Profiling of DHOK targets through transcriptome sequencing. Profiling of DHOK targets through transcriptome sequencing

DHOK (14,15β-dihydroxyklaineanone) is a novel diterpene isolated from roots of Eurycoma longifolia Jack, a traditional herb widely applied in Southeast Asia. It is reported that DHOK has cytotoxic effect on cancer cells, but its anti-cancer mechanism has still been not clear. To elucidate the anti-cancer mechanism of DHOK, we used transcriptome sequencing (RNA-seq) to identify cancer-associated gene signatures in human CRC cells treated with DHOK. The differential expression of genes in SW620 cells treated with or without DHOK (12.5 μM, 24 h) was significant. Compared with the control, there were totally 4083 genes with their expression levels significantly altered following DHOK treatment. Among these differentially expressed genes, 2857 genes were downregulated and 1226 genes were upregulated. Subsequently, we performed GO (gene ontology) analysis of DHOK targets. It was shown that the distribution of these targets was in different parts of the cell, and especially in the nucleus, cytoplasm, mitochondria, lysosome, endoplasmic reticulum, Golgi apparatus etc.To understand the inhibitory effect of DHOK on human CRC cells, the differentially expressed genes identified were subjected to functional enrichment analysis. The significantly enriched GO terms were mainly associated with autophagy, including autophagosome formation, lysosomal transport, mitophagy, regulation of autophagy, metabolic stress etc. KEGG (kyoto encyclopedia of genes and genomes) analysis showed that the targets of DHOK participated in various signaling pathways, including VEGF (vascular endothelial growth factor), mTOR, P53, TNF-α (tumor necrosis factor), MAPK (mitogen activated protein kinase), Ras, Wnt, FoxO (forkhead box) etc. Overall design: Examination of a compound on cell transcriptome.

DHOK（14,15β-二羟基klaineanone）是一种从广泛应用于东南亚的传统药用植物长叶苦木（Eurycoma longifolia Jack，又名东革阿里）根部分离得到的新型二萜类化合物。已有研究表明，DHOK对癌细胞具有细胞毒性，但其抗癌作用机制仍未明确。为阐明DHOK的抗癌作用机制，本研究采用转录组测序（RNA-seq）技术，在经DHOK处理的人结直肠癌细胞（colorectal cancer, CRC）中筛选癌症相关基因特征。经12.5 μM DHOK处理24小时的SW620细胞与未处理对照组相比，基因的差异表达极为显著。与对照组相比，经DHOK处理后共有4083个基因的表达水平发生显著改变。在这些差异表达基因中，2857个基因表达下调，1226个基因表达上调。随后，我们对DHOK的作用靶点开展了基因本体论（Gene Ontology, GO）分析。结果显示，这些靶点分布于细胞的不同区域，尤其富集于细胞核、细胞质、线粒体、溶酶体、内质网、高尔基体等部位。为探究DHOK对人结直肠癌细胞的抑制作用，本研究对筛选得到的差异表达基因进行了功能富集分析。显著富集的GO条目主要与自噬相关，涵盖自噬体形成、溶酶体运输、线粒体自噬、自噬调控、代谢应激等过程。京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）分析结果显示，DHOK的作用靶点参与了多条信号通路，包括血管内皮生长因子（vascular endothelial growth factor, VEGF）、mTOR、P53、肿瘤坏死因子α（tumor necrosis factor-α, TNF-α）、丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）、Ras、Wnt、叉头框蛋白（forkhead box, FoxO）等。实验整体设计：探究化合物对细胞转录组的影响。