The Role Of LncRNA-AF117829.1 In The Immune Pathogenesis Of Severe Aplastic Anemia

ObjectiveIn this study,RNA-seq technology was used to detect the expression of messenger RNA(m RNA)and long non-coding RNA(lncRNA)in CD8 + T cells of peripheral blood from patients with severe aplastic anemia(SAA)and healthy controls.The target genes of lncRNAs were predicted by bioinformatics,Gene Ontology(Go)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis,including the key biological processes and signal pathways involved in target genes.This study aims to further verify and clarify the function of key lncRNAs involved in the mechanisms of SAA,and provide a new potential therapeutic target for immunotherapy of SAA.To investigate the quantity and function of CD56 bright NK cells in patients with Immune-related pancytopenia.Study on the high efficient culture technology of NK cells in vitro in order to obtain the best cell expansion effect.Methods Part ? The expression of m RNAs and lncRNAs in peripheral blood CD8 + T cells from SAA patients and normal controls were detected by RNA-seq technology,while the differentially expressed m RNAs and its biological function or signal pathway were analyzed by GO and KEGG cluster analysis.At the same time,the target genes of differentially expressed lncRNAs were analyzed by GO and KEGG to predict the function of lncRNA in SAA.Part ? Real time quantitative PCR(RT-PCR)was used to verify the expression changes of the key lncRNAs and their predicted target genes m RNAs.Flow cytometry(FCM)was used to detect the expression of perforin and granzyme B in CD8 + T of SAA patients.Part ? The expression of lncRNA-AF117829.1 was correlated with perforin and granzyme B expression of CD8+ T cells.lncRNA AF117829.1 in CD8 + T cells of SAA patients and healthy controls were overexpressed by lentivirus vector.Detection of lncRNA AF117829.1 and m RNA RIP2 in CD8 + T cells were tested by RT-PCR.Changes of perforin and granzyme B expression in CD8 + T cells were detected by FCM.Protein changes of RIP2 signaling pathway were detected by Western blot.Part ? GSK583,a RIP2 kinase inhibitor,was used to intervene CD8 + T cells in peripheral blood of SAA patients in vitro.Western blot was used to detect the expression of RIP2 signaling pathway protein after GSK583 intervention.Changes of perforin and granzyme B in CD8 + T cells after GSK583 intervention were detected by flow cytometry.Part ? The quantitative and functional changes of CD56 bright NK cells in peripheral blood of patients with IRP were detected by using Flow Cytometry(FCM)before and after immunosuppressive therapy(IST).The expressions of activating receptor(NKG2D,NKp46,NKp44),inhibitory receptor(NKG2A,CD158 a,CD158b)and perforin and granzyme B were detected by FCM.IL-2 and IL-18 levels in serum were detected by ELISA.The correlation between the changes of quantities and function of CD56 bright NK cells and clinical indicators of IRP patients were evaluated.Part ? High purity NK cells were extracted from 6 untreated patients with IRP using the immunomagnetic beads sorting which were then cultured in RPMI-1640 medium containing 20% FBS,with or without NK cell expansion agents,and were used to amplify high-purity NK cells on the basic of recombinant IL-2 and recombinant interleukin(IL)-15.The expression of the activated receptors NKG2 D and NKp46 and the inhibitory receptors CD158 a and NKG2 A before and after expansion of NK cells were detected by flow cytometry.Results Part ?The expression profile of lncRNAs and m RNAs in CD8 + T cells in newly diagnosed SAA patients and healthy controls was analyzed by RNA-seq technology(a Fold change?2,and P < 0.05).A total of 2099 m RNA expression changes(included 1104 up-regulated m RNA and 995 down-regulated m RNA).The results of GO analysis indicated that differentially expressed m RNAs were involved in biological processes,such as catalysis,transduction activity,molecular function regulation,cell metabolism,immune system and so on.KEGG analysis showed that the differentially expressed m RNAs were mainly enriched in JNK,RAS,NOD2 / RIP2 and MAPK signaling pathways.A total of 194 lncRNAs were changed in SAA patients including up-regulated in 107 and down regulated in 87.Go analysis showed that the differential expression of lncRNAs was related to biological processes,such as organelle components,catalytic activity,response to stimulation,signal transduction inside and outside cells,biological regulation,and metabolic process.KEGG analysis showed that the differential expression of lncRNAs was mainly related to ubiquitin mediated proteomics,antigen processing and presentation,NOD and other signaling pathways.Part ?In this part,the predicted target gene of lncRNA AF117829.1 is RIP2,in which the m RNA RIP2 meets the differential expression condition of high-throughput histochemical detection(a Fold change ?2,and P < 0.05).The results of qRT-PCR showed that the expression of lncRNA AF117829.1 in SAA patients was decreased,while the expression of m RNA RIP2 was increased.The results were consistent with those of gene chip.At the same time,Western blot results showed that RIP2 signaling pathway related proteins were significantly higher in SAA.LncRNA AF117829.1 was negatively correlated with perforin and granzyme B expression in CD8+ T cells.Part ?After overexpression of lncRNA-AF117829.1 in CD8 + T cells of SAA patients,the expression of m RNA-RIP2 was lower than that of the control group.The results of flow cytometry showed that the expression of perforin and granzyme B in CD8 + T cells decreased.Western blot results showed that the expression of RIP2 signaling pathway related proteins was decreased.Part ?After GSK583 intervened the CD8+T cells in the peripheral blood of SAA patients in vitro,Western blot results showed that RIP2 signaling pathway related protein decreased.Flow cytometry results showed that the expression of perforin and granzyme B in CD8 + T cells decreased.Part ?The percentage of CD56 bright NK cells in the peripheral blood of patients with IRP was significantly higher than that of the control group.The expression level of NKG2 D in IRP patients was higher than that in normal control group,and CD158 a was lower than that in normal control group.The product of CD56 bright NK cell percentage and granzyme B in peripheral blood of patients with IRP was significantly higher than that in the control group.The serum levels of IL-2 and IL-18 in IRP patients were higher than those in normal control group.Part ?NK cell expansion agent combined with the stimulation of recombinant IL-2 and recombinant IL-15 could increase the number whilst maintaining the purity of NK cells.There were no significant changes in the expression of NKG2 D,NKp46,NKG2 A and CD158 a in patients with IRP before and after NK cell culture.Conclusions1.The study systematically described the expression changes of lncRNA and m RNA in CD8+ T cells in SAA,including their possible biological functions and signal pathways.The low expression of lncRNA AF117829.1 in CD8 + T cells in SAA could regulate the function of CD8 + T cells by promoting the expression of RIP2.The results were helpful to understand the molecular mechanism of immune pathogenesis and provided potential targets for diagnosis and treatment of SAA.2.The increased quantities and function of cd56 bright NK cells may play an important role in the pathogenesis of IRP.3.The amplification method of NK cell amplification agent combined with recombinant IL-2 and recombinant IL-15 can increase the number of NK cells on the basis of maintaining the purity of NK cells,and has no obvious effect on NK cell function,thus laying a foundation for the study of NK cell immunotherapy.