外泌体miR-25-3p和miR-425-5p对持续ALT正常慢性乙肝患者肝脏炎症的早期诊断价值及机制研究

Chronic hepatitis B (CHB) is fatal. A large number of studies have shown that there are significant liver injuries in some patients with persistent normal ALT (PNALT). Early detection of liver injury and antiviral therapy is very important. Our previous study showed that exosome-derived miR-25-3p / miR-425-5p in PNALT CHB patients by high-throughput sequencing was closely related to the degree of liver inflammation, and confirmed that the downstream target gene of miR-25-3p was liver inflammation and immune-related genes such as TLR3, TRAF6 and EGR2. In this study, we first expand the clinical sample size to explore the feasibility of these two miRNAs in the diagnosis of early liver injury; then analyzed the effects of exosome miRNAs on hepatocyte inflammatory injury and immune signal regulation followed by fluorescence labeling of exosomes in liver cell transport, co-culture of exosomes and recipient hepatocytes. Finally, constructed the chronic liver injury model mice to observe the mechanism of exogenous miRNAs affecting hepatic inflammatory injury and the anti-hepatitis B virus immunotherapy in vivo. Thereby, the study elucidate the significance of exosomal miR-25-3p/miR-425-5p as novel candidate for early liver injury and treatment of PNALT.

慢性乙型肝炎（CHB）危害严重，大量研究证实部分持续ALT正常（PNALT）患者存在显著肝损伤，尽早发现这类患者的肝脏损伤并给予抗病毒治疗是关键。本课题前期在PNALT的CHB 患者中通过高通量测序筛选出外泌体miR-25-3p/miR-425-5p与肝脏炎症程度密切相关，并确认了miR-25-3p的下游靶基因为肝脏炎症和免疫相关基因TLR3、TRAF6和EGR2。本研究拟首先扩大临床样本量，探讨这两个miRNAs对肝脏炎症早期诊断的可行性；接着通过荧光标记的手段研究外泌体在肝细胞中的转运，共培养外泌体与受体肝细胞，分析外泌体miRNAs对肝细胞炎症损伤和免疫信号调节的影响；最后构建慢性肝损伤小鼠模型，观察外泌体miRNAs影响肝脏炎症的机制及抗乙肝病毒的免疫治疗作用。阐明外泌体miR-25-3p/miR-425-5p对PNALT的早期肝损伤的诊断价值，为该类患者抗病毒治疗提供新思路。

本项目在对慢性乙型肝炎持续肝功能正常（PNALT）患者外周血外泌体进行高通量测序及qRT-PCR验证后，发现外泌体来源的miR-25-3p表达水平与肝组织炎症分级密切相关。按照研究计划，通过细胞实验验证外泌体的功能，qRT-PCR、凋亡实验、Transwell和细胞划痕试验等，详细分析外泌体miR-25-3p的功能，通过干预miR-25-3p的表达，确认其影响肝癌细胞凋亡及增殖的作用。并验证其可靶向调控FBXW7，调节PI3K/Akt通路，另一方面，发现miR-25-3p也可调控SIK1 ，调节 Wnt/β-catenin通路。动物成瘤实验也证实外泌体miR-25-3p通过上述通路影响肝癌细胞的炎症凋亡。全面系统阐述外泌体来源的miR-25-3p可能是PNALT患者由慢性乙型肝炎发展为肝癌的潜在机制miR-25-3p可以促进肝癌的细胞转移和增殖，同时抑制它们的凋亡。.本研究一方面证实外泌体来源miR-25-3p对PNALT慢性乙肝肝脏炎症早期诊断的可靠性； 同时通过体内体外实验，详细分析外泌体分泌miR-25-3p影响肝脏炎症的作用及具体分子机制；通过干预miR-25-3p的表达，分析并证实其影响肝细胞凋亡、侵袭转移等的机制，为肝脏炎症提供新的分子诊断标志，并为开发基于miRNA的新型抗HBV免疫药物提供依据。

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