Identification of translational microRNA biomarker candidates for ketoconazole-induced liver injury using next-generation sequencing

Drug induced liver injury (DILI) is a leading cause of acute liver failure. Reliable and translational biomarkers are needed for early detection of DILI. microRNAs (miRNAs) have received wide attention as a novel class of potential DILI biomarkers. However, it is unclear how DILI drugs other than acetaminophen may influence miRNA expression or which miRNAs could serve as useful biomarkers in humans. We selected ketoconazole (KCZ), a classic hepatotoxin, to study miRNA biomarkers for DILI as a proof-of-concept for a workflow that integrated in vivo, in vitro, and bioinformatics analyses. We examined hepatic miRNA expression in KCZ-treated rats at multiple doses and durations using miRNA-sequencing and correlated our results with conventional DILI biomarkers such as liver histology. Significant dysregulation of rno-miR-34a-5p, rno-miR-331-3p, rno-miR-15b-3p, and rno-miR-676 was associated with cytoplasmic vacuolization, a phenotype in rat livers with KCZ-induced injury, which preceded the elevation of serum liver transaminases (ALT and AST). Between rats and humans, miR-34a-5p, miR-331-3p, and miR-15b-3p were evolutionarily conserved with identical sequences, whereas miR-676 showed 73% sequence similarity. Using quantitative PCR, we found that the levels of hsa-miR-34a-5p, hsa-miR-331-3p, and hsa-miR-15b-3p were significantly elevated in the culture media of HepaRG cells treated with 100 mM KCZ (a concentration that could induce cytotoxicity). Additionally, we computationally characterized the miRNA candidates for their gene targeting, target functions, and miRNA/target evolutionary conservation. In conclusion, we identified miR-34a-5p, miR-331-3p, and miR-15b-3p as translational biomarker candidates for early detection of KCZ-induced liver injury with a workflow applicable to computational toxicology studies.药物性肝损伤（Drug induced liver injury, DILI）是急性肝衰竭的首要致病原因。亟需可靠且可转化的生物标志物以实现DILI的早期检测。微小RNA（microRNAs, miRNAs）作为一类新型潜在的DILI生物标志物，已受到广泛关注。然而，目前尚不明确除对乙酰氨基酚（acetaminophen）外的其他DILI药物如何影响miRNA表达，亦不清楚哪些miRNA可作为人类中有效的DILI生物标志物。本研究选取经典肝毒素酮康唑（ketoconazole, KCZ）作为研究对象，探索DILI相关miRNA生物标志物，以此验证整合了体内、体外及生物信息学分析的研究流程的可行性。我们通过miRNA测序（miRNA-sequencing）检测了不同剂量与暴露时长的KCZ处理大鼠的肝脏miRNA表达水平，并将研究结果与肝脏组织学等常规DILI生物标志物进行关联分析。结果显示，rno-miR-34a-5p、rno-miR-331-3p、rno-miR-15b-3p及rno-miR-676的显著异常表达，与KCZ诱导损伤的大鼠肝脏中的细胞质空泡变性表型密切相关，该表型出现于血清肝转氨酶（ALT和AST）水平升高之前。在大鼠与人类之间，miR-34a-5p、miR-331-3p及miR-15b-3p的序列具有进化保守性，二者序列完全一致；而miR-676的序列相似性为73%。通过定量PCR（quantitative PCR）检测，我们发现经100 mM KCZ（该浓度可诱导细胞毒性）处理的HepaRG细胞培养液中，hsa-miR-34a-5p、hsa-miR-331-3p及hsa-miR-15b-3p的水平显著升高。此外，我们通过计算生物学手段对候选miRNA的基因靶向性、靶标功能及miRNA/靶标的进化保守性进行了表征。综上，本研究通过适用于计算毒理学研究的流程，鉴定出miR-34a-5p、miR-331-3p及miR-15b-3p作为可用于早期检测KCZ诱导肝损伤的转化生物标志物候选物。