【作者】 刘兴；

【导师】 周莉；

【作者基本信息】 重庆医科大学 ， 流行病与卫生统计学， 2023， 硕士

【摘要】 背景:目前,乙型肝炎病毒(hepatitis B virus,HBV)感染仍是全球共同关注的重大公共卫生问题,而中国涵盖了世界上三分之一的HBV感染者。众所周知,HBV感染是导致慢性乙型肝炎(chronic hepatitis B,CHB)发生的最主要原因,且CHB可能会进一步发展为肝纤维化、肝硬化以及肝癌等严重的疾病。因此,加强CHB患者的治疗是防治HBV感染的重要任务。临床上最常用于治疗CHB患者的药物主要包括干扰素(IFN)和核苷类似物(NAs)。虽然NAs药物能有效抑制CHB患者的HBV DNA水平,但它们对血清乙型肝炎表面抗原(HBsAg)和乙型肝炎E抗原(HBe Ag)的抑制作用十分有限。相比NAs单独治疗,IFN与NAs的联合用药不仅能有效抑制血清HBV DNA水平,还能实现更高的HBsAg和HBe Ag清除率。但是,IFN药物在临床上的应用受到其副作用和CHB患者低应答率的限制。且国内外研究者对于IFN药物治疗CHB患者的作用机制尚未阐明。因此,我们需要更多关于CHB患者接受IFN治疗的研究来指导CHB患者的抗病毒治疗,以减轻中国居民疾病负担,提高中老年人生命质量,实现乙型肝炎的防治目标。第一部分基于生信分析构建慢性乙型肝炎患者对干扰素治疗应答相关的miRNA-mRNA调控网络目的:本研究拟利用GEO数据库中CHB患者接受IFN治疗的数据集,筛选与IFN治疗应答相关的潜在标志物,通过生信分析探索影响IFN治疗应答的潜在机制,为CHB患者的抗病毒治疗研究提供新的靶点和思路。方法:本研究从GEO数据库中下载了GSE29911和GSE27555的系列矩阵文件,应用R软件的“limma”程序包对差异表达的miRNA(DEmiRNA)和mRNA(DEG)进行鉴定。然后,我们利用随机森林和k-最近邻(KNN)算法筛选预测因子并构建预测模型,在star Base数据库中预测miRNA的靶基因。本研究通过R软件的“cluster Profiler”程序包进行基因功能注释和通路富集分析,利用String数据库和Cytoscape软件构建交互网络。结果:本研究在GSE29911中共检测到18个差异表达的miRNAs,在GSE27555中共检测到700个DEGs。接下来,我们从DEmiRNA中筛选出具有预测IFN应答潜力的4个miRNAs(miR-873,miR-200a,miR-30b,let-7g),并且以miRNA为预测因子建立干扰素应答的预测模型,预测模型的准确率为76.92%。我们将4个miRNAs的5204个靶基因与700个DEGs相交获得了106个mRNAs。功能注释和通路富集分析显示,106个mRNAs主要参与调节白细胞跨内皮迁移,PI3K-Akt,JAK-STAT,白细胞介素,IL4和IL13信号传导等通路。然后,我们构建了106个mRNAs的PPI网络,并从中鉴定了10个枢纽基因(ACTA1,IQGAP1,ACTG2,GSN,ANXA2,FLNA,CD44,FGF2,MMP2和MKI67),构建了提示IFN应答机制的miRNA-mRNA网络。结论:本研究通过生物信息学方法发现了4个预测CHB患者接受IFN治疗时是否发生应答反应的潜在生物标志物(miR-873,miR-200a,miR-30b和let-7g),构建了一个关于IFN应答的miRNA-mRNA调控网络,提示了IFN治疗CHB患者的作用机制,为进一步筛选和研究IFN应答的有效生物标志物提供了数据参考和实验方向。第二部分实验验证miR-873参与IFN下调HBV表达的过程目的:现今关于IFN发挥抗HBV作用的具体靶位点和IFN治疗CHB患者作用机制的研究还不够充足,本研究拟在体外实验中进一步验证4个miRNAs与HBV感染和IFN治疗的关系,旨在探讨miRNA在IFN治疗过程中的作用,为CHB患者的抗病毒治疗提供数据支持和研究思路。方法:本研究在细胞中转染p HBV1.1质粒48h模拟CHB患者的生理过程,用重组人IFN-α-2b处理转染了p HBV1.1质粒的细胞48h模拟CHB患者接受IFN治疗过程。我们采用实时定量聚合酶链反应(RT-q PCR)检测人群和细胞中miRNA的表达水平,利用酶联免疫吸附试验(ELISA)检测细胞培养上清中乙型肝炎病毒表面抗原(HBsAg)和乙型肝炎病毒E抗原(HBe Ag)的水平。结果:RT-qPCR结果显示,miR-873,miR-200a,miR-30b和let-7g在CHB患者中的表达水平显著高于健康对照人群,并且在转染p HBV1.1质粒的细胞中的表达水平显著高于对照组细胞。相比未用IFN处理的细胞,用IFN处理转染了p HBV1.1质粒的细胞中miR-873的表达水平更低。ELISA结果显示,相比对照组,用miR-873拮抗剂处理的细胞中HBsAg水平降低,而用miR-873模拟物处理的细胞中HBsAg水平升高。本研究中,干扰素处理会导致转染了p HBV1.1质粒的细胞上清中HBsAg和HBe Ag水平下调,当用IFN和miR-873的抑制剂一同处理细胞时HBsAg和HBe Ag的水平进一步降低,而IFN和miR-1287-5p模拟物同时处理细胞会导致HBsAg和HBe Ag的水平升高,差异均有统计学意义。我们进一步探索了miR-873对ANXA2的调控作用,miR-873会下调的ANXA2水平。相比对照组,ANXA2在转染了p HBV1.1的肝癌细胞中表达水平上调,在IFN处理细胞中表达水平下调。结论:本研究在人群和细胞中验证了miR-873与HBV感染和IFN治疗的相关性,发现了miR-873参与IFN下调HBV表达的作用,进一步提示了miR-873作为干扰素应答生物标志物的潜力。更多还原

【Abstract】 Background: At present,hepatitis B virus(HBV)infection is still a major public health concern worldwide,while China covers one-third of HBV infections in the world.Meanwhile,HBV infection is the most important cause of chronic hepatitis B(CHB).Clinically,the most commonly used drugs for the treatment of CHB patients mainly include interferons(IFN)and nucleoside analogues(NAs).Although NAs can effectively suppress HBV DNA levels in CHB patients,their inhibition of hepatitis B surface antigen(HBs Ag)and hepatitis B e antigen(HBe Ag)is limited.Compared with NAs monotherapy,the combination of IFN and NAs could not only stably inhibit the serum HBV DNA level,but also increase rates of HBs Ag and HBe Ag seroclearance.However,the clinical application of IFN is limited by its side effects and low response rate in CHB patients.At present,the response and mechanism of interferon drugs in the treatment of CHB patients have not been elucidated by domestic and foreign researchers.Therefore,more studies on the treatment of CHB patients with interferon are inevitable requirements to reduce the burden of disease in Chinese residents,improve the quality of life of middle-aged and elderly people,and achieve the goal of hepatitis B prevention and treatment.Part 1 Constructing MiRNA-mRNA Regulatory Network of Response to Interferon Treatment in Chronic Hepatitis B Patients Based on Bioinformatics AnalysisObjective: This study aims to use the data set of CHB patients treated with IFN from GEO database to screen the potential markers associated with IFN treatment response.We explored the potential mechanisms affecting response to IFN treatment through bioinformatics analysis,provided new targets and ideas for antiviral therapy in CHB patients.Methods: A series of matrix files of GSE29911 and GSE27555 were downloaded from GEO database.The differentially expressed miRNAs(DEmiRNAs)and differentially expressed genes(DEGs)were identified by using the “limma” package of R software.Then,random forest and K-nearest neighbor(KNN)algorithm are used to establish the prediction model.The target genes of 4 miRNAs were predicted by star Base v2.0.Next,functional annotations and pathway enrichment analysis are performed through the “cluster Profiler” package of R software.Interactive networks were built using the String database and Cytoscape.Results: A total of 18 DEmiRNAs were detected in GSE29911 and700 DEGs were detected in GSE27555.Then,4 miRNAs(miR-873,miR-200 a,miR-30 b,and let-7g)with higher predictive potential for IFN response were screened from 18 DEmiRNAs.The prediction model of interferon response was established with an accuracy of 76.92%.We obtained 106 mRNAs by crossing 5204 target genes of 4 miRNAs with700 DEGs.Functional annotation and pathway enrichment analysis revealed that 106 mRNAs were mainly involved in regulating signaling of leukocyte transendothelial migration,PI3K-Akt,JAK-STAT,interleukin,IL4 and IL13 pathway.We identified 10 hub genes(ACTA1,IQGAP1,ACTG2,GSN,ANXA2,FLNA,CD44,FGF2,MMP2,and MKI67)from the PPI network and constructed a miRNA-mRNA network suggestive of IFN response mechanisms.Conclusion: This study has identified 4 potential biomarkers(miR-873,miR-200 a,miR-30 b,and let-7g)that predict response to IFN treatment in CHB patients by molecular epidemiology.We constructed a miRNA-mRNA regulatory network on IFN response,which indicated the mechanism of IFN treatment in CHB patients,it provides a data reference and experimental direction for further screening effective biomarkers of IFN response.Part 2 MiR-873 was Verified to be Involved in IFN Down-regulation of HBV ExpressionObjective: At present,the specific target sites and mechanism of IFN treatment for CHB patients have not been clarified.We intend to verify the relationship between the 4 miRNAs and HBV or IFN treatment in vitro experiments.The aim of this study was to explore the targets of IFN and to provide data support for antiviral treatment of CHB patients.Methods: The pHBV1.1 plasmid was transfected into cells for 48 h to simulate the physiological process of CHB patients.Cells transfected with pHBV1.1 plasmid were treated with recombinant human IFN-α-2b for 48 h to simulate the IFN treatment process of CHB patients.Real-time quantitative polymerase chain reaction(RT-q PCR)was used to detect the expression of miRNA in human and cell.The contents of hepatitis B surface antigen(HBs Ag)and hepatitis B e antigen(HBe Ag)in cell culture supernatant were detected by enzyme linked immunosorbent assay(ELISA).Results: The results of RT-q PCR showed that the expression levels of miR-873,miR-200 a,miR-30 b,and let-7g in CHB patients were significantly higher than those in healthy controls.Compared with the control group,the expression level of 4 miRNAs in the cells transfected with pHBV1.1 plasmid was significantly increased.The expression level of miR-873 was lower in cells transfected with the pHBV1.1 plasmid and IFN treatment than in cells not treated with IFN.ELISA results showed that HBs Ag levels were decreased in cells treated with miR-873 antagonists compared with controls,whereas HBs Ag levels were increased in cells treated with miR-873 mimics.In this study,IFN treatment resulted in down-regulation of HBs Ag and HBe Ag levels in the supernatants of cells transfected with the pHBV1.1 plasmidthe.levels of HBs Ag and HBe Ag were further reduced when cells were treated with both IFN and miR-873 inhibitor,whereas co-treatment of cells with IFN and miR-873 mimics resulted in increased levels of HBs Ag and HBe Ag.We explored the regulatory effect of miR-873 on ANXA2.Compared with the control group,ANXA2 was up-regulated in cells transfected pHBV1.1 plasmid and down-regulated in cells treated with IFN.Conclusion: In this study,we found the relationship between miR-873 and HBV or IFN in human and cells.Then,we identified the role of miR-873 in the down-regulation of HBV expression by IFN,and suggested the potential of miR-873 as a biomarker of interferon response.更多还原