Screening,validation And Interaction Network Analysis Of Differential Expression Of CircRNA In Peripheral Blood Of DVT Patients

ObjectivesDeep vein thrombosis（DVT）is a common complication after major orthopedic surgery,especially after hip and knee replacement.In the absence of anticoagulant therapy,the incidence of DVT is significantly increased due to venous blood stasis in the legs,endothelial injury and abnormal activation of coagulation cascade.Many studies have been carried out at home and abroad on the regulatory mechanism of DVT at the molecular biological level,and some microRNAs（miRNAs）have been found to be related to the occurrence and development of DVT,but its regulatory network needs to be further explored and improved.With the in-depth study of non-coding RNA（ncRNA）,it is found that circRNA has a strong innovative significance in the field of thrombotic diseases,providing a new research direction for clinical diagnosis and treatment.In this study,circRNA expression in peripheral blood of DVT patients and control group was detected by using circRNA chip technology,and the differentially expressed circRNA was screened out.Real-time quantitative reverse transcription polymerase chain reaction（qRT-PCR）was used to select circRNA for verification of the chip results.Through bioin-formatics,including gene ontology（GO）analysis and gene and genome encyclopedia（KEGG）pathway analysis,the function of predicting differential expression of circRNA was predicted.A circRNA-miRNA-mRNA prediction network was constructed for candidate circRNA to enrich miRNA data.It provides a basis for searching for molecular markers with high specificity and sensitivity to predict the occurrence of DVT,monitor the progress of DVT,and evaluate the condition and prognosis.Methods1.According to inclusion and exclusion criteria,10 patients with DVT who were admitted to the First Affiliated Hospital of Kunming Medical University from February 2019 to December 2019 were selected as the DVT group（experimental group）,including 6 males and 4 females,with an average age of 66.5 ± 3.27 years.Ten patients without DVT who visited the First Affiliated Hospital of Kunming Medical University during the same period were collected as the control group,including 5 males and 5 females,with an average age of 65.1 ± 2.54 years.DVT includes thrombosis of venae tibiales anteriores,venae tibiales posteriores,peroneal veins,popliteal vein and femoral vein.2.Three patients in the DVT group and three in the control group were randomly selected to collect peripheral venous blood and extract total RNA from peripheral blood samples of the two groups.After ribosomal RNA and linear RNA were removed,circRNA chip technology was used to detect circRNA expression in samples of the DVT group and the control group.3.The results of the two groups of samples were normalized for statistical analysis,and the differences in expression between the two groups of samples were compared.Stratified clustering analysis and visualization of differentially expressed circRNA genes between the two groups were performed.4.The top 5 circRNAs with the most significant differential expression were selected to verify the chip detection results.Specifically,7 remaining peripheral venous blood of subjects in DVT group and control group were collected,total RNA was extracted,and qRT-PCR experiment was conducted to detect the expression of circRNA in the samples of the two groups,so as to verify the circRNA chip test results.5.The circRNA with the most significant difference among the 5 circRNAs that have been verified was selected,and the circRNA/miRNA/mRNA action network of the circRNA was constructed by Arraystar based on miRanda and TargetScan database,as well as miRNA target prediction software,and its function was analyzed by bioinformatics.6.SPSS 19.0 software was used for statistical data,and the measurement data were expressed as mean± standard deviation（SD）.The significance of the results was evaluated by t-test,and p<0.05 indicated that the difference between the two groups was statistically significant.Results1.Six samples（i.e.3 DVT group samples and 3 control group samples）were used to screen the differences in circRNA expression between the two groups.The normalized data intensity was approximately the same distribution,and the distribution of log2-ratio was similar for the six samples.2.Stratified cluster analysis of genes revealed differences in circRNA expression between the two groups.3.Compared with the control group,circRNA showed different expression patterns in DVT patients.Statistical analysis showed that the differences in circRNA expression between the two groups were statistically significant（fold change>2.0,p<0.05）.4.Compared with the control group,303 circrnas in the DVT group were dysregulated,of which 83 were up-regulated and 220 were down-regulated.5.Most of the differentially expressed circRNA were exon type circRNA,and the main transcription sources were chr1,chr2,chr12 and chr10.6.Qrt-pcr results showed that the expression levels of has_circ₀00455,has_circ₁01515 and has_circ₁01212 in the DVT group increased,and the expression levels of has_circ₀00185 and has_circ₀01982 decreased.7.Bioinformatics analysis of differentially expressed circRNA showed that the terms closely related to biological processes,cell components,and molecular functions were protein transport,cytoplasmic,and ATP binding,respectively.The most significant enrichment pathways were thymic hormone signaling pathway,tumor endocytosis,proteoglycan,fc-r mediated phagocytosis,focal adhesion kinase signaling pathway,insulin signaling pathway,p53 signaling pathway,antibiotic biosynthesis,epithelial bacterial invasion,and AMPK signaling pathway.8.The predicted miRNA targets of has_circ₀00455 were hsa_miR₂0b₃p,hsa miR₂2₃p,hsa_miR₂2₅p,hsa_miR₁38₅p and hsa miR 559.9.Qrt-pcr results showed that the expressions of hsa_miR₂0b₃p,hsa_miR₂2₃p and hsa_miR 559 in the DVT group were down-regulated10.MiRNA target prediction software predicted that there were 652 targets interacting with hsa circ RNA 000455,among which 313 target genes were interacting with hsa-mir-138-5p,with the largest interaction network.The 229 target genes interact with hsa-mir-22-3p in the second largest interaction network.Conclusions1.The most significant peripheral blood differentially expressed circRNA in DVT patients:the expression levels of circ₀00455,circ₁01515 and circ₁01212 increased,and the expression levels of circ₀00185 and circ₀01982 decreased.2.The predicted target miRNA of circ₀00455 in peripheral blood of DVT patients:the expression levels of miR 22 5P and miR₁38₅p were increased,and the expression levels of miR₂0b₃p,miR₂2₃p and miR₅59 were decreased.3.Bio informatics analysis showed that circRNA₀00455/mir-22-3p/NLRP3 may be involved in the development of DVT.