再生障碍性贫血患者RNA互作网络分析及潜在治疗药物预测

Analysis of RNA Interaction Network and Prediction of Potential Therapeutic Agentsin Patients with Aplastic Anemia

目的探究再生障碍性贫血(aplastic anemia,AA)患者骨髓T细胞差异表达的微小核糖核酸(microRNA,miRNA)和信使RNA(messenger RNA,mRNA)基因,构建互作调控网络并筛选药物,随后评估其治疗方案疗效。方法在基因表达数据库检索并进行综合分析,使用R语言对AA相关miRNA与mRNA进行差异表达分析,并通过miRNet预测差异表达miRNAs(DE-miRNAs)的靶向mRNAs,并与差异表达mRNAs(DE-mRNAs)进行比较与负相关配对,得到失调mRNAs,再对失调mRNAs进行功能基因注释(GO)分析与通路富集(KEGG)分析,构建AA的miRNA-mRNA互作调控网络。采用自主研发的表观精准治疗平台(EpiMed)进行潜在治疗药物预测,并将盐酸二甲双胍纳入新型联合治疗方案中,对入组的40例难治性AA进行临床疗效评估,评估主要指标包括血红蛋白、中性粒细胞绝对值、血小板计数及输血依赖等。结果差异分析最终获得26个DE-miRNA与168个靶向DE-mRNA,对其进行构建调控网络。GO和KEGG分析结果显示,AA相关失调mRNA可能参与T细胞分化、造血调控、物质代谢等过程,与病毒感染、细胞脂向分化、细胞凋亡以及物质代谢等信号通路相关。通过EpiMed预测得到环孢素A、吗替麦考酚酯、雷帕霉素、二甲双胍、维生素B、白藜芦醇等药物可能具有治疗AA的作用。研究共纳入难治性再生障碍性贫血患者40例,男17例,女23例,年龄14~83岁,治疗前后血红蛋白浓度、中性粒细胞绝对值计数、血小板计数分别为83.5 g/L vs.119.0 g/L、1.600×10^(9)/L vs.1.875×10^(9)/L、32.5×10^(9)/L vs.46.0×10^(9)/L,差异具有统计学意义(均P<0.05)。治疗后1、3、6、12个月分别有28例(70%)、28例(70%)、32例(80%)、35例(88%)患者获得血液学反应。结论本研究构建的T细胞miRNA-mRNA调控网络为研究AA提供了新的视角,临床研究初步验证含盐酸二甲双胍联合环孢素A方案治疗难治性AA安全有效,副作用可接受,为临床治疗再生障碍性贫血提供了新的方法。

Objective To explore the microRNA and mRNA related differential gene expression in patients with aplastic a-nemia.To construct an interactive regulatory network and screen out potential therapeutic agents,and subsequently evaluate the efficacy of treatment regimens.Methods The gene expression database was searched and analyzed.R language was used for dif-ferential analysis between AA-related miRNA and mRNA.DE-miRNAs were predicted by miRNet and targeted mRNAs were obtained.mRNAs were compared with DE mRNAs by negative correlation pairing,and Gene Ontology(GO)analysis and K yoto Encyclopedia of Genes and Genomes(KEGG)analysis were performed on abnormal mRNAs.The miRNA mRNA interactive regulatory network of AA was constructed.Self developed epigenetic precision therapy platform(EpiMed)was used to predict potential therapeutic agents,and Metformin hydrochloride was incorporated into a novel combination therapy regimen.The clinical efficacy among 40 cases of refractory AA enrolled in the study was evaluated.The main assessment indicators included he-moglobin,absolute neutrophil value,platelet count and transfusion dependence.Results In all,26 miRNAs with 168 targeted DE-mRNAs were obtained from differential analysis,and regulatory network was constructed.GO and KEGG analysis showed that AA-related dysregulated mRNAs were involved in T cell differentiation,hematopoietic regulation,substance metabolism and other processes,and were related to signaling pathways such as viral infection,cell lipid differentiation,apoptosis and sub-stance metabolism.Cyclosporin A,Mycophenolate mofetil,Rapamycin,Metformin,vitamin B and Resveratrol were predicted as potential therapeutic agents by EpiMed prediction.Among the 40 patients with refractory AA,there were 17 males and 23 females who were aged from 14 to 83 years old.The concentration of hemoglobin,the absolute value of neutrophil count,and platelet count of patients before and after treatment were 83.5 g/L us.119.0 g/L,1.600×10^(9)/L rvs.1.875×10^(9)/L,32.5×10^(9)/L us.46.0×10^(9)/L,respectively(P<0.05).Hematologic responses were observed in 28(70%),28(70%),32(80%),and 35(88%)patients at 1,3,6 and 12 months after treatment.Conclusion The miRNA-mRNA regulatory network of T cells constructed in this study provides a new perspective for studying AA.Clinical studies preliminarily verified that Metformin hydrochloride combined with Cyclosporine A was safe and effective in the treatment of refractory AA with acceptable side effects,providing a new approach for the clinical treatment of aplastic anemia.