The Role And Mechanism Of LncRNA GUSBP5-AS In Regulating The Biological Function Of Endothelial Progenitor Cells And Recanalization Of Deep Vein Thrombosis

Venous thromboembolism(VTE)includes deep vein thrombosis(DVT)and pulmonary embolism(PE),which is the third most common vascular disease in the world,after acute coronary syndrome and stroke,and seriously threatens the lives of patients.The incidence of DVT is increasing year by year,and it increases with age.DVT can cause serious complications of post-thrombotic syndrome(PTS),and even cause fatal pulmonary embolism,which may lead to patient death.The purpose of the treatment of DVT is to prevent death caused by pulmonary embolism,spread of thrombosis,and recurrence of venous thromboembolism.Rapid thrombolysis can reduce valve damage and venous hypertension,and reduce long-term complications.At present,the standard clinical treatment for DVT is anticoagulation,but anticoagulation can only slow down the continued growth of thrombus,not accelerate the resolution of existing thrombus and eliminate existing thrombus,nor can it reduce the incidence of post-thrombotic syndrome.And it will increase the risk of bleeding in patients.After anticoagulation therapy,about 20%-50%of DVT patients still develop post-thrombotic syndrome,with intractable edema,pain,heaviness,pigmentation and even venous ulcers in the affected limb,which seriously affects the patient’s survival and quality of life.In addition,due to delays in diagnosis and treatment,some DVT patients have been treated with poor results of anticoagulation therapy.Therefore,it is of great value in clinical practice to find new methods for early diagnosis,effective prevention and treatment of DVT.Endothelial progenitor cells(EPCs)are a type of pluripotent cells that differentiated into mature endothelial cells,which were first isolated from adult peripheral blood by Asahara and others in 1997.EPCs can also secrete vascular growth factors and cell growth factors,playing an important role in the regeneration and repair of blood vessels.With the increase in research on EPCs,it has been regarded as the focus of regenerative medicine,and has shown potential application therapeutic value in vascular tissue engineering and cell therapy,especially in the treatment of cardiovascular and angiogenesis-related diseases.EPC derived from bone marrow is first released into the microcirculation,therefore,it has a relatively higher content in veins.EPCs can repair damaged or missing endothelium,increase the formation of new blood vessels,and promote the venous revascularization of thrombus.Therefore,EPCs can be used as potential seed cells for biological treatment of DVT.Emerging studies have shown that EPCs are recruited to the thrombus site,through the secretion of vascular growth factors,cytokines and other factors to accelerate the dissolution and recanalization of thrombus,and it also plays an important role in adult physiology and pathological angiogenesis.In addition,the ability of EPCs to proliferate,migrate and angiogenesis by differentiation into endothelial cells,and has recently become a promising treatment method for DVT-related diseases in patients with poor therapeutic effects in current treatment regimens.EPCs have shown their potential therapeutic value,however,there are still many challenges in their clinical application.Adverse conditions in the microenvironment affect the number and function of circulating EPCs,including smoking,advanced age,diabetes,cardiovascular risk factors,ischemic diseases,and graft vascular disease.Therefore,the development of methods to improve the recruitment of EPCs to thrombus and enhance its angiogenesis is of vital importance to the use of EPCs in the treatment of DVT.Long non-coding RNA(LncRNA)is a type of non-coding RNA with more than 200 nucleotides.It regulates molecules through a variety of mechanisms,including epigenetic modification and transcription and translation regulation.Studies have shown that lncRNA can be used as miRNA competitors or sponge molecules to indirectly regulate gene expression,and lncRNA has now become an important regulator of angiogenesis,development,differentiation,metabolism and autophagy.Studies have found that autophagy is one of the most important degradation pathways in eukaryotes,and it plays an important role in maintaining cell homeostasis and the development of vascular diseases.In addition,a variety of lncRNAs are dysregulated in various diseases including vascular diseases,and may participate in the regulation of disease progression,providing promising new targets for vascular disease treatment.However,the effects of lncRNAs on DVT resulation and recanalization remain unclear.In the previous research of this group,the differential expression of lncRNA in EPCs of DVT patients and EPCs of healthy people was detected by lncRNA chip.We found that the lncRNA GUSBP5-AS(enst00000511042)in the EPCs of DVT patients was significantly up-regulated,compared with healthy controls.We speculate that lncRNA GUSBP5-AS may be involved in regulating the angiogenesis of EPCs and in the resolution and recanalization of venous thrombosis.This study focuses on the role of lncRNA GUSBP5-AS in the biological functions of EPCs,DVT resolution and recanalization,and explores its potential molecular mechanisms.These findings may help to develop an effective new method for the treatment of DVT.This study will be discussed in four parts,the following are the purpose,methods,results and conclusions.Part Ⅰ.Effects of lncRNA GUSBP5-AS on the biological function of endothelial progenitor cellsObjective:To clarify the role of lncRNA GUSBP5-AS in the proliferation,apoptosis,migration,invasion and angiogenesis in endothelial progenitor cell in vitro.Methods:1.Lentiviruses infected EPCs.Cells were divided into four groups:interference control group(lentivirus delivery LV3-NC vector plasmid into EPCs),sh-lncRNA GUSBP5-AS group,overexpression control group(lentivirus delivery LV5-NC vector plasmid into EPCs),lncRNA GUSBP5-AS overexpression group.Immunofluorescence microscope was used to observe the fluorescence intensity,and RT-qPCR was used to detect the infection efficiency;2.CCK8 experiment was used to detect the changes in the proliferation ability of EPCs in each group;3.Flow cytometry was used to detect cell apoptosis in each group;4.Scratch test and Transwell test were performed to detect changes in the migration and invasion functions of EPCs in each group;5.In vitro tube formation experiment was performed to test the changes in the angiogenesis ability of EPCs in each group.Resluts:1.After each group of lentiviruses infected EPCs,their cell morphology and growth status were good.The infection efficiency effect of each group was more than 95%,and the results of RT-qPCR showed that the infection rate of the lentivirus is high,and the infection effect is stable and reliable;2.Down-regulation of lncRNA GUSBP5-AS in EPCs significantly reduced the proliferation ability of EPCs,compared to the LV3-NC group.On the contrary,overexpression of lncRNA GUSBP5-AS significantly enhanced the proliferation ability of EPCs,compared to the LV5-NC group.There is no significant difference between the LV3-NC group and the LV5-NC group;3.Compared with the LV3-NC group,down-regulation of lncRNA GUSBP5-AS in EPCs significantly significantly promoted the apoptosis of EPCs.On the contrary,compared with the LV5-NC group,overexpression of lncRNA GUSBP5-AS significantly inhibited the apoptosis of EPCs.And there is no significant difference between the LV3-NC group and the LV5-NC group;4.Down-regulation of lncRNA GUSBP5-AS in EPCs significantly reduced the ability of migration and invasion in EPCs,compared to the LV3-NC group.On the contrary,overexpression of lncRNA GUSBP5-AS significantly enhanced the ability of migration and invasion in EPCs,compared to the LV5-NC group.There is no significant difference between the LV3-NC group and the LV5-NC group;5.Down-regulation of lncRNA GUSBP5-AS in EPCs significantly reduced the angiogenesis ability of EPCs,compared to the LV3-NC group.On the contrary,overexpression of lncRNA GUSBP5-AS significantly enhanced the angiogenesis ability of EPCs,compared to the LV5-NC group.There is no significant difference between the LV3-NC group and the LV5-NC group.Conclusion:LncRNA GUSBP5-AS played a key role in the regulation of the biological functions of EPCs.Down-regulation of lncRNA GUSBP5-AS significantly reduced the proliferation,migration,invasion and angiogenesis capabilities of EPCs,and promoted their apoptosis;overexpression of lncRNA GUSBP5-AS had the opposite effect.Part Ⅱ.Effects of lncRNA GUSBP5-AS on angiogenesis in vivo and deep vein thrombosisObjective:To clarify the effects of lncRNA GUSBP5-AS on angiogenesis in vivo and its use in endothelial progenitor cells for the treatment of deep vein thrombosis.Methods:1.EPCs mixed with Matrigel plugs were injected subcutaneously into nude mice to simulate angiogenesis in vivo,and the effect of lncRNA GUSBP5-AS on the angiogenesis ability of endothelial progenitor cells in each group was tested;2.HE staining was performed to observe the formation of new blood vessels in each group;3.The nude mouse DVT model was established.EPCs were injected.One week later,the thrombus was obtained,and the thrombus length and dry weight were weighed,and the DVT size was statistically analyzed;4.Observe the migration of EPCs in the thrombus under a fluorescence microscope;5.Immunofluorescence staining was used to detect the expression of MMP2 and CD34 in venous thrombosis,and analyze the thrombolysis and revascularization.Resluts:1.Compared with the LV3-NC group,the down-regulation of lncRNA GUSBP5-AS group significantly reduced new blood vessels;compared with the LV5-NC group,the lncRNA GUSBP5-AS overexpression group significantly enhanced angiogenesis in vivo;There is no significant difference between the LV3-NC group and the LV5-NC group;2.The results of HE staining showed that there were fewer new blood vessels in Matrigel plugs injected with EPCs that down-regulated lncRNA GUSBP5-AS.In Matrigel plugs injected with EPCs overexpressing lncRNA GUSBP5-AS,there are more new blood vessels,and there are blood cells in the official cavity;3.EPCs overexpressing lncRNA GUSBP5-AS significantly reduced the size of deep vein thrombosis;4.Overexpression of lncRNA GUSBP5-AS significantly enhanced the migration ability of EPCs to deep vein thrombosis sites;5.Overexpression of lncRNA GUSBP5-AS promoted the resolution and revascularization of DVT,thus playing an important role in the treatment of deep vein thrombosis.Conclusion:LncRNA GUSBP5-AS played an important role in regulating the angiogenesis of EPCs in vivo and the resolution and recanalization of DVT.Overexpression of lncRNA GUSBP5-AS significantly enhanced the homing and angiogenesis ability of EPCs,and promoted the dissolution and revascularization of DVT.Part Ⅲ.The studies on mechanisms of lncRNA GUSBP5-AS in regulation of the biological function in endothelial progenitor cellsObjective:To explore the molecular mechanisms of lncRNA GUSBP5-AS regulating the biological function in endothelial progenitor cells.Methods:1.The mRNA differential expression profiles in endothelial progenitor cells regulated by lncRNA GUSBP5-AS were detectd by high-throughput sequencing,and cluster analysis was used to screen mRNA in LV5-NC EPCs and lncRNA GUSBP5-AS EPCs;2.Perform GO enrichment analysis and KEGG analysis to screen genes closely related to cell migration and angiogenesis;3.Biological information analysis was used to analyze the regulatory network of lncRNA-miRNA-mRNA,comprehensive analysis and screening of lncRNA GUSBP5-AS targets;qRT-PCR was performed to verify the differentially expressed miRNAs and mRNA levels in EPCs regulated by lncRNA GUSBP5-AS;The dual luciferase reporter gene experiment was used to verify the target miRNA of lncRNA GUSBP5-AS,and RT-qPCR was performed further to teste its regulatory effect;4.Perform GO enrichment analysis and KEGG analysis to screen genes closely related to cell migration and angiogenesis;5.Synthesize miRNA mimics,miRNA inhibitor and its empty plasmid lentivirus,and co-infect EPCs with lncRNA GUSBP5-AS,EPCs were divided into four groups:NC group,lncRNA GUSBP5-AS overexpression group,lncRNA GUSBP5-AS+miR-223-3p co-infection group and miR-223-3p mimics group,and check the infection efficiency;6.In vitro tube formation experiment was performed to test the changes in the angiogenesis ability of EPCs in the four group;7.The scratch test and Transwell test were performed to detect the changes in the migration and invasion abilities of the four groups in EPCs;8.Starbase,TargetScan,miRanda and miRDB databases were used to predict the downstream target genes of miR-223-3p;9.The dual luciferase reporter gene experiment was used to detect the relationship between miR-223-3p and FOXO1,and RT-qPCR was used further verified its regulatory effect;10.Synthesize FOXO1 overexpression,sh-FOXO1 and its empty plasmid lentivirus,EPCs wree infect lentivirus,And cells were divide into three groups:NC group,FOXO1 overexpression group and sh-FOXO1 group,and test the infection efficiency;11.In vitro tube formation experiment was performed to test the changes in the angiogenesis ability of EPCs in the three group;12.The scratch test and Transwell test were performed to detect the changes in the migration and invasion abilities of the three groups in EPCs;13.The downstream pathways regulated lncRNA GUSBP5-AS in EPCs were tested via western blot;14.The downstream pathway regulated by lncRNA GUSBP5-AS targeting miR-223-3p in EPCs was verify through western blot;15.Immunofluorescence was used to detect the effect of lncRNA GUSBP5-AS on the expression of F-actin in EPCs.Resluts:1.2,430(52%)genes in the lncRNA GUSBP5-AS overexpression group were up-regulated,while 2,220(48%)genes were down-regulated,compared to the control group;2.LncRNA GUSBP5-AS is mainly closely related to metabolic process,stress,cell cycle,migration,and gene expression;3.MiR-223-3p had a complementary binding site for the 3’untranslated region(3’UTR)of lncRNA GUSBP5-AS.The dual luciferase reporter gene assay confirmed that miR-223-3p bound to lncRNA GUSBP5-AS;4.The dual luciferase reporter gene experiment showed that miR-223-3p was the direct target miRNA of lncRNA GUSBP5-AS;RT-qPCR results showed that the overexpression of lncRNA GUSBP5-AS down-regulated the expression of miR-223-3p,and lncRNA GUSBP5-AS could attenuate the overexpression levels of miR-223-3p;5.The effects of cells infected by lentiviruses in each group are stable,the infection efficiency is high,and the cell morphology and growth state are good;6.The lncRNA GUSBP5-AS in EPCs significantly enhanced the angiogenesis ability of EPCs,compared with the NC group,and the overexpression of miR-223-3p significantly reduced the angiogenesis ability.Compared with the lncRNA GUSBP5-AS group,the angiogenesis ability of the lncRNA GUSBP5-AS+miR-223-3p mimics group was significantly inhibited,and there was no significant difference compared with the NC group;7.The lncRNA GUSBP5-AS in EPCs significantly enhanced the migration and invasion abilities of EPCs,compared with the NC group,and the overexpression of miR-223-3p significantly reduced the migration and invasion abilities.Compared with the lncRNA GUSBP5-AS group,the migration and invasion abilities of the lncRNA GUSBP5-AS+miR-223-3p mimics group were significantly inhibited,and there was no significant difference compared with the NC group;8.The downstream target genes of miR-223-3p predicted by Starbase,TargetScan,miRanda and miRDB databases and the comprehensive analysis based on the results of RNAseq suggested that FOXO1 was the predicted target of miR-223-3p,which may play an important role in regulating the biological functions of EPCs9.FOXO1 was the direct target of miR-223-3p via the dual luciferase reporter gene assay and RT-qPCR further confirmed that miR-223-3p down-regulated the expression level of FOXO1;10.EPCs infected with FOXO1 over-expressed and down-regulated lentivirus had good morphology and growth;the infection effect is stable and the infection efficiency is high;11.Overexpression of FOXO1 significantly enhanced the angiogenesis ability of EPCs,compared to the NC group,while downregulation of FOXO1 had the opposite effect;12.Overexpression of FOXO1 significantly enhanced the migration and invasion capabilities of EPCs,compared to the NC group,while downregulation of FOXO1 had the opposite effect;13.Overexpression of lncRNA GUSBP5-AS increased the protein expression levels of FOXO1,p-Akt,FGF2,MMP2 and MMP9,while downregulation of lncRNA GUSBP5-AS had the opposite effect;14.MiR-223-p inhibitor significantly enhanced the expression of FOXO1,p-Akt,FGF2,MMP2 and MMP9,while miR-223-p mimics inhibited their expression levels;15.Overexpression of lncRNA GUSBP5-AS significantly enhanced the expression of F-actin in EPCs,while down-regulation of lncRNA GUSBP5-AS impaired the expression of F-actin.Conclusion:LncRNA GUSBP5-AS interacted with miR-223-3p,regulated FOXO1 mRNA and protein expression levels,activated the Akt pathway,and enhanced fibroblast growth factor 2(FGF2),matrix metalloproteinase-2/9(MMP2/9)and F-actin expression,thereby enhancing the homing and angiogenesis ability of EPCs.Part Ⅳ.Clinical application of lncRNA GUSBP5-AS in deep vein thrombosisObjective:To explore the clinical value of lncRNA GUSBP5-AS in deep vein thrombosis.Methods:1.Collect peripheral blood of DVT patients and healthy adults,induced and cultured EPCs,and detectd the expression levels of lncRNA GUSBP5-AS,miR-223-3p and FOXO1 in each group by RT-qPCR,and analyzed the correlation between them;2.The regulatory effect of miR-223-3p on lncRNA GUSBP5-AS in EPCs was tested by RT-qPCR;3.The regulatory effect of lncRNA GUSBP5-AS on FOXO1 in EPCs was tested by RT-qPCR.Resluts:1.Spearman correlation analysis showed that in the EPC samples of 15 DVT patients,lncRNA GUSBP5-AS was negatively correlated with miR-223-3p,FOXO1 was negatively correlated with miR-223-3p expression,and lncRNA GUSBP5-AS was positively correlated with FOXO1;2.MiR-223-3p mimics and inhibitor attenuated and enhanced the expression level of lncRNA GUSBP5-AS in EPCs,respectively;3.Knockdown and overexpression of lncRNA GUSBP5-AS reduced and increased the expression level of FOXO1 mRNA in EPCs,respectively.Conclusion:LncRNA GUSBP5-AS,miR-223-3p and FOXO1 had a certain correlation in the EPCs of DVT patients,providing a new biomarker and promising new targets for DVT patients in the diagnosis,prevention and treatment of DVT.