抗结核药物性肝损伤相关环状RNA鉴定及其作用机制研究

The specific molecular mechanism of anti-tuberculosis drug-induced liver injury (ADLI) is still not clear. Epigenetics provides the new ideas for the studies of disease mechanism in recent years. Circular RNA (circRNA)，a member of Epigenetics, become an advanced research hotspot in RNA study area, which was involved in many important signal transduction pathway, embryogenesis regulation and disease progression. Based on ours early research on Epigenetics, we screened 3415 differential expression circRNA by RNA chip, in plasma of ADLI patients with pulmonary tuberculosis who were in a follow-up cohort. And 101 differential expressions reached more than 10 times. This study will ①detect the key circRNA biomarkers for ADLI in pulmonary tuberculosis patients’ plasma and hepatocytes lines LO2 through bioinformatics prediction, qRT-PCR and other methods; ② reveal the regulating function of the key circRNA for microRNA, protein and signal path of oxidative stress, inflammation, and apoptosis through Co-immunoprecipitation, gene clone and RNA interference. In the circRNA field, we expect to provide new ideas and experimental bases for pathogenesis, preventing and therapeutic methods of ADLI.

抗结核药物性肝损伤 (anti-tuberculosis drug-induced liver injury, ADLI) 的具体分子机制仍不明确。表观遗传学的快速发展为疾病的发生机制研究提供了新思路，其中环状RNA参与了多种重要的信号转导通路，调控胚胎发育及疾病进程，成为RNA领域最新的研究热点。我们在前期表观遗传学研究基础上，利用环状RNA表达谱芯片在肺结核随访队列ADLI患者血浆中筛选到3415个差异表达环状RNA，差异表达10 倍以上者达到101个。本课题拟①利用生物信息学预测、qRT-PCR验证等在肺结核患者血浆、LO2人肝细胞系确定表征ADLI的关键性环状RNA生物标志物；②利用免疫共沉淀、基因克隆、RNA干扰等手段揭示关键性环状RNA对microRNA、蛋白以及氧化应激、炎症、凋亡等信号通路的调控作用。期望在环状RNA领域为ADLI发病机制及其防治研究提供新的思路和实验依据。

抗结核药物性肝损伤（anti-tuberculosis drug-induced liver injury, ADLI）的具体分子机制仍不明确。表观遗传学的快速发展为疾病的发生机制研究提供了新思路，其中环状RNA参与了多种重要的信号转导通路，调控胚胎发育及疾病进程，成为RNA领域最新的研究热点。本项目先后进行了差异表达环状RNA筛选、验证、生物信息学预测、诊断价值研究、在ADLI中的表达水平分析、功能及其作用机制研究。主要结果如下：①利用环状RNA表达谱芯片在肺结核随访队列ADLI患者血浆中以及模拟临床用药诱导的肝细胞损伤模型中筛选出差异表达的环状RNA共6661个，并在ADLI患者血浆中验证了其中的7条环状RNA与ADLI的关系，确定了其基因组定位。②发现hsa_circ_0010996、hsa_circ_0082152、hsa_circ_0027252（circMARS）在ADLI患者血浆中表达上调，与ALT、AST、TNF-α、IL-1β、SOD、CAT、Bcl-2等呈正相关；hsa_circ_0011940、hsa_circ_007769、hsa_circ_0093884、hsa_circ_0064044（circITPR1）在ADLI患者血浆中表达下调，与ALT、AST、NF-κB、TNF-α、IL-6等呈负相关。③利用生物信息学软件预测了hsa_circ_0011940、hsa_circ_0027252、hsa_circ_0093884和hsa_circ_0077692的靶基因分别为miRNA-21、NF-κB、SIRT1和HDAC2。在构建的ADLI细胞模型中利用免疫共沉淀、质粒过表达、RNA干扰等手段揭示了这四种关键性环状RNA对miRNA、蛋白以及氧化应激、炎症、凋亡等信号通路的调控作用。④发现了circMARS—miR-6808-5p/-6874-3p/-3157-5p—KMT2C—EGFR功能轴参与ADLI。⑤证明了hsa_circ_0027252和hsa_circ_0064044可作为ADLI早期诊断生物标志物，二者联合应用ROC曲线下面积为0.88，灵敏度和特异度分别为75.34%和82.06%。本项目从结核病患者血浆和细胞模型研究两方面探讨了关键环状RNA在ADLI中的表达及其分子机制，为ADLI的防治研究提供了新的研究思路和实验依据。