Loss of histone macroH2A1 in hepatocellular carcinoma cells promotes paracrine-mediated chemoresistance and CD4+CD25+ T regulatory cells activation

We recently described the phenotype of HepG2 and Huh-7, hepatocellular carcinoma cells, knocked down for histone variant macroH2A1. Both cell lines acquire a cancer stem cell phenotype (Lo Re O et al., Hepatology 2017, PMID: 28913935; Lo Re O et al., Epigenetics 2018, PMID: 30165787). We found that short hairpin RNA-mediated macroH2A1 knockdown induced acquisition of CSC-like features, including the growth of significantly larger and less differentiated tumors when injected into nude mice. MacroH2A1-depleted HCC cells also exhibited reduced proliferation, resistance to chemotherapeutic agents, and stem-like metabolic changes consistent with enhanced hypoxic responses and increased glycolytic pathways. As macroH2A1 is a potent transcriptional modifier we asked how KD of this histone variant might affect patterns of gene expression, and whether we could identify potential mechanistic links to the observed in vitro and in vivo HCC phenotypes. Here we used RNA-Seq to gain deep mechanistic insight into the distinct functions of macroH2A1 KD and conditioned medium (CM)-exposed Huh-7 cells. Heatmap analysis of the differentially expressed genes between the three groups revealed a similar transcriptomic profile between KD and CM, compared to the control condition. Genes were considered differentially expressed between groups if their expression values significantly differed by >2 fold. Statistical differences in gene expression were assessed by the ANOVA test. Correction for multiple test was achieved by the Benjamini-Hochberg procedure. The significance threshold was set to 0.05. Using a cut-off of 2 fold change, assessment of differentially expressed genes for Huh-7 macroH2A1 KD or Huh-7 CM KD versus CTL showed no transcriptional overlap between the different Huh-7 cell lines. 783 and 987 genes were instead significantly differentially-expressed in macroH2A1 KD or CM KD versus control cells, respectively. Interestingly, the significantly enriched transcripts over-represented in KD and CM-exposed cells compared to control cells were implicated in a number of functions and diseases that were also shared between CM versus control comparisons. Specifically, Ingenuity pathway analysis (IPA) identified categories of cancer, gastrointestinal diseases, lipid metabolism, cell-to-cell signaling, nucleic acid metabolism, cell death & survival and others, in common between KD versus CTL and CM versus CTL. These data support a paracrine modulation of gene expression by macroH2A1 KD in HCC cells. Profiling the transcriptome of hepatocellular carcinoma cells Huh-7 control, knocked-down (KD) for macroH2A1, or exposed to conditioned medium from KD cells, using RNA-Seq.

本团队此前曾报道了组蛋白变体macroH2A1（histone variant macroH2A1）敲低的肝癌细胞（hepatocellular carcinoma cells）HepG2与Huh-7的表型特征。上述两种细胞系均获得了癌干细胞（cancer stem cell）表型（Lo Re O等，《Hepatology》2017年，PMID：28913935；Lo Re O等，《Epigenetics》2018年，PMID：30165787）。本研究发现，短发夹RNA（short hairpin RNA, shRNA）介导的macroH2A1敲低可诱导细胞获得癌干细胞样特征，具体表现为接种至裸鼠体内后可形成体积显著更大、分化程度更低的肿瘤。经macroH2A1敲低的肝癌细胞（HCC cells）同时表现出增殖能力下降、对化疗药物产生耐药性，以及与增强的缺氧应答和糖酵解通路激活相符的干细胞样代谢改变。鉴于macroH2A1是一种强效的转录调控因子，本研究旨在探究该组蛋白变体的敲低如何影响基因表达模式，以及能否明确与体外、体内观察到的肝癌表型之间的潜在机制关联。本研究通过RNA测序（RNA-Seq），对macroH2A1敲低的Huh-7细胞以及经条件培养基（conditioned medium, CM）处理的Huh-7细胞的独特功能开展深入的机制解析。对三组细胞的差异表达基因进行热图分析后发现，与对照组相比，macroH2A1敲低组与条件培养基处理组的转录组谱具有相似性。当基因表达量差异倍数≥2倍时，即判定为组间差异表达基因。基因表达的统计学差异通过方差分析（ANOVA test）进行评估，多重检验校正采用本杰明尼-霍赫贝格（Benjamini-Hochberg）校正法，显著性阈值设为0.05。以2倍表达变化作为筛选阈值，对Huh-7细胞macroH2A1敲低组、条件培养基处理组与对照组（control, CTL）的差异表达基因进行分析后发现，两类处理组之间不存在转录组重叠。其中，macroH2A1敲低组与条件培养基处理组分别有783个和987个基因与对照组相比出现显著差异表达。值得注意的是，与对照组相比，macroH2A1敲低组与条件培养基处理组中显著富集的转录本所涉及的功能与疾病类别，与条件培养基处理组vs对照组的分析结果存在重叠。具体而言，经IPA（Ingenuity pathway analysis）分析发现，macroH2A1敲低组vs对照组、条件培养基处理组vs对照组两组共有的富集类别包括癌症、胃肠道疾病、脂质代谢、细胞间信号传导、核酸代谢、细胞死亡与存活等。上述结果证实，肝癌细胞中macroH2A1敲低可通过旁分泌方式调控基因表达。本研究通过RNA测序对以下三类肝癌细胞Huh-7的转录组进行了分析：对照组、macroH2A1敲低组以及经敲低细胞的条件培养基处理组。