Analysis Of Differential Expression Profile Of Serum Exosomes MiRNA In Patients With Liver Metastasis Of Colorectal Cancer

Objective:The differential expression profile of miRNA in serum exocrine of healthy human-colorectal cancer patients-colorectal cancer patients with liver metastasis was established by miRNA high-throughput sequencing.The serum exocrine miRNA molecules which play an important role in the occurrence and development of colorectal cancer liver metastasis were screened,and their possible biological functions,regulatory molecular pathways and key targets were predicted.The purpose of this study is to explore the important molecular mechanism in the process of colorectal cancer liver metastasis.To find sensitive methods for early diagnosis and to improve the existing treatment model to provide new ideas.Methods:From November 2021 to November 2022,patients with colorectal cancer diagnosed by imaging and pathological diagnosis(CRC,C group)and colorectal cancer liver metastasis group(CRLM,L group)were selected as the case group.Serum was collected from all patients prior to tumor intervention.In addition,healthy persons of similar age and sex(Normal,N group)were selected as the control group.The serum exocrine was extracted by molecular size exclusion combined with ultrafiltration.Transmission electron microscopy(TEM)was used to examine the morphology of exosomes particles,nano-flow detector(Nano FCM)was used to measure average particle size and the major peak of particle size,and the exosomes marker proteins were identified by Western blotting(WB).The total RNA of bleeding exocrine was isolated by miRNeasy (?)Minikit.The expression of exocrine miRNA in serum of healthy controls(n =3),CRC patients(n =3)and CRLM patients(n =3)was detected by Illumina second generation sequencing.The differential expression profile of miRNA in serum exocrine of CRLM patients was constructed,and the miRNA with significant difference was screened.The target genes of the selected mirnas were predicted by miRanda and RNAhybrid software,and the obtained target gene sequences were compared with NR,Swiss-Prot and other databases by BLAST software to obtain the annotation data of the target genes.R-package cluster Profiler was used to annotate and enrich the biological functions(GO function)and regulated signaling pathways(KEGG pathway)of differentially expressed miRNA target genes,and to evaluate their enrichment in biological processes(BP),molecular functions(MF),cell components(CC)and molecular pathways Have a biological function;Cytoscape3.9.1 software was used to visualize the PPI network data between target genes obtained from String database to further understand the relationship between differentially expressed miRNA target genes.Results:1.The quality of serum exocrine and exocrine miRNA extracted from 1Normal group,CRC group and CRLM group conformed to the standard,and high-throughput sequencing and data analysis were performed.2.The extracted exosomes were disc-shaped or double concave disc-shaped structures with bilateral membranes observed by TEM,and the serum exosomes were observed by Nano FCM,The average particle size was 101.19 nm,and it ranged from 50 to 200 nm,with the majority of the particles falling within the range of 80 nm.The TSG101,ALIX,and CD9 positive marker proteins were expressed by the isolated exosomes,according to the results of the WB test,but not the Calnexin exosome negative marker proteins.3.(1)Compared with Normal group,there were 18 high expression and 10 low expressions miRNA in serum exosomes of CRC group.There were 29 high expression and 23 low expressions miRNA in serum exosomes of CRLM group.(2)Compared with CRC group,there were 16 high expression and 14 low expressions miRNA in serum exosomes of CRLM group.(3)there were 7 differentially expressed miRNA,4 up-regulated and 3down-regulated in CRC group and CRLM group compared with Normal group,between the Normal group and the CRLM group,there was no discernible change in their expression.(4)there were 23 differentially expressed miRNA,13 up-regulated and 10 down-regulated in CRLM group compared with Normal group and CRC group,between the Normal group and CRLM group,there was no discernible change in their expression4.The differential expression of miRNA in serum exosomes mainly affects the occurrence and development of CRLM,such as chemokine signal pathway,RAP1 signal pathway,VEGF signal pathway,glutathione metabolism,phospholipase D signal pathway,base excision repair,proteasome and negative regulation of oxidative stress.5.As target genes,CFL1,DNM2 and PSMD8 play an important role in the occurrence and development of CRC.6.As target genes,PIK3R1,HRAS,EGFR,CDC42 and CBL play an important role in the occurrence and development of CRLM.Conclusions:1.There are significant differences in the expression of serum exocrine miRNA among healthy people,patients with colorectal cancer and patients with liver metastasis of colorectal cancer,suggesting that serum exocrine miRNA may be involved in the occurrence and development of liver metastasis of colorectal cancer.2.Serum exosomes hsa-miR-133a-3p,hsa-miR-223-3p,hsa-miR-15a-5p,hsa-miR-4306,hsa-miR-451 a and hsa-miR-429 may be key genes in the occurrence and development of colorectal cancer liver metastases,and hsa-miR-143-3p may affect the chemotherapy of colorectal cancer patients Sensitivity.3.CFL1,DNM2,PSMD8,PIK3R1,HRAS,EGFR,CDC42 and CBL may be important targets in the occurrence and development of liver metastases in colorectal cancer,among which PSMD8,DNM2 and HRAS are newly predicted key targets.