Study On The Effect Of Mmp13 In Venous Endothelial Cells On Deep Venous Thrombosis

Deep venous thrombosis(DVT)is a common complication after major orthopedic surgery.It has a high incidence and is difficult to predict and diagnose in the early stage,which seriously affects the quality of life of patients.The pathogenesis of DVT is complex,involving many processes such as activation of venous endothelialcells,inflammatory response and imbalance of coagulation/anticoagulation system.Among them,gene regulation of Klfs,Mmps,IL and TF in venous endothelial cells is closely related to thrombosis.In the early stage of the research group,through the detection of RNA-seq in the inferior vena cava tissue of normal group and DVT model group,it was found that the expression of Mmp13 was up-regulated in DVT group.In addition,after tail vein injection of siRNA interfering with the expression of Klf15,the expression of Mmp13 in venous endothelial cells was up-regulated and the wet weight of thrombus increased,suggesting that Klf15 may regulate the expression of downstream Mmp13 and then affect thrombosis.In this study,we conducted an experimental study on the expression changes and role of Mmp13 in Klf15 gene knockout homozygous mice DVT model,and further verified the effect of Klf15 on Mmp 13 expression.Objective:1.The target genes regulated by KLF15 were screened by chromatin co-immunoprecipitation sequencing.2.To determine whether Mmp 13 plays a role in the formation of deep veous thrombosis and to further verify whether Klf15 affects thrombosis by regulating the expression of Mmp 13 in C57BL/6 mice with wild type and Klf15 gene knockout homozygotes,we observe the thrombosis and the changes of Mmp13 expression in vein endothelium of wild type and homozygous mice.Methods:Part Ⅰ:Screening target genes regulated by klfl5 by chromatin immunoprecipitation sequencingThe KLF15 overexpression plasmid was constructed and transformed into HUVECs.Target genes bound by KLF15 in HUVECs were enriched by chromatin immunoprecipitation.Combined with high-throughput sequencing,the DNA sites specifically binding to transcription factor KLF15 were analyzed in the whole genome,and the differential genes were analyzed by GO and KEGG.Part Ⅱ:The DVT model of Klf15 gene knockout homozygous mice was constructed by stenosis method.siRNA was injected into the tail vein to interfere with the expression of Mmpl3 in mice.The expression changes and role of Mmp13 in mouse DVT model were analyzed.1.Experiment 1:20 wild-type C57BL/6 mice were randomly divided into Blank-WT group(n=10)and DVT-WT group(n=10).Ten Klfl5 gene knockout homozygous C57BL/6 mice were selected and randomly divided into Blank-KO group(n=5)and DVT-KO group(n=5).All the above mice were raised in SPF grade,regardless of gender,with a body weight of 25～26g.The thrombus model was established by the method of inferior vena cava stenosis in both DVT-WT and DVT-KO groups.24 hours after operation,the thrombus and the corresponding venous endothelial tissue were taken out,and the wet weight and length of thrombus were observed and measured.The thrombus formation rate was calculated,and the expression of Mmp13 in vein endothelial cells was detected by Q-PCR.2.The experiment 2:12 wild-type C57BL/6 mice were randomly divided into NC-WT group(n=6)and Mmp 13-siRNA-WT group(n=6).Twelve Klfl5 gene knockout homozygous C57BL/6 mice were selected and randomly divided into NC-KO group(n=6)and Mmp13-siRNA-KO group(n=6).All the above mice were raised in SPF grade,male or female,and their body weight was about 25～26g.200μl saline was injected into tail vein 24 hours before modeling in NC-WT group and NC-KO group,and 200μl Mmp13-siRNA was injected into tail vein in Mmp13-siRNA-WT group and Mmp13-siRNA-KO group.24 hours after the establishment of the model,the thrombus and the corresponding venous endothelial tissue were removed,the weight and length of thrombus were observed and measured,the thrombus formation rate was calculated,and the expression of Mmp13 in vein endothelial cells was detected by Q-PCR.RNA sequencing technique was used to detect the differential gene expression in venous endothelial tissue of NC-WT group and Mmp13-siRNA-WT group,and GO and KEGG analysis were performed to study its effect on thrombosis.3.GraphPadPrism7 statistical software was used to analyze the experimental data,comprehensively analyze the effect of Mmp13 on deep venous thrombosis,and verify the regulatory relationship between Klf15 and Mmp 13.Results:Part ⅠpLVX-mCMV-ZsGreenl-Puro-DX-KLF15 overexpression plasmid was successfully constructed.Chromatin co-immunoprecipitation sequencing showed that a large number of KLF15 target genes were selectively enriched in KLF15 overexpressed HUVECs and involved in a variety of signal pathways.Part ⅡExperiment 11.Survival rate and thrombosis prevalence of mice:there was no death in the DVT group and the death rate was 0.the thrombosis prevalence was 0;In DVT-WT group,one mouse died,the mortality rate was 10%,and the thrombosis prevalence was 77.77%.In DVT-KO group,one mouse died,the mortality rate was 20%,and the thrombosis prevalence was 75%.2.The weight of inferior vena cava thrombosis:no thrombosis was found in Blank-WT group and Blank-KO group;The weight of thrombus in DVT-WT group was 16.50±3.68 mg,the weight of thrombus in DVT-KO group was 24.70±4.69mg.The weight of thrombus in DVT-KO group was significantly higher than that in DVT-WT group(P<0.05).3.The expression of Mmp 13 in mouse inferior vena cava endothelial cells was detected by Q-PCR.Compared with Blank-WT group,the expression of Mmp 13 in DVT-WT group was significantly increased(P<0.05).Compared withDVT-WTgroup,the expression of Mmp 13 in DVT-KO group was significantly increased(P<0.05).Experiment 21.Tail vein injection:Four groups were given tail vein injection,The success rate of NC-WT group and Mmp13-siRNA-KO group was 83.3%;The success rate was 100%in Mmp13-siRNA-WT group and NC-KO group.2.Survival rate and thrombosis prevalence of mice:DVT model was established by inferior vena cava stenosis method.In NC-WT group,0 mice died,the mortality was 0,and the thrombosis prevalence was 100%;In Mmp13-siRNA-WT group,one mouse died,the mortality was 16.6%,and thrombosis prevalence was 83.3%;In NC-KO group,one mouse died,the mortality was 16.6%,and the thrombosis prevalence was 66.7%;In Mmp13-siRNA-KO group,0 mice died,the mortality was 0,and the thrombosis prevalence was 80%.3.The weight of IVC thrombosis in mice:The weight of thrombus in NC-WTgroup,Mmp13-siRNA-WTgroup,NC-KOgroup and Mmp13-siRNA-KO group was 17.60±1.01mg、9.02±2.16mg、25.13±1.18 mg and 13.83 ±2.31mg.The weight of thrombus in Mmp 13-siRNA-WT group was significantly lower than that in NC-WT group(P<0.05).Compared with NC-KO group,the wet weight of thrombus in Mmp13-siRNA-KO group was lighter(P<0.05).4.The expression of Mmp 13 in mouse inferior vena cava endothelial cells:Compared with NC group,the expression of Mmp13 in Mmp13-siRNA group was significantly decreased after tail vein injection of Mmp13-siRNA(P<0.05).5.RNA-seq results:A total of 1855 differentially expressed genes were identified in Mmp 13-siRNA-WT group and NC-WT group,of which 932 genes were up-regulated and 923 genes were down-regulated(|log2(FoldChange)|>2,padj<0.05).The differentially expressed genes Nlrp3,IL-1βand Cxcr2,Cxcl2 related to thrombosis were screened out,which may affect thrombus formation through inflammatory reaction and immune regulation.Conclusion(s):1.Mmp13 promotes thrombosis in venous endothelial cells of mouse DVT model.2.Mmp13 may affect thrombosis by regulating Nlrp3,IL-1β and Cxcr2,Cxcl2.3.Klf15 may affect thrombosis by regulating the expression of Mmp13.