The Role And The Mechanisms Of Long Non-coding RNA WTAPP1 In Regulating The Migration And Angiogenesis Of Endothelial Progenitor Cells

Deep vein thrombosis(DVT)is a common disease in the clinical with an annual incidence range from 0.01% to 0.27%.Nowadays,the treatments of DVT mainly contained: 1.Preventing the symptomatic pulmonary embolism and the propagation of thrombosis,alleviating pain and edema in the affected extremities;2.Removing the thrombus timely,such as thrombolysis via urokinase,thrombectomy by surgery;3.Preventing thrombosis recurrence and post-thrombotic syndrome(PTS)through long term anticoagulation.PTS is one of the long term complications of DVT even if the patients got a strict anticoagulation.The incidence of PTS at the 2 years of DVT is as high as 20%-50%.Ulcer was found in about 15% affected extremities.The incidence of venous claudication is as high as 40%.There are 15% patients who will suffered mobility limitation and 100% patients whose life quality is affected.The effects of the therapies in PTS are poor.Thus,it is very necessary to find out a new diagnosis,safe and efficient treatments,accurate evaluation of prognosis via deep studies in the molecular mechanisms of DVT.It will bring good news to the patients and is the necessary of the national scientific and technical key task.Stem cell therapy,with a brilliant prospect,is a new science and technology.It brings a hope for several intractable diseases.It has been got some positive results in pre-clinical and clinical studies of cardiovascular diseases.Treatment with stem cells in DVT and in the prevention of PTS is a new way,having a profound significance.Endothelial progenitor cells(EPCs),a kind of multipotent stem cells,mainly stored in bone marrow.The number of EPCs increases as soon as 24 hours after DVT and peaks at 48 hours after DVT.The mobilized EPCs are released into the vein firstly and play an important role in the recanalization of thrombus,especially in the aspects of angiogenesis and repairment of endothelium.However,the mobilized of EPCs was limited and subjected to many risk factors,including age,smoke and diabetic mellitus et al.Thus promotion of the homing and angiogenesis of EPCs is very important.In the present study,mononuclear cells were isolated from peripheral blood via density gradient centrifugation.These cells were cultured in the special EGM-2 MV medium.The cellular morphology,cell surface markers and the results of staining with Dil-ac-LDL and FITC-UEA were fit to the characteristic of EPCs.A long non-coding RNA,named WTAPP1,was selected from the previous lnc RNA gene array examination.CCK8 was performed to detect the proliferation of EPCs after up-regulation and down-regulation with lentivirus.Wound healing assay and invasive assay to detect the capacity of migration,tube formation assay to detect the capacity of angiogenesis were performed.Finding the targeted gene through bioinformatics analysis,we found that metal matrix proteinase 1(MMP1)was the targeted gene of lnc RNA WTAPP1.MMP1 plays an important role in cell migration and angiogenesis.Meanwhile,we found a related mi RNA,which could target to MMP1,via mi RNA gene array.Furthermore,we also tested the relationship among the lnc RNA WTAPP1 and autophagy,Akt/PI3K/m TOR signal pathway.The result above lays a theoretical foundation to promote the capacity of EPCs.This may provide an experimental evidence of the treatment with EPCs for DVT and ischemic diseases.Besides,this could also provide a new perspective and method for the clinical treatment of DVT.There were three parts in this study.We will discuss them separately.Part 1.Culture and identification of endothelial progenitor cells isolated from peripheral bloodPurpose: To provide cells for the further study via identification after density gradient centrifugation and cultureMethods: Peripheral blood was collected from healthy human-being.And then mononuclear cells were isolated via density gradient centrifugation.These cells were inoculated into a six-well plate which was coated with type 1 collagen.After 7 days' culture with EGM-2 MV,half of the medium was changed to newer.When the cells grew enough cover the 90% of the well.Cell morphology was observed under light microscope.Cell identification was performed via flow cytometry and fluorescence staining with Dil-Ac-LDL and FITC-UEA-1.Result: The cells grew like polygon,fusiformis and oval in a colony way.Cells of the two to three passages grew like a flagstone.These cell morphologies fit with the classic characteristic of EPCs.The results of fluorescence staining showed that these cells had the ability of taking Dil-Ac-LDL and bonding with FITC-UEA-1.Flow cytometry detected the cell surface marker,including CD31,CD34,CD45,CD133 and CD309,finding that the results fit with the characteristic of EPCs.Conclusion: Cells isolated from the peripheral blood via density gradient centrifugation and cultured with EGM-2 MV fit with the characteristic of EPCs in cell morphologies.These cells could take Di I-Ac-LDL and bonding with FITC-UEA-1.The results of flow cytometry showed that the cells were EPCs which could be used in further experiment.Part 2 The role of long non-coding RNA WTAPP1 in the regulation of migration and angiogenesis of endothelial progenitor cellsPurposes: To explore the role of long non-coding RNA WTAPP1 in the regulation of EPCs migration and angiogenesis.Methods: 1.Lentiviruses of si RNA and overexpression of lnc RNA WTAPP1 were manufactured to infect EPCs.Infection efficiency was measured by fluorescence microscope.The effect of lnc RNA WTAPP1 was detected by q RT-PCR;2.Cell groups: normal EPCs,vector infected EPCs,lnc RNA WTAPP1 si RNA lentivirus infected EPCs,lnc RNA WTAPP1 overexpression lentivirus infected EPCs;3.CCK8 was performed to detect the proliferation of the different cell groups;4.Wound healing assay and invasive assay were performed to detect the capacity of migration in EPCs;5.The capacity of angiogenesis of the different groups of EPCs was detected by tube formation in vitro and in vivo.Results: 1.Infection efficiency of the every group of lentivirus infection was more than 90%.The results of q RT-PCR showed that different lentivirus group could regulate the expression of lnc RNA WTAPP1;2.There were no differences among the four groups of EPCs.P > 0.05.These results showed lnc RNA WTAPP1 had no effects on cell proliferation;3.The results of wound healing assay showed that the number of EPCs in the lnc RNA WTAPP1 si RNA group was less than the number of EPCs in the vector group(116.7±19.9 VS 182.0±13.7,P < 0.01).The cell number of migration in the group of lnc RNA WTAPP1 overexpression were more than in the vector group(249.0±28.0 VS 182.0±13.7,P < 0.01);The results of invasive assay showed that the cell migration in the lnc RNA WTAPP1 si RNA group was less than the number of EPCs in the vector group(18.7±2.5 VS 33.0 ±2.0,P < 0.001).The cell migration in the lnc RNA WTAPP1 overexpression group was more than the number of EPCs in the vector group(45.7± 4.2 VS 33.0 ±2.0,P < 0.01);4.Tube formation in vitro showed that lnc RNA WTAPP1 si RNA could decrease the number of tubes(28.5 ± 4.8 VS 43.5±3.7,P < 0.001),while lnc RNA WTAPP1 could enhance the number of tubes,compared with tubes in vector groups(63.3±5.0 VS 43.5±3.7,P < 0.001).In vivo tube formation assay showed a similar result.Lnc RNA WTAPP1 si RNA could decrease the area of tubes(0.30 ± 0.04 VS 0.43 ± 0.09,P < 0.05),while lnc RNA WTAPP1 could enhance the area of tubes(0.75±0.05 VS 0.43 ± 0.09,P < 0.001),compared with tubes in vector groups.Conclusion: lnc RNA WTAPP1 overexpression could promote the capacities of migration and angiogenesis of EPCs,while downregulation of lnc RNA WTAPP1 could inhibit these functions of EPCs.Part 3 The study on the mechanisms of long non-coding RNA WTAPP1 in the regulation of migration and angiogenesis of endothelial progenitor cellsPurposes: To explore the mechanisms of long non-coding RNA WTAPP1 in the regulation of migration and angiogenesis of endothelial progenitor cells.Methods: 1.Detect the effect of lnc RNA WTAPP1 on MMP1 by q RT-PCR.The groups of EPCs were divided into four groups: normal EPCs,vector infected EPCs,lnc RNA WTAPP1 si RNA lentivirus infected EPCs,lnc RNA WTAPP1 overexpression lentivirus infected EPCs;2.Detect the effect of lnc RNA WTAPP1 on MMP1 protein by Western Blot.The groups of EPCs were divided into four groups: normal EPCs,vector infected EPCs,lnc RNA WTAPP1 si RNA lentivirus infected EPCs,lnc RNA WTAPP1 overexpression lentivirus infected EPCs;3.To explore the role of mi RNA in the regulation of MMP1 by lnc RNA WTAPP13.1 To detect all the mi RNAs expressed differently in normal EPCs and EPCs with lnc RNA WTAPP1 si RNA lentivirus infection via mi RNA array examination;3.2 To select a mi RNA which has the potential role in regulating MMP1;3.3 To test the result of mi RNA array by q RT-PCR;3.4 To make the lentiviruses targeted to regulate the mi RNA,infecting to EPCs and then examining the efficiency of infection;3.5 To detect the role of mi R-3120-5P in the regulation of MMP1 by q RT-PCR and western blot;3.6 To test the role of mi R-3120-5P in the migration and angiogenesis by invasive assay and tube formation assay;4.To explore the relationship between the expression of lnc RNA WTAPP1 and mi R-3120-5P.Pearson correlation analysis was performed between the expression of lnc RNA WTAPP1 and the expression of mi R-3120-5P in the EPCs with lnc RNA WTAPP1 overexpression;5.To explore the relationship among lnc RNA WTAPP1?MMP1 m RNA and mi R-3120-5P via bioinformatic analysis;According to the results of Blast among the gene sequence of lnc RNA WTAPP1?MMP1 m RNA and mi R-3120-5P,the molecular regulation mechanisms were explained;6.To find out the related signal pathways according to coding-noncoding gene co-expresion analysis of lnc RNA WTAPP1;7.To explore the role of Akt/PI3K/m TOR signal pathways in the regulation of MMP1 by lnc RNA WTAPP1 via Western blot to detect the phosphorylation of Akt,PI3 K and m TOR.8.To explore the role of autophagy in the regulation of MMP1 by lnc RNA WTAPP1.Rapamycin and Atg5 si RNA transfection were added into the lnc RNA WTAPP1 overexpression and si RNA lentivirus infection respectively.MMP1 protein was detected by Western blot.Results: 1.The expression of MMP1 was attenuated in the group of lnc RNA WTAPP1 si RNA via q RT-PCR examination.There was a significant difference when compared with vector lentivirus infection(p<0.001).The results of MMP1 protein expression detected by western blot were similar to the results of q RT-PCR(P<0.05).Pearson correlation analysis found that the expression of MMP1 m RNA were positive correlation.It also had a significant difference(p<0.05).2.Mi RNA array found that there were 17 mi RNAs with a significant difference between lnc RNA WTAPP1 si RNA infection groups and vector groups.Among the 17 mi RNAs,three were up-regulation and 14 were down-regulation.The expression of mi R-3120-5P in the lnc RNA WTAPP1 si RNA infected EPCs was as high as 3.3 times than the EPCs with vector infection.This result fit with the results of q RT-PCR.There was a negative correlation between lnc RNA WTAPP1 and mi R-3120-5P analyzed by Pearson correlation analysis(p<0.001).It was also found that MMP1 had some complementary fragments with mi RNA at the site of 3' UTR predicted by Target Scan 7.0.3.Comparison of the nucleotide sequence among mi R-3120-5P,lnc RNA WTAPP1 and MMP1,we found that both MMP1 and lnc RNA WTAPP1 had the complementary sequences with mi R-3120-5P.4.The results of Western blot showed that MMP1 could be inhibited by mi R-3120-5P.5.Invasive assay for mi R-3120-5P showed that minics of mi R-3120-5P could reduce the numbers of invasive cells compared with vector infected EPCs(24.7 ± 3.5 VS 38.0 ± 6.2,P < 0.01).While mi R-3120-5P inhibitor could enhance the number of EPCs invasion compared with vector infection(59.0 ± 4.6 VS 38.0 ± 6.2,P < 0.01).There was a significant difference.The results of tube formation showed that mi R-3120-5P minics could inhibited the capacity of angiogenesis compared with vector infection(20.7+4.0 VS 30.3+2.5,P < 0.05).While mi R-3120-5P inhibitor could promote the capacity of angiogenesis compared with vector infection(41.3+4.1 VS 30.3+2.5,P < 0.01).6.The results of CNC analysis found that there were a lot m RNAs with a similar expression pattern.Most of the m RNAs had something with cell differentiation and angiogenesis.And most of them were related with Akt/PI3K/m TOR signal pathway and autophagy pathway.7.The phosphorylation of Akt,PI3 K and m TOR,which was regulated by lnc RNA WTAPP1,was detected by Western blot examination,finding that lnc RNA WTAPP1 overexpression could enhance the phosphorylation these proteins.8.Lnc RNA WTAPP1 overexpression in EPCs could impair the expression of LC3 B and Atg5 but promote the expression of P62.And vice versa.Conclusion : The mechanisms in the EPCs migration and angiogenesis regulated by lnc RNA WTAPP1 included: lnc RNA WTAPP1 could promote the expression of MMP1 via mi R-3120-5P and autophagy which played an important role in the regulation of cell migration and angiogenesis.Furthermore,Akt/PI3K/m TOR might also be involved.