Abstract: Objective　To analyze the differential expression of non⁃coding RNAs (ncRNAs) from liver tissues adjacent to hepatic cystic echinococcosis (CE) lesions and distant normal liver tissues using whole transcriptome sequencing, and perform functional annotations of differentially expressed ncRNAs, so as  to explore the potential role of ncRNAs in the pathogenesis of CE. Methods　Intraoperative liver tissue specimens adjacent to hepatic CE lesions and distant normal liver tissue specimen were sampled from patients with hepatic CE, and the expression profiles of microRNAs (miRNAs), circular RNAs (circRNAs), and long non⁃coding RNAs (lncRNAs) were detected using whole transcriptome sequencing. Differentially expressed genes were identified, and functional annotations were performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. In addition, a circRNA/lncRNA⁃miRNA⁃messenger RNA (mRNA) competing endogenous RNA (ceRNA) network was constructed using the Cytoscape software, and the expression of hub miRNAs in the network was validated using real⁃time quantitative reverse transcription PCR (RT⁃qPCR) assay.  Results　A total of 41 differentially expressed miRNAs were identified between the adjacent and distal tissues of hepatic CE lesions, including 8 up⁃regulated and 33 down⁃regulated miRNAs, which were significantly enriched in biological processes of Ras signaling and neutrophil activation. Five differentially expressed circRNAs were detected, including 3 up⁃regulated and 2 down⁃regulated circRNAs, which were significantly enriched in molecular functions of hormone signaling pathways and RNA transcription regulation. A total of 447 differentially expressed lncRNAs were identified, including 200 up⁃regulated and 247 down⁃regulated lncRNAs, which were involved in cell proliferation, immune regulation, and extracellular matrix remodeling pathways. MiRNA target analysis predicted hsa⁃miR⁃27a⁃5p, hsa⁃miR⁃21⁃3p, and hsa⁃miR⁃181b⁃2⁃3p as hub nodes in the ceRNA network. RT⁃qPCR assay detected that the relative expression levels of ENSG00000253736, HAS2⁃AS1, PCSK6, hsa⁃miR⁃21⁃3p, hsa⁃miR⁃27a⁃5p, MIR23AHG, VIPR1⁃AS1, LINC02910, and hsa⁃miR⁃181b⁃2⁃3p were 3.00 ± 0.25, 2.75 ± 0.33, 1.01 ± 0.51, 2.65 ± 0.41, 1.01 ± 0.29, 1.10 ± 0.31, 1.05 ± 0.27, 0.25 ± 0.49, and 2.56 ± 0.35 in adjacent tissues of hepatic CE lesions, normalized to that in distant tissues from hepatic CE lesions,  respectively (t = 6.21, 5.83, 7.51, 7.46, 6.12, 6.65, 7.13, 1.87 and 7.81, all P values  < 0.01), which was consistent with whole transcriptome sequencing results. Conclusions　Differentially expressed ncRNAs from adjacent and distal liver tissues of hepatic CE lesions may contribute to the pathological mechanisms of CE through mediating cell proliferation, immune evasion, and inflammatory responses, in which hsa⁃miR⁃27a⁃5p and hsa⁃miR⁃21⁃3p may serve as hub miRNAs.

Key words: Cystic echinococcosis, Liver, Whole transcriptome sequencing, Non?coding RNA, MicroRNA, Circular RNA, Long non?coding RNA, Competing endogenous RNA network

摘要： 目的　采用全转录组测序技术分析肝细粒棘球蚴病（cystic echinococcosis，CE）病灶毗邻组织与远端正常组织的非编码RNA（non⁃coding RNA， ncRNA）差异表达，并对差异表达ncRNA进行功能注释，以探讨ncRNA在CE发病中的潜在作用。方法　收集肝CE患者术中毗邻病变组织及远端正常肝组织样本，利用全转录组测序技术检测微小RNA（microRNA，miRNA）、环状RNA（circular RNA，circRNA）、长链ncRNA（longncRNA，lncRNA）表达谱，筛选差异表达基因，并进行基因本体论（Gene Ontology，GO）和京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）功能注释分析。同时，借助Cytoscape软件构建circRNA/lncRNA⁃miRNA⁃信使RNA（messenger RNA，mRNA）竞争性内源RNA（competitive endogenous RNA，ceRNA）网络，并通过实时荧光定量PCR（real⁃time quantitative reverse transcriptionPCR，RT⁃qPCR）验证该网络中的关键miRNA。结果　在肝CE病灶毗邻组织与远端正常组织间共检测到41个差异表达miRNA，其中8个上调miRNA、33个下调miRNA，主要富集于Ras信号传导和中性粒细胞活化等生物学过程；检测到5个差异表达circRNA，其中3个上调circRNA、2个下调circRNA，主要富集于激素信号通路及RNA转录调控功能；检测到447个差异表达lncRNA，其中200个上调lncRNA、247个下调lncRNA，涉及细胞增殖、免疫调节及细胞外基质重塑等通路。miRNA靶向分析发现，hsa⁃miR⁃27a⁃5p、hsa⁃miR⁃21⁃3p和hsa⁃miR⁃181b⁃2⁃3p为ceRNA网络中的关键节点。RT⁃qPCR验证结果显示，以CE病灶远端组织中上述基因相对表达量为1，CE病灶毗邻组织中ENSG00000253736、HAS2⁃AS1、PCSK6、hsa⁃miR⁃21⁃3p、hsa⁃miR⁃27a⁃5p、MIR23AHG、VIPR1⁃AS1、LINC02910、hsa⁃miR⁃181b⁃2⁃3p 的相对表达量分别为3.00 ± 0.25、2.75 ± 0.33、1.01 ± 0.51、2.65 ± 0.41、1.01 ± 0.29、1.10 ± 0.31、1.05 ± 0.27、0.25 ± 0.49、2.56 ± 0.35，不同组织中上述基因相对表达量差异均有统计学意义（t = 6.21、5.83、7.51、7.46、6.12、6.65、7.13、1.87、7.81，P均< 0. 01），以上结果与测序结果一致。结论　肝CE病灶毗邻及远端组织中差异表达的ncRNA可能通过调控细胞增殖、免疫逃逸和炎症反应参与CE发病，hsa⁃miR⁃27a⁃5p和hsa⁃miR⁃21⁃3p可能是关键miRNA。

关键词: 细粒棘球蚴病, 肝脏, 全转录组测序, 非编码RNA, 微小RNA, 环状RNA, 长链非编码RNA, 竞争性内源RNA网络