Study On The Effect Of Colorectal Cancer Resistance To Oxaliplatin On The Genome Three-dimensionalspatial Structure Based On DLO Hi-C Technology

Colorectal cancer(CRC)is the fourth deadliest cancer in the world,second only to lung cancer,liver cancer and stomach cancer,with nearly 900,000 people deaths every year.In China,the incidence of CRC is increasing year by year as people's living standards continue to improve and more and more people are turning to Western diets and lifestyles.The development of basic disciplines such as pathophysiology has improved people's understanding of the disease,so that the treatment of CRC has changed from endoscope,surgical local resection,extensive surgery for metastatic disease to local ablation therapy for metastatic lesions,palliative chemotherapy,targeted therapy and personalized treatment such as immunotherapy.Although these new treatments have improved the overall survival(OS)of CRC;mortality in patients with metastatic,relapse and drug-resistance CRC has not been significantly improved.Oxaliplatin was introduced in the treatment of metastatic CRC in 2000.Prior to this,cisplatin and carboplatin have been shown to be ineffective.The results showed that oxaliplatin not only significantly improved the objective response rate,improved the survival percentage of metastatic resection,but also improved the overall survival of the patient.Therefore,oxaliplatin is widely used as a standard first-line chemotherapy drug for metastatic CRC.Unfortunately,acquired resistance to oxaliplatin-based single therapy or combination therapy is the main reason for treatment failure,and the development of chemical resistance has limited its effectiveness in clinical treatment.Therefore,it is important to clarify the root cause of this phenomenon and find a better anti-cancer method.At present,many studies focus on revealing the molecular' mechanism of oxaliplatin resistance from the horizontal structure of the genome,but it is still unknown how the three-dimensional spatial structure of the cancer cell genome changes before and after drug resistance occurs,and how the three-dimensional spatial structure regulates the acquisition of drug resistanceIn this project,we explored the differences in the three-dimensional spatial structure of CRC cell lines before and after acquiring resistance and screened some resistance-related genes to regulate the acquisition of CRC resistance through chromatin conformation.The main research content can be divided into the following three parts:1.Construction of oxaliplatin-resistant colorectal cancer cell line HCT1160xR:The CRC cell line HCT116 was transformed into oxaliplatin-resistant cell line HCT1160xR using a concentration gradient method;CCK-8 method was used to detect the IC50 values of oxaliplatin in two cell lines;using cell migration,invasion,scratching methods and detection of epithelial-mesenchymal transition(EMT)markers confirmed that HCT1160xR was more malignant than HCT116,which further proved the successful construction of HCT1160xR cell line.2.Study on the effect of oxaliplatin on the three-dimensional space of colorectal cancer cell lines based on DLO Hi-C technology:the application of digestion-ligation-only high-throughput chromosome conformation capture(DLO Hi-C)analysis of the three-dimensional spatial structure changes of the CRC cell line HCT116 after oxaliplatin resistance.The chromatin compartment representing transcriptional activity was highly conserved,with approximately 20%compartments occurring conversion;there was no statistically significant difference in the size or number of topologically associated domains(TADs);the number and the interactions intensity of chromatin loops decreased significantly.3.Three-dimensional genomics combined with RNA-seq and ChIP-seq for screening the key genes regulating drug resistance by chromatin conformation:RNA-seq technology was used to detect the differential genes of the two cell lines and enrich the KEGG signaling pathway.Chromatin immunoprecipitation sequencing(ChIP-seq)was used to detect the intensity of H3K4me1,H4K4me3,H3K27me3,and H3K27ac,which represent transcriptional activity.Enrichment analysis of compartment A-B/B-A and RNA-seq down-regulation/up-regulation revealed GSTA1,HBB,CYP2C9,LEF1 and PRDX6 and other key genes,and hypothesis:The acquisition of oxaliplatin resistance in tumor cells was due to the reduction of reactive oxygen species(ROS)and the enhancement of metastatic capacity mediated by the change of active chromatin conformation.In HCT1160xR cells,the expression of LEF1 was reduced by RNAi technology.Reducing the expression of LEF1 could weaken the resistance of drug-resistant cells and affect the invasion and metastasis ability of drug-resistant cells by cell migration,invasion methods and detecting of the expression of EMT-related molecules.In summary,this topic clarified that the CRC cell line has an effect on the three-dimensional spatial structure of the genome after acquiring drug resistance.For example,20%of the genome realized A/B region conversion,the TAD domain was highly conserve and the number of loops was significantly reduced.The key genes such as GSTA1,HBB,CYP2C9,LEF1 and PRDX6 regulated the acquisition of drug resistance through three-dimensional conformational changes of the genome;it was confirmed that reducing the expression of LEF1 in HCT1160xR could reverse tumor drug resistance.