| Background and purpose Aplastic anemia is a hematological disease characterized by pancytopenia and bone marrow failure.The incidence rate of aplastic anemia is significantly higher in China than in Europe and America,and the proportion of acquired aplastic anemia is a relatively high in China.Aplastic anemia is an acute onset,rapid progress and poor clinical prognosis disease.At present,the clinical treatments are immunosuppressive therapy with antithymocyte globulin and cyclosporine or hematopoietic stem cell transplantation.However,the above treatments have some limitations,and the two treatments are only effective for part of patients,there are still a small proportion of patients who have no response to the treatments.Therefore,it is urgent to explore new methods for the treatment of aplastic anemia.Non-codingRNA is a kind ofRNA whose structure is similar to mRNA but not translated for protein.Non-codingRNA includes microRNA(miRNA),long noncodingRNA(lncRNA),circularRNA(circRNA)and other noncodingRNAs.Studies have proved that non-codingRNA has important biological functions and is closely related to the occurrence and development of human diseases.However,the relationship between noncodingRNA and aplastic anemia is not fully understood.This study aims to identify important miRNA,lncRNA and their regulatory networks involved in the pathogenesis of aplastic anemia,and preliminarily explore the relationship between relevant non-codingRNAs and the pathogenesis of aplastic anemia,so as to lay foundations for futher study about the role of relevant noncodingRNAs in the pathogenesis of aplastic anemia,and to provide a theoretical basis for the use of relevant noncodingRNAs as biomarkers or therapeutic targets of aplastic anemia in the future.Methods1 Identify core miRNAs and their regulatory networks involved in the T cell abnormalities of aplastic anemia patients(1)The miRNA and mRNA microarray data(GSE82095 and GSE3807)of bone marrow T cells from aplastic anemia patients and healthy controls in GEO database were retrieved and obtained,and the corresponding microarray annotation files were obtained.(2)Using the Affy chip analysis package in R,the background correction,normalization and log2 correction of the data were carried out by RMA method,KNN method was used for data supplemented and the gene expression matrix and the grouping matrix were constructed.After the linear model fitting,Bayesian t test was used for the differential genes and the differential genes were screened by P <0.05 and|log2FC|>1.(3)Mi RNet was used to predict the differential mRNA regulated by differential miRNA.Venn diagram was used to take the overlap between the target gene of differential miRNA and differential mRNA,and the mRNA of the overlapping part was negatively correlated with the miRNA that may target it,so as to construct the miRNA-mRNA regulation pairs.(4)Cluster Profiler and enrichplot packages in R were used to analyze and visualize the GO and KEGG functions of differential mRNAs.(5)The protein-protein interaction network of differential mRNA was constructed by STRING database.The core mRNAs in the network that may be involved in the pathogenesis of aplastic anemia was identified by MCODE in Cytoscape,core mRNAs were associated with the above miRNA-mRNA regulation pairs,and the core miRNA-mRNA regulation networks were constructed.2 Identify the key lncRNAs and their regulatory networks involved in the pathogenesis of aplastic anemia(1)The peripheral blood of 5 aplastic anemia patients and 5 healthy controls were collected.The totalRNA was extracted by Trizol method,the c DNA library was constructed,and the transcriptome was sequenced.(2)The original sequencing data were compared to the reference genome through HISAT2,the expression of each gene was calculated by RPKM method,and the expression differences of lncRNAs and mRNAs between aplastic anemia patients and healthy controls were calculated by DEGseq 和 DESeq2.The differences were screened by adj.P <0.05 and |log2FC|>1.(3)Cluster Profiler and enrichplot packages in R were used to analyze and visualize the GO and KEGG functions of differential mRNAs.(4)For differentially expressed lncRNA and mRNA,lncRNA-mRNA cis-regulation network,lncRNA-TF-mRNA trans-regulation network and lncRNA-miRNA-mRNA competitive regulation network were constructed.(5)Through qPCR,the differentially expressed lncRNAs and mRNAs were verified.3 Analyze the effect of overexpression lncRNA-AC007991.2 on the biological characteristics of human bone marrow mesenchymal stem cells(1)The low expressed lncRNA-AC007991.2 in aplastic anemia children was selected as the research object.The lncRNA-AC007991.2 overexpression plasmid was constructed,293 T cells were transfected with the plasmid,the virus was collected and infected the human bone marrow mesenchymal stem cells.The overexpression of lncRNA-AC007991.2 in human bone marrow mesenchymal stem cells was verified by qPCR.(2)The ffects of overexpressed lncRNA-AC007991.2 on the proliferation of human bone marrow mesenchymal stem cells were detected by CCK8 method.The effects of overexpressed lncRNA-AC007991.2 on cell cycle,apoptosis and mitochondrial membrane potential of human bone marrow mesenchymal stem cells were analyzed by flow cytometry.After being induced by osteogenic or adipogenic differentiation medium for 14 days,the cells were stained with alizarin or oil red to observe the effects of overexpressed lncRNA-AC007991.2 on osteogenic and adipogenic differentiation of human bone marrow mesenchymal stem cells.(3)Human bone marrow mesenchymal stem cells and Jurkat T cells were co-cultured at the ratio of 1:10 for 96 hours.The proliferation,cell cycle and apoptosis of Jurkat T cells were detected by CCK8 method and flow cytometry.The effect of overexpressed lncRNA-AC007991.2 on the immunomodulatory ability of human bone marrow mesenchymal stem cells were shown by the apoptosis of co-cultured Jurkat T cells.(4)After the totalRNA of human bone marrow mesenchymal stem cells was extracted by Trizol method,the c DNA library was constructed and the transcriptome was sequenced.The effects of overexpressed lncRNA-AC007991.2 on the expression of genes related to proliferation,cell cycle,apoptosis,osteogenic differentiation,adipogenic differentiation and immunomodulatory ability of human bone marrow mesenchymal cells were shown with heatmap.GO,KEGG and GSEA enrichment analysis were used to show the effect of overexpressed lncRNA-AC007991.2 on the transcriptome of human bone marrow mesenchymal cells.Results1 Core miRNAs and their regulatory networks involved in the T cell abnormalities of aplastic anemia patients(1)A total of 40 differentially expressed miRNAs(including 21 up-regulated and 19down-regulated)and 1511 differentially expressed mRNAs(including 695up-regulated and 816 down-regulated)were identified by the expression profile analysis of bone marrow T cells in aplastic anemia patients and healthy controls.(2)Using these differentially expressedRNA,303 miRNA-mRNA regulatory relationship pairs(including 25 differentially expressed miRNAs and 199 differentially expressed mRNAs)were constructed.(3)GO and KEGG analysis showed that the functions of 199 differentially expressed mRNA regulated by differential miRNA were mainly concentrated in the nucleus and chromosome,participate in the transcriptional regulation signal pathway,and might be involved in the differentiation and plasticity of T cells.(4)The established core miRNA-mRNA regulatory network showed that hsa-mir-34a-5p,hsa-mir-195-5p and hsa-mir-424-5p might be the core miRNAs with abnormal T cells in aplastic anemia patients.The target genes of these three core miRNAs were histone methylation gene KMT2 D and histone related genes.2 Key lncRNAs and their regulatory networks involved in the pathogenesis of aplastic anemia(1)A total of 60 differentially expressed lncrnas(including 6 up-regulated and 54down-regulated)and 364 differentially expressed mRNAs(including 41 up-regulated and 323 down-regulated)were identified by the expression profile analysis of peripheral blood cells of aplastic anemia patients and healthy controls.(2)GO and KEGG enrichment analysis showed that compared with the healthy control,the differentially expressed genes in aplastic anemia patients were mainly enriched on the signal pathways such as cell membrane,platelet activation,platelet dysfunction and cell adhesion.(3)By constructing the cis-,trans-and endogenous competitive lncRNA-mRNA regulated network,lncRNA-AC007991.2,AC007922.2,AC111000.4,AC147651.1,RHOXF1P1,AC007556.1 were identified as key lncRNAs in the pathogenesis of aplastic anemia.They can regulate the expression of IDO1,SEMA7 A,GFI1B,DHRS9,HRH4 and PDGFA through networks.3 Effect of overexpression lncRNA-AC007991.2 on the biological characteristics of human bone marrow mesenchymal stem cells(1)Human bone marrow mesenchymal stem cells were divided into lncRNA overexpressed group(infected with lncRNA-AC007991.2 overexpressed plasmid constructed virus),empty plasmid group(infected with empty plasmid constructed virus)and blank control group(no virus infection).Expression of lncRNA-AC007991.2 in those groups was verified by qPCR.(2)Compared with the blank control group,the empty plasmid group showed disordered cell morphology,decreased proliferation ability(P < 0.05),no obvious change of cell cycle,decreased mitochondrial membrane potential(60.39% vs 8.74%),enhanced osteogenic differentiation ability and weakened adipogenic differentiation ability.(3)Compared with Jurkat T cells co-cultured with blank control group,the cell cycle of Jurkat T cells co-cultured with empty plasmid group did not change significantly but the proportion of apoptotic Jurkat T cells decreased(43.71% vs 64.78%).(4)Compared with the empty plasmid group,the lncRNA overexpressed group had no significant changes in cell proliferation(P > 0.05),cell cycle and apoptosis,osteogenic differentiation ability,while their adipogenic differentiation ability decreased.(5)Compared with Jurkat T cells co-cultured with empty plasmid group,the cycle changes of Jurkat T cells co-cultured with lncRNA overexpressed group was not obvious,but the proportion of apoptotic Jurkat T cells increased(58.00% vs 43.71%).(6)The expression profiles of lncRNA overexpressed group and empty plasmid group showed that the two groups of cells could not be clustered according to the gene expression profiles related to cell proliferation,cell cycle,apoptosis and osteogenic differentiation,but could be correctly clustered according to the gene expression related to adipogenic differentiation or immunomodulatory function.Go,KEGG and GSEA analysis of differentially expressed genes were enriched in mesenchymal stem cell differentiation,adipogenic differentiation,immune regulation,inflammatory response,cytokines and chemokines pathways.Conclusions1 Hsa-mir-34a-5p,hsa-mir-195-5p and hsa-mir-424-5p are the core miRNAs involved in the abnormality of T lymphocytes in patients with aplastic anemia.Hsa-mir-34a-5p,hsa-mir-195-5p and hsa-mir-424-5p are the core miRNAs in the constructed core network,which may participate in the differentiation and plasticity of T lymphocytes in patients with aplastic anemia by regulating histone methylation gene KMT2 D and histone related genes.2 LncRNA-AC007991.2,AC007922.2,AC111000.4,AC147651.1,RHOXF1P1,AC007556.1 are the key lncRNAs involved in the pathogenesis of aplastic anemia.They may regulate the expression of IDO1,SEMA7 A,GFI1B,DHRS9,HRH4 and PDGFA through networks,so as to participate in the pathogenesis of aplastic anemia.Patients with aplastic anemia may have abnormal activation of platelets.The differentially expressed genes between aplastic anemia patients and healthy controls were mainly enriched in platelet activation pathways,suggesting that patients with aplastic anemia may have abnormal activation of platelets.Abnormally activated platelets may further activate innate immunity and adaptive immunity through cascade reaction,which may participate in the pathogenesis of aplastic anemia.3 Lentivirus infected human bone marrow mesenchymal stem cells showed disordered morphology,decreased in proliferation,decreased in mitochondrial membrane potential,enhanced in osteogenic capacity,weakened in adipogenic capacity and weakened in immunosuppressive capacity against Jurkat T cells.Overexpressing lncRNA-AC007991.2 has no significant effect on the proliferation,apoptosis and osteogenic differentiation of human bone marrow mesenchymal stem cells,but can weaken its adipogenic differentiation ability and enhance its immunosuppressive ability on Jurkat T cells.LncRNA-AC007991.2may participate in the pathogenesis of aplastic anemia by affecting the adipogenic differentiation ability and immunosuppressive ability of human bone marrow mesenchymal stem cells. |
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