Preliminary Study Of Non-coding RNAs Of Platelet In Patients With Immune Thrombocytopenia

Part One Investigation of platelet apoptosis in adult patients with chronic immune thrombocytopeniaBackground and objective: Immune thrombocytopenia(ITP)is an acquired and heterogeneous autoimmune-mediated haematological disease typically characterized by a low platelet count.ITP in adults typically has a chronic course(persistent thrombocytopenia with disease durations longer than12 months),and many will require some form of ongoing therapy.Currently,the precise mechanism of thrombocytopenia remains unclear.Emerging evidence over the past several years suggests that platelet biogenesis and ageing are regulated,at least in part,by apoptotic mechanisms.Apoptosis,a programmed process of cell death,is an important process whereby cells are intentionally marked for clearance from the body and is the main mechanism that regulates the life span of the cell and the elimination of damaged or infected nucleated cells.In recent years,it has become increasingly apparent that apoptotic-like events occur in platelets and are linked to the production of platelets and their subsequent life span.However,the association between decreased platelets and apoptosis in ITP patients is poorly understood.In the current study,we extensively examined whether apoptosis contributed to the decreased platelet count observed in patients with ITP.Research on the role of platelet apoptosis will provide important insight into the pathogenesis of ITP,the etiopathogenesis of which has not yet been clarified.Methods:To better understand the role of platelet apoptosis in ITP pathophysiology,we investigated apoptotic markers in platelets acquired from 40 chronic ITP patients.Furthermore,the results of ITP patients were compared to those from 40 healthy individuals.Markers of apoptosis,including phosphatidylserine(PS)exposure and mitochondrial inner membrane potentials(??m),were examined using flow cytometry.The expression of pro-apoptotic molecules such as Bak and Bax and anti-apoptotic molecules such as Bcl-x L were determined using quantitative real-time PCR(q RT-PCR)and Western blotting.Results: Our study demonstrated that the platelet mitochondrial membrane depolarization in chronic ITP patients(5.62�0.78%)tended to be higher than in healthy controls(3.23�0.32%)(P<0.0001).Additionally,the proportion of platelets with surface-exposed PS in the chronic ITP group(9.75�1.63%)was significantly higher(P < 0.0001)than that of healthy controls(4.32�0.15%).The m RNA expression levels of Bcl-x L in platelets from chronic ITP patients(0.68�0.03)were significantly lower when compared to the healthy controls(0.93�0.13,P<0.0001).The protein expression levels of Bcl-x L were also lower in platelets from chronic ITP patients than in controls,however,this difference was not significant.In addition,the results showed that the m RNA expression levels of Bak and Bax were found to be significantly higher(P<0.0001)in ITP patients(1.42�0.12 and 1.83�0.05,respectively)when compared to those in healthy controls(0.95�0.07and1.02�0.06,respectively).The protein expression levels of Bak and Bax were not significantly different between the ITP and control groups.Moreover,the ratio of Bcl-x L to Bak and Bcl-x L to Bax appeared to be decreased in ITP patient platelets when compared to control platelets(P < 0.05,respectively).Conclusions: In conclusion,our study indicates that the enhancement of platelet apoptosis observed in patients with chronic ITP may be one of the pathogenic mechanisms of chronic ITP.Better insight into these mechanisms might lead to novel therapeutic approaches and better patient selection for the treatment of ITP.Part two Micro RNA profiling of platelets in patients with immune thrombocytopeniaBackground and objective:Immune thrombocytopenic purpura(ITP)is currently defined as an autoimmune disease,characterized by a decreased platelet count(due to autoantibodies mediating platelet destruction and insufficient platelet production),purpura and increased bleeding tendency.The pathogenesis of ITP haven't beencompletely clarified.Micro RNAs(mi RNAs)is a class of noncoding RNAs,which can negatively regulate gene expression after transcription,thus it plays pivotal roles in diverse biologic processes.Recent results showed that platelets contain an abundance and diversity of microRNAs(mi RNAs),which are known as key regulators of gene expression at the post-transcriptional level.Mi RNAs are present in platelets in variable quantities and are associated with specific human phenotypes and disease states.There are few studies about the mi RNA pattern and their roles in patients with ITP.Whether mi RNAs are involved in the pathogenesis of thrombocytopenia in ITP is still unknown.The aims of this study were to identify differentially expressed mi RNAs in the platelet of ITP and to predict their functions and signaling pathways.Methods:Platelets were collected from 22 ITP patients and 8 healthy controls.The mi RNA expression was first screened by mi RNA microarray.Then the expression of selected differentially expressed mi RNAs was validated by quantitative real-time reverse transcription-polymerase chain reaction(q RT-PCR).Finally,using mi RWalk databases,we predicted the target genes of the down-regulated mi RNAs(top 20)and up-regulated mi RNAs(top 20).GO functional and KEGG pathway enrichment analyses were performed for themi RNA-target gene pairs.Results:In this study,302 mi RNAs were found differentially expressed between platelets from ITP patients and control groups(>3 fold changes,P < 0.05).Total of 65 mi RNAs were found only expressed in ITP,while 73 mi RNAs only in control group.Bioinformatics analysis predicted that mi R-548a-5p,mi R-1185-2-3p,mi R-30a-3p,mi R-6867-5p,mi R-765,and mi R-3125 were highly correlated with platelet apoptosis and adhesion of ITP.The increased mi R-296-5p and mi R-125 a might participate in the platelet adhesion of ITP through targeting the CD40 L and PECAM1.Conclusions: Our research indicated that the abnormal expression of mi RNAs may be involved in the pathogenesis of ITP.Our findings may contribute to understand the molecular mechanisms of ITP.Part three Lnc RNAs profiling of platelets in patients with immune thrombocytopenic purpura.Background and objective: Immune thrombocytopenic purpura(ITP)is an autoimmune disease characterized by thrombocytopenia due to autoantibodies mediating platelet destruction and insufficient platelet production.Currently,the precise mechanism of thrombocytopenia remains unclear.Emerging evidence over the past several years suggests that platelet biogenesis and ageing are regulated,at least in part,by apoptotic mechanisms.In recent years,the systematical researchers are concentrating on long non-coding RNAs(Lnc RNAs),which are defined as transcripts greater than 200 nucleotides in length without obvious protein coding functions.Long noncoding RNA have received wide attention recently as key molecules that mediate a variety of physiological and pathological processes by regulating gene expression,including cell growth,cell cycle progression,differentiation,and apoptosis.Furthermore,a large and growing body of literature has indicated the involvement of Lnc RNAs in a variety of diseases,especially cancer.There are few studies about the Lnc RNA pattern and their roles in patients with ITP.Whether Lnc RNAs are involved in the pathogenesis of thrombocytopenia in ITP is still unknown.The aims of this study were to identify differentially expressed Lnc RNAs in the platelet of ITP and to predict their functions and signaling pathways.Recent study found that platelet contained an abundance and diversity of mi RNAs,which are known as key regulators of gene expression at the post-transcriptional level,but the regulatory mechanism of mi RNAs is not very clear.By RNA sequencing and bioinformatics analysis,we found numerous Lnc RNAs existed in platelet,and partial sequence of some Lnc RNAs are the same as the sequence of mi RNA.Whether can these Lnc RNAs regulate mi RNAs and affect the apoptosis of platelet? These results provide new insight into the molecular mechanisms of affecting differential platelet gene regulation,which may increase understanding of the role of platelet apoptosis in ITP.Methods:In this project,to investigate the expression of Lnc RNAs in platelet of ITP,total RNA was extracted from highly purified platelet of ITP and control.The Lnc RNAs expression was first screened by RNA-SEQ.Then the expression of selected differentially expressed Lnc RNAs was validated by quantitative real-time reverse transcription-polymerase chain reaction(q RT-PCR).Finally,the Lnc RNAs associated with apoptosis were selected and the corresponding Lnc RNA-mi RNA-m RNA signaling pathways were found out by bioinformatics analysis.Results: In this study,43 Lnc RNAs were found relatively highly expressed in platelets from ITP patients compared with control groups,and 58 Lnc RNAs were significantly down-regulated.Bioinformatics analysis predicted that n382037/mi R-15 a,mi R-16/BCL2 were highly correlated with platelet apoptosis of ITP.According to the identification of RT-q PCR,we find the expression levels of Lnc RNA n382037 was significantly higher in ITP patients than in healthy controls;mi R-15/mi R-16 were relatively highly expressed in ITP,however,the target gene BCL2 expression levels were significantly decreased in the platelets of ITP patients compared to healthy controls.Conclusions: Our research indicated that the abnormal expression of Lnc RNAs may be involved in the pathogenesis of ITP.Data collected here indicate that the overexpression of Lnc RNA n382037 could up-regulate the levels of mi R-16/mi R-15 in platelet,inhibited BCL2 expression and contributed to platelet apoptosis in ITP.Our findings may contribute to understand the molecular mechanisms of ITP.