Global assessment of Antrodia cinnamomea induced microRNA and mRNA transcriptomic alterations in hepatocarcinoma cells

Antrodia cinnamomea (Ac), a traditional medicine and an endemic fungus in Taiwan, has been used in cancer research. Recent research has revealed decreased cell proliferation after treatment of Ac on tumor. In this study, we profiled the 2 hours and 4 hours genome-wide miRNA and mRNA transcriptome by next-generation sequencing techniques to report the early apoptotic effect on Ac fruiting body extract treated human hepatocarcinoma cells, SK-HEP-1, instead of prolonged treatment. Results showed that miRNAs were globally downregulated during the first 2-4 hours in, and solely in, AcFBE-treated SK cells. The inhibition of miRNAs imposed no discrimination against any particular miRNA species, but oncogenic miR-21, miR-191 and two oncogenic clusters miR-17-92 and miR-106b-25 were among the most significantly inhibited miRNAs. In addition to miRNA expression, mRNA transcriptome data indicated the association of apoptosis mechanism with AcFBE treatment. Western blotting indicated a decrease in key proteins Drosha and Dicer required for miRNA biogenesis, and an increase of XRN2 involved in miRNA degradation. Our results suggest that miRNAs appeared to be the prime targets of Ac in disrupting multiple miRNA regulatory pathways and global disruption of miRNA transcriptome resulting in activation of extrinsic and intrinsic (mitochondrial) pathways. Overall design: Human liver SK-Hep-1 cells with or without Antrodia cinnamomea treatment at 2 hours and 4 hours were sequenced by SOLiD 3 and SOLiD 5500xl to obtain miRNA profiles; mRNA profiles also were profiled by SOLID 3. Mouse liver BNL CL.2 cells with or without Antrodia cinnamomea treatment at 2 hours and 4 hours were sequenced by SOLiD 3 to obtain miRNA profiles.

牛樟芝（Antrodia cinnamomea, Ac）是台湾特有的药用真菌，同时也是传统中药材，已被广泛应用于癌症相关研究。已有研究证实，经Ac处理后肿瘤细胞的增殖能力显著降低。本研究采用下一代测序技术，对经牛樟芝子实体提取物（Ac fruiting body extract, AcFBE）处理的人肝癌细胞SK-HEP-1，在2小时和4小时两个时间点的全基因组microRNA（miRNA）和信使RNA（mRNA）转录组进行测序分析，旨在阐明其早期凋亡效应，而非长期处理后的效应。结果显示，在处理后的2-4小时内，经AcFBE处理的SK细胞中miRNA整体呈现下调趋势，且该下调现象仅存在于此处理组中。该miRNA抑制效应并未表现出对特定miRNA分子的选择性，但致癌性miR-21、miR-191以及两个致癌miRNA簇miR-17-92和miR-106b-25是受抑制最为显著的miRNA。除miRNA表达谱外，mRNA转录组数据也显示AcFBE处理与细胞凋亡机制存在显著关联。蛋白质免疫印迹（Western blotting）实验结果显示，参与miRNA生物合成的关键蛋白Drosha和Dicer的表达水平显著下降，而参与miRNA降解的XRN2蛋白表达量则有所升高。本研究结果提示，miRNA或许是Ac发挥抗癌作用的首要靶标：Ac通过干扰多条miRNA调控通路、引发miRNA转录组的全局紊乱，最终激活细胞外源性和内源性（线粒体）凋亡通路。实验整体设计如下：分别对经或未经Ac处理的人源肝脏SK-Hep-1细胞，在2小时和4小时两个时间点，采用SOLiD 3和SOLiD 5500xl平台进行测序以获取miRNA表达谱；同时采用SOLiD 3平台获取其mRNA表达谱。此外，对经或未经Ac处理的小鼠肝脏BNL CL.2细胞，在2小时和4小时两个时间点，采用SOLiD 3平台测序以获取miRNA表达谱。