in cancer tissues than in normal tissues;The expression of transcription factor ETV4 was further verified in cells,and we found that the expression of ETV4 in SW480,HCT116 and SW620 was significantly higher than its expression in HCo Epi C.（2）THZ2 had strong toxic effects on all four kinds of cells.The IC₅₀of THZ2 to HCo Epi C was 0.4225±0.0475（μM）,the IC₅₀ of THZ2 to SW480 was 0.1031±0.0003（μM）,the IC₅₀ of THZ2 to HCT116 was0.1515±0.0119（μM）,and the IC₅₀ of THZ2 to SW620 was0.0573±0.0145（μM）;THZ2 treatment significantly reduced CDK7 levels in three cancer cells at the m RNA level,but did not change the expression of CDK7 protein levels;THZ2 treatment significantly reduced the expression of ETV4 in HCT116 and SW480 cells at the m RNA and protein levels;THZ2 could significantly reduce the ability of colony formation,migration and invasion in colorectal cancer cells;THZ2combined with cisplatin could significantly reduce cell viability,and in most cases,the two play a synergistic effect;THZ2 treatment reduced the expression of cancer stem cell-associated genes including SOX9,Nanog,SOX2,and OCT4.Conclusion:In colorectal cancer,the transcription factor ETV4 is regulated by super-enhancers,and THZ2 could significantly reduce the expression of ETV4;THZ2 plays a strong anticancer effect in colorectal cancer cells,including inhibiting cell proliferation,migration and invasion,as well as inhibiting the expression of cancer stem cell-associated genes.Compared with cisplatin alone,THZ2 combined with cisplatin had stronger anticancer effect.