Supplementary Material for: miRNome profiling analysis reveals novel hepatocellular carcinoma diagnostic, prognostic and treatment-related candidate biomarkers: post-hoc analysis of SORAMIC trial

Introduction
Early diagnosis of hepatocellular carcinoma as well as evaluation of prognosis and prediction of treatment efficacy remain challenging due to the missing specific non-invasive biomarkers. The aim of this study is to identify disease-specific microRNA (miRNA) patterns for diagnosis, prediction of prognosis and treatment response in patients with hepatocellular carcinoma (HCC).
Methods
The study population included 42 HCC patients from SORAMIC clinical trial: 22 patients received sorafenib monotherapy, 20 patients underwent 90Y radioembolization in combination with sorafenib. 20 individuals were included in the control group. Hepatocellular carcinoma patients underwent collection of plasma samples before and 7-9 weeks after the beginning of the treatment. Isolation of circulating miRNAs, preparation of small RNA sequencing libraries and next-generation sequencing were performed. Association analysis for novel diagnostic, prognostic and treatment-related candidate biomarkers was performed.
Results
A total of 42 differentially expressed (16 up-regulated and 26 down-regulated) miRNAs were identified comparing baseline and control group plasma samples. hsa-miR-215-5p and hsa-miR-192-5p were down-regulated, while hsa-miR-483-5p and hsa-miR-23b-3p were up-regulated comparing baseline and 7-9 weeks post-sorafenib monotherapy samples. hsa-miR-215-5p was the sole down-regulated miRNA in the same combination therapy comparison. hsa-miR-183-5p, hsa-miR-28-3p and hsa-miR-1246 were found to be significantly up-regulated comparing non-responders versus responders to sorafenib. High hsa-miR-215-5p expression was significantly associated with worse HCC patients’ prognosis.
Conclusions
Systematic miRNA profiling of highly characterized samples from SORAMIC study revealed a subset of potential miRNA biomarkers for hepatocellular carcinoma diagnosis and prognosis of sorafenib-treated patients’ survival.

引言
由于缺乏特异性无创生物标志物，肝细胞癌（hepatocellular carcinoma, HCC）的早期诊断、预后评估以及治疗疗效预测仍面临诸多挑战。本研究旨在筛选可用于肝细胞癌患者诊断、预后预测及治疗应答评估的疾病特异性微小RNA（microRNA, miRNA）表达谱。
方法
本研究队列纳入来自SORAMIC临床试验的42例肝细胞癌患者：其中22例接受索拉非尼单药治疗，20例接受90Y放射性栓塞联合索拉非尼治疗；另纳入20名个体作为对照组。肝细胞癌患者分别于治疗开始前及治疗开始后7~9周采集血浆样本。后续完成循环miRNA分离、小RNA测序文库构建及下一代测序，并针对潜在的新型诊断、预后及治疗相关候选生物标志物开展关联分析。
结果
对比基线与对照组血浆样本，共筛选得到42个差异表达miRNA，其中16个表达上调、26个表达下调。对比基线与索拉非尼单药治疗后7~9周的血浆样本，hsa-miR-215-5p与hsa-miR-192-5p表达下调，而hsa-miR-483-5p及hsa-miR-23b-3p表达上调。在联合治疗组的对比分析中，仅hsa-miR-215-5p表达下调。对比索拉非尼治疗的无应答者与应答者，hsa-miR-183-5p、hsa-miR-28-3p及hsa-miR-1246的表达显著上调。hsa-miR-215-5p高表达与肝细胞癌患者不良预后显著相关。
结论
对SORAMIC研究中经过严格表征的样本进行系统性miRNA表达谱分析，筛选得到一组可用于肝细胞癌诊断以及索拉非尼治疗患者生存预后评估的潜在miRNA生物标志物。