| Background and objectiveDeep vein thrombosis?DVT?refers to the abnormal clot formation within the deep vein,mostly of the lower extremities.Pulmonary embolism?PE?,a potentially life-threatening condition,occurs when a thrombus detaches from the DVT original site and travels to the lung.Post-thrombotic syndrome?PTS?is the most common long-term complication of DVT,seriously affecting the quality of patients'life.Venography is the gold standard for the diagnosis of DVT with lower exstremities,which has a high accuracy.However,it is invasive and some patients may be allergic to radiography agent.D-dimer is widely used to rule out of suspected DVT clinically.D-dimer assay is high sensitive,but has a poor specificity.In the diagnosis of lower extremities DVT,the specificity of P-selectin is rather high.However,the testing methods and optimal cutoffs for P-selectin vary a lot in different laboratories.Therefore,novel candidates or complementary biomarkers are still imperative in DVT diagnosis.Circulating mi RNAs play roles in the development,diagnosis and prognosis in various diseases.Ciculating miRNA-320b had been proved to be differentially expressed in patients suffering from venous thrombosis.However,its expression in patients with acute DVT of lower extremities wasn't clear.Consequently,we detected the expression of plasma miRNA-320b as well as D-dimer and P-selectin in DVT and PTS patients,hoping to provide more information for the development and diagnosis of DVT.Methods1.Patient recruitment:Plasma samples were recruited from patients with acute DVT of lower extremities and PTS to be as DVT group and PTS group,separately.During the same period,consecutive healthy volunteers with age and gender matched were enrolled as the control group.2.Plasma preparation and preservation:Fasting peripheral venous blood was extracted from each study subject at the next morning after admission.After centrifugation,plasma was separated and immediately stored for further analysis.3.Detection of the relative expression of plasma miRNA:The relative expression of mi RNA-320b and miRNA-16 were evaluated by qRT-PCR assay.The individual relative expression of miRNA-320b was calculated by the equation of2–?Cp(?Cp=Cp miRNA-320b–Cp miRNA-16),and 2–??Cp was expressed as fold change???Cp=?Cp patients–?Cp controls?.4.Detection of plasma D-dimer:Immunoturbidimetric assay was performed to assess plasma D-dimer levels.5.Detection of plasma P-selectin:ELISA assay was used to determine the concentrations of plasma P-selectin.6.Targets identification of miRNA-320b:Target gene prediction softwares were conducted to identify the predicted target genes of miRNA-320b.After that,we performed GO and KEGG pathway analysis of the putative target genes.7.Statistical analysis:Statistical analyses were conducted by SPSS statistics software version 21.0.Normal continuous variables were presented as mean±standard deviation,while the non-normally distributed data were presented as median and inter-quartile range.Categorical values were expressed as counts and percentages.One-way ANOVA was performed to compare the differences among three cohorts.Correlations were evaluated using Spearman correlation.ROC curves,AUCs,sensitivities and specificities of these markers were constructed to determine the diagnostic power and accuracy for DVT.All the analyses were two-tailed and P values less than 0.05 were deemed statistically significant.Results1.Compared with control group,the relative expression of plasma miRNA-320b in patients with acute DVT of lower extremities was significantly elevated?P<0.001?and the fold change was 1.79.The relative expression of plasma miRNA-320b between PTS group and controls showed no significant difference?P=0.091?.2.Compared with control group,the levels of D-dimer in patients with acute DVT of lower extremities were significantly higher?P<0.001?.While,the levels of D-dimer between PTS patients group and control group showed no significant difference?P=0.139?.3.Compared with control group,the levels of P-selectin in patients with acute DVT of lower extremities were significantly higher?P<0.001?.While,the levels of P-selectin between PTS patients group and control group showed no significant difference?P=0.118?.4.In the acute lower extremities DVT group,the correlation between plasma miRNA-320b and D-dimer was positively correlated?r=0.358,P=0.001?.While,the correlation between plasma miRNA-320b and P-selectin was not statistically significant?r=-0.072,P=0.524?.5.ROC curve analyses showed that the AUC for miRNA-320b was 0.787?95%CI:0.710-0.864?;the AUC for D-dimer was 0.882?95%CI:0.821-0.943?;the AUC for P-selectin was 0.825?95%CI:0.754-0.895?.The AUC for miRNA-320b and D-dimer was 0.886?95%CI:0.828-0.944?;the AUC for miRNA-320b and P-selectin was 0.904?95%CI:0.853-0.954?;the AUC for P-selectin and D-dimer was 0.935?95%CI:0.893-0.978?;the AUC for mi RNA-320b,P-selectin and D-dimer was 0.953?95%CI:0.920-0.986?.6.Bioinformatics analysis showed that the biological function of miRNA-320b predicted target genes involved angiogenesis,blood vessel development and blood vessel morphogenesis.KEGG enrichment terms were mainly converged on PI3K-Akt signaling pathway,TGF-beta signaling pathway and Cytokine-cytokine receptor interaction.Conclusions1.The relative expression of plasma miRNA-320b in patients with acute DVT of lower extremities was significantly upregulated,indicating that it might have a potential diagnostic value for DVT.2.D-dimer and P-selectin were significantly increased in patients with acute DVT of lower extremities compared with controls,and the levels of plasma miRNA-320b and D-dimer were positively correlated.3.The simultaneous detection of abnormal plasma miRNA-320b,D-dimer and P-selectin could improve the accuracy of DVT diagnosis and might contribute to the early dectection of DVT. |
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