| Objective: In this study,the whole exome sequencing(WES)was used to detect colorectal cancer(CRC)metastasis-associated genes and their regulatory network analysis was performed to explore the relationship between gene mutation and CRC metastasis.Methods: A total of 22 CRC primary tumors and paired metastases were collected.Then,the DNA was extracted from the samples for WES detection and mutation sites identification.The effects of different location,metastasis time and metastatic site on the mutation spectrum were compared,and the mutant genes in liver metastasis samples,which accounted for 77% of metastasis samples,were further analyzed such as tissue specific expression,CNV and regulatory network,and finally the important genes related to the occurrence and development of CRC metastasis were screened out.Results: 1)A total of 9669 mutant genes were found in primary tumors,among which 628 genes were mutated in more than half of the samples,and 365 genes were differentially expressed in the CRC expression profile,and 6 genes(CDC25B,KCTD9,ATIC,PSMD14,NAT10,and MIPEP)showed statistical differences in all CRC-related expression profiles and methylation profiles.2)A total of 7199 mutant genes were detected in CRC metastases,among which 613 genes were mutated in more than half of the samples,66 genes(including MUC12,MUC4,MUC2,MUC5 B,NBPF1,AHNAK,GPRIN2,SPON1,and UGT2B4)were differential expressed in CRC metastasis profile,and these genes were involved in biological processes related to immune responses,lipid transport,and cell maturation.3)There were 407 same mutant genes(including KRAS and NBPF1)between CRC primary and metastatic lesions,among which 67 mutant genes were differential expressed,and these genes were enriched in DNA replication,recombination,and cell cycle-related pathways.4)The mutational spectrum of CRC primary tumors in different parts was disparate.The number of mutation sites and mutant genes in the rectum was the highest,followed by descending colon and colonic colon.The three common mutation genes were involved in cancer related biological processes such as DNA repair,osteoblast proliferation,maintenance of gastrointestinal epithelium,and PPAR signaling pathways.5)The mutational spectrum of CRC primary tumors at different metastatic times was different.The number of mutation sites and genes in sexual metastasis was much more than that in metachronous metastasis.These simultaneous metastatic specific mutations were related to the process of cell-matrix adhesion,adhesion connection and adhesion plaque.6)The mutation profiles of different metastatic sites were different.The number of high frequency mutant genes in liver metastases,lung metastases and ovarian metastases was higher than that of the corresponding primary tumors,and the intersections of mutated genes were related to processes such as cell adhesion molecules and the regulation of actin cytoskeleton.7)In comparison with CRC primary lesions,liver metastases showed a decrease in CNV on 1q21.1,3q29,7q22.1,15q11.2,16p11.2,and 19p12,including the second exon of MUC4 of the mucin family.The differentially expressed CNVs related genes were associated with gamma-aminobutyric acid signaling pathway,DNA metabolism,actin movement,sulfur metabolism and other processes.8)Among the liver metastasis-specific mutant genes,CASP12 had the most types of mutations(missense mutation,splice mutation,and termination codon acquired mutation),followed by SPTA1(94%),HRNR(76%),VPS41(71%),and FAM86B1(71%).Liver metastasis-specific mutant genes were mainly involved in immune-related biological processes.REAC pathway analysis revealed that these genes were significantly enriched in the GALNT12 deficiency leading to colorectal cancer 1(CRCS1)pathway(including MUC5 B,MUC4,MUC3 A,MUC16,MUC6 and MUC5AC).9)The expression level of liver metastasis-specific mutant gene in liver was higher than those in other tissues.AKR1C4,GC,AGT,FMO5,SEC14L2,ABCC2,ECHS1,and HLF had the highest specific expression index in liver tissues.10)The network module mining and functional analysis of liver metastasis-specific genes identified four sub-networks.The hub genes of the first gene cluster were CREB3L1,E2F5 and RANBP2,which were mainly related to cell cycle and apoptosis.The hub genes of the second gene cluster were KLF9,RBBP5 and SMC3,which were involved in processes such as cell repair,metastasis and nucleic acid metabolism.The hub genes of the third gene cluster were HLF and SIN3 B,which were involved in cell viability,migration and invasion.The hub genes of the fourth gene cluster were MYLK and TNS1,which were related to cell adhesion,migration and microtubule movement.Conclusion: We found CRC primary tumors and metastases(especially liver metastases)related mutations genes,such as mucin family and CRC shift related to the level of gene mutation,by WES.Meanwhile,it also showed the CRC primary focal gene mutation spectrum was influenced by location and transfer time.Finally,we determined the key genes in the liver metastases based on regulatory network analysis.The results of this study confirmed the effectiveness of WES in identifying tumor related genes and provided clues for future studies on CRC metastasis. |
