慢性乙型肝炎环状RNA表达谱的鉴定与分析

Identification and analysis of circular RNA expression profiles in chronic hepatitis B

摘要

目的 分析慢性乙型肝炎（chronic hepatitis B，CHB）患者与健康对照人群的外周血单核细胞（peripheral blood mononuclear cells，PBMCs）中环状RNA（circular RNA，circRNA）表达谱，筛选验证差异表达的circRNA，并研究差异表达circRNA的特征、通路及下游基因。 方法 利用高通量芯片技术鉴定CHB患者和健康对照组中异常表达circRNA。随后对差异circRNA来源基因进行基因本体论和京都基因百科全书富集分析。基于竞争性内源RNA理论，利用多个数据库预测差异circRNA结合的微小RNA（microRNA，miRNA）和miRNA靶mRNA，并构建circRNA-miRNA-mRNA调控网络。最后利用实时荧光定量PCR（quantitative real-time PCR，qRT-PCR）对芯片结果进行验证。 结果 与健康对照组相比，CHB患者PBMCs中存在321个表达上调和549个表达下调的circRNAs。大多数差异表达circRNAs来源于外显子剪切，主要分布于1号和2号染色体。生物信息学分析表明差异circRNAs的来源基因主要参与免疫、炎症反应及疾病相关通路。circRNA-miRNA-mRNA调控网络包括11个circRNAs、17个miRNAs和212个mRNAs。qRT-PCR结果显示circRNA表达趋势与芯片结果一致。与健康对照组比较，hsa_circ_0018744表达在CHB患者组中明显上调（F=4.382，P<0.01），hsa_circ_0085744表达明显下调（F=4.906，P<0.01）。 结论 与健康对照组相比，CHB患者PBMCs中存在差异表达的circRNAs。进一步功能分析表明，这些差异表达circRNAs可以通过某些通路参与CHB发生发展。

Abstract

Objective To analyze the expression profiles of circular RNAs（circRNAs） in peripheral blood mononuclear cells （PBMCs） in patients with chronic hepatitis B（CHB） and healthy controls，select and validate differentially expressed circRNAs，and study the characteristics，pathways，and downstream genes of differentially expressed circRNAs. Methods We identified differentially expressed circRNAs between patients with CHB and controls using high-throughput microarray technology； performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses on the origin genes of the differentially expressed circRNAs； based on the competing endogenous RNA hypothesis，predicted the target micro RNAs（miRNAs） of the differentially expressed circRNAs and the target messenger RNAs（mRNAs） of the miRNAs using multiple databases to construct a circRNA-miRNA-mRNA regulatory network；and finally，validated the microarray results using quantitative real-time PCR（qRT-PCR）. Results A total of 321 upregulated circRNAs and 549 downregulated circRNAs were found in PBMCs of patients with CHB compared with healthy controls. Most of the differentially expressed circRNAs were derived from exon splicing，and were mainly distributed on chromosomes 1 and 2. The enrichment analyses demonstrated that the origin genes of the differentially expressed circRNAs were mainly involved in immune，inflammatory response，and disease-related pathways. The established circRNA-miRNA-mRNA regulatory network consisted of 11 circRNAs，17 miRNAs，and 212 mRNAs. The qRT-PCR results demonstrated that circRNA expression trends were consistent with the microarray results. Notably，in the CHB group，hsa_circ_0018744 was significantly upregulated（F=4.382，P<0.01），and hsa_circ_0085744 was significantly downregulated（F=4.906，P<0.01），as compared with healthy controls. Conclusion The identified differentially expressed circRNAs in PBMCs between patients with CHB and healthy controls may play a role in the development and progression of CHB through certain pathways.

关键词

慢性乙型肝炎 / 环状RNA / 高通量芯片 / 实时荧光定量PCR

Key words

chronic hepatitis B / circular RNA / high-throughput microarray / quantitative real-time PCR

基金资助

重庆市渝中区自然科学基金资助项目(20210139)

重庆医科大学未来医学青年创新计划资助项目(W0177)

重庆市自然科学基金资助项目(CSTB2024NSCQ-MSX0169)