Study On Abnormal Expression Of ITP Genes And Their Functional Pathways Based On High-throughput Sequencing Technology

Primary immune thrombocytopenia(pITP),also known as idiopathic thrombocytopenic purpura(ITP),is an autoimmune-mediated bleeding disorder,and the clinical symptoms are a reduction in platelets in the peripheral blood,mucocutaneous haemorrhage,and visceral haemorrhage.The potential bleeding risk seriously affects the quality of life of patients.First-line treatment of ITP mainly includes glucocorticoids,anti-D immunoglobulin and intravenous immunoglobulin,but the long-term effect of treatment is not satisfactory,and many patients still cannot achieve sustained.In addition to the destruction of platelet caused by antibody mediation,abnormal cellular immunity also affects the pathogenesis of ITP.At present,immune imbalance caused by autoimmune tolerance deficiency can be regarded as the central link of ITP.Therefore,it is of great significance to explore the factors leading to ITP immune imbalance for understanding the pathogenesis of ITP.DNA methylation is one of the most common forms of epigenetic modification.Abnormal DNA methylation can affect the expression of related genes and cytokines,resulting in changes in T cell differentiation and reactivity,which is closely related to the pathogenesis of immune diseases.Abnormal expression of related genes induced by methylation defects can be involved in autoreactive immune responses and ultimately accelerate the progression of immune thrombocytopenia.In addition,apoptosis is also the focus of ITP pathogenesis research in recent years.In-depth study of apoptosis-related proteins that play a key role in ITP will lay an important foundation for exploring the mechanism of ITP and seeking therapeutic approaches.Therefore,in this study,methylation sequencing and proteomics were used to analyze and verify the abnormal methylated genes and apoptosis-related proteins in ITP.Part Ⅰ:Explore key methylated genes and pathways in ITP through methylation analysis.Objective:As DNA methylation is known to play an important role in the occurrence and development of ITP,we further explored the key methylated genes and related pathways of ITP,providing new ideas for subsequent precision therapy.Methods:Genome-wide methylation sequencing was performed on 21 ITP patients and 9 normal control samples to obtain the differentially expressed methylation sites and regions and annotate them.The annotated results of differential methylation regions were cross-analyzed with Phenopedia database and DisGeNET database to obtain ITP-related differential methylation genes.These genes were analyzed for GO and KEGG enrichment to search for key genes and signaling pathways.Results:A total of 16 differentially methylated genes related to ITP were obtained,which were ABCC3,AQP1,C4BPB,DNMT3B,FGFR3,NOTCH1,RET,SH2B2,SLC19A1,SLC4A11,SNRNP70,TYK2 and ZMPSTE24.TPO,TCIRG1,and LTA.KEGG enrichment analysis of these 16 genes revealed an important immune-related pathway in ITP-Th1 and Th2 cell differentiation pathway.Meanwhile,hypermethylated NOTCH1 is involved in the expression of this pathway and regulates many downstream cytokines.Conclusions:The abnormal hypermethylated gene NOTCH1 regulates many downstream cytokines through Thl and Th2 cell differentiation pathways to influence ITP.Part Ⅱ:Demethylation agents act to treat ITP by affecting NOTCH1 and related pathways.Objective:The key gene NOTCH1 is known to express abnormal hypermethylation in ITP,and it is clear that the demethylation drug(decitabine)has a therapeutic effect on ITP by reducing NOTCH1 methylation and regulating downstream Thl and Th2 cytokines.Methods:The expression of NOTCH1,Thl and Th2 cytokines in ITP was detected by RT-PCR and Luminex technology,which can verify the decreased expression of hypermethylated NOTCH1 and influence the expression of downstream cytokines.In addition,bone marrow mononuclear cells were extracted from ITP patients and added with decitabine and PBS respectively for cell culture.The expression of NOTCH1 and related cytokines was detected by RT-PCR,ELISA and flow cytometry to verify the effectiveness of decitabine in treating ITP..Results:Compared with normal control group,the expression levels of NOTCH1 and its downstream Th2 cytokines in ITP group were significantly decreased and positively correlated.The expression level of Th1 cytokine increased significantly and was negatively correlated with NOTCH1.Meanwhile,the use of the drug decitabine reversed the expression of NOTCH1 and its downstream cytokines in ITP,and the expression of NOTCH1 and Th2 cytokines was significantly increased and Th1 cytokine expression was significantly decreased in the ITP group compared with the ITP group treated with PBS.Conclusion:Abnormally low expression of hypermethylated NOTCH1 results in down-regulation of Th2 cytokines and up-regulation of Th1 cytokines.Decitabine can increase the expression of NOTCH1,thus improving the imbalance between Thl and Th2 cytokines,and has a good therapeutic effect on ITP.Part Ⅲ:Proteomic analysis found abnormal expression of apoptosis-related proteins and pathways in ITP.Objective:To explore new key apoptotic proteins and related signaling pathways in ITP,and to discover new mechanisms of apoptosis in ITP.Methods:Bone marrow samples from 20 ITP patients and 20 healthy controls were collected and proteomic techniques were used to analyze abnormally differentially expressed proteins and protein interaction networks to discover apoptosis-related proteins and their relationships in ITP.Parallel response monitoring(PRM)was used for quantitative analysis of identified target proteins to validate proteomic results.Results:A total of 829 proteins were identified.Compared with the normal control,26 proteins in ITP group were up-regulated and 69 proteins were down-regulated.Bioinformatics analysis showed that down-regulated differentially expressed proteins(HSPA6,HSPA8,ITGB3,YWHAH,and PRDX6)were associated with apoptosis and could affect ITP through PI3K-Akt signaling pathway.And protein interaction networks show that these proteins can interact and communicate with each other.At the same time,PRM results confirmed that apoptosis related proteins(HSPA6,HSPA8,ITGB3,YWHAH,and PRDX6)were significantly reduced in ITP patients.Conclusion:Down-regulated apoptosis-related proteins(HSPA6,HSPA8,ITGB3,YWHAH,and PRDX6)are closely related to the pathogenesis of ITP through PI3K-Akt signaling pathway.