Expression Of Circular Rna In Serum Exosome Of Colorectal Cancer With Liver Metastases Patients And Preliminary Study On Cerna Interaction Network

Objective:To explore the difference in the expression of circRNA in plasma exosomes of patients with liver metastasis from colorectal cancer and patients with colorectal cancer,and to analyze the diagnostic efficacy of early diagnosis of liver metastasis from colorectal cancer;combining the previous experimental results and biological information Analysis,prediction of the circRNA-miRNA-mRNA regulatory network involved in differentially expressed circRNA,and preliminary verification of the predicted competitive endogenous RNA（ceRNA）network using clinical plasma tissue samples and cell function experiments.Methods:（1）Using gene chip technology,analyze the differential expression profiles of circRNA in serum exosomes of 4 primary patients with rectal cancer and 4 patients with liver metastases from colorectal cancer Exo-circRNA.In plasma,multiple candidate Exo-circRNA such as Hsa_circ₀001296 and hsa_circ₀006773 were verified by PCR.Using three circRNA-miRNA binding site prediction databases to screen out miRNAs combined with differentially expressed circRNA in plasma exosomes of primary colorectal cancer patients and colorectal cancer liver metastasis patients;using three miRNA target gene prediction databases,Predict miR-509-3p target genes,perform GO（Gene Ontology）analysis and KEGG（Kyoto Encyclopedia of Genes and Genomes）pathway analysis on the predicted target genes,and finally initially construct circRNA-miRNA-mRNA in colorectal cancer liver metastasis Interaction network.（2）By comparing the clinical plasma samples of 60 primary colorectal cancer patients with 60 colorectal cancer liver metastasis patients,verify whether the screened circRNA expression is consistent with the trend of the chip results,and use ROC curve to evaluate its diagnostic value;use real-time Fluorescence quantitative PCR（qRT-PCR）technology was used to compare the expression and related trends of circRNA,miRNA,and mRNA between 30 primary colorectal cancer tissues and 17 liver metastasis tissues.（3）Detect the expression of hsa_circ₀001296 in 5 colorectal cancer cell lines including normal intestinal epithelial cells,transiently transfect hsa_circ₀001296 inhibitor into the highest interference cell line,and analyze hsa_circ₀001296 by CCK8,scratch test,Transwell invasion test Effect on the function of colorectal cancer cells;use qRT-PCR to detect the expression of hsa_circ₀001296 and miR-509-3p in transfected cells and analyze the relationship between the two;the circRNA-miRNA constructed by the double luciferase reporter gene experiment-Whether there is a combination of mRNA interaction networks.RESULT:（1）A total of 7048 differentially expressed circRNAs were detected in this study,of which 3752 were up-regulated and 3296 were down-regulated in patients with colorectal cancer liver metastasis;the top 10 up-regulated ones were selected for PCR verification and found hsa_circ₀001296 Compared with CRC,hsa_circ₀006773 was significantly up-regulated in CRLM samples.Finally,Hsa_circ₀001296 and hsa_circ₀006773 were selected for follow-up research.Based on the previous experimental results,chip analysis data and bioinformatics analysis,a hsa_circ₀001296?miR-509-3p?AXL interaction network was preliminarily constructed.（2）Compared with primary colorectal cancer patients,it was found that the expression of hsa_circ₀001296 in plasma exosomes of patients with colorectal cancer liver metastases was significantly up-regulated（P<0.05）,which was consistent with the trend of gene chip results,and the area under the ROC curve was 0.664 AUC,With a certain diagnostic efficacy;compared with the primary tumor tissue of colorectal cancer,miR-509-3p expression was down-regulated,hsa_circ₀001296 expression was up-regulated,and AXL expression was up-regulated（P<0.001）.3p expression level has a negative correlation trend.（3）Successfully interfered with hsa_circ₀001296 in colorectal cancer cell lines;CCK8 results showed that after interfering with hsa_circ₀001296 expression,the cell proliferation ability was enhanced（P<0.001）;the scratch test used the healing rate to evaluate the cell migration ability after transfection and showed that it interfered with hsa_circ₀001296 expression After that,the cell migration ability was enhanced（P<0.01）;Transwell analysis of cell migration ability showed that the cell invasion ability was enhanced after interference with hsa_circ₀001296 expression（P<0.05）;qRT-PCR experiments showed that compared with the control group,the interference miR-After 99b-5p expression,hsa_circ₀001296 expression was up-regulated（P<0.01）,AXL expression was up-regulated（P<0.001）,dual luciferase reporter gene experiment found that wild-type plasmid vector and miRNA-509-3p mimic,miRNA-509-3p After co-transfection of mimic NC,the fluorescence value decreased（P<0.001）.After co-transfection of the mutant plasmid vector with miRNA-509-3p mimic and miRNA-509-3p mimic NC,there was no significant difference in fluorescence value（P>0.05）.Conclusion:（1）The expression of hsa_circ₀001296 in serum exosomes of patients with colorectal cancer is up-regulated and has certain diagnostic efficacy.（2）The expression levels of hsa_circ₀001296 and AXL in primary and liver metastases of colorectal cancer showed a negative correlation with miR-509-3p.（3）Interfering or enhancing the expression of miR-509-3p in cells,affecting the proliferation,migration and invasion of colorectal cancer cells.（4）The expression of miR-509-3p can affect the expression level of has_circ₀001296 and AXL.The dual luciferase reporter gene experiment confirmed that has_circ₀001296 can target miRNA-509-3p.（5）Preliminary verification of hsa_circ₀001296?miR-509-3p? AXL has a regulatory role in colorectal cancer.