Ding Xiao-Lei, Yang Xiaojing, Liang Gangning, Wang Kai
Co-Innovation Center for Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing, Jiangsu, 210037, China.
Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, CA 90033, USA.
Sci Rep. 2016 Apr 19;6:24545. doi: 10.1038/srep24545.
DNA methylation in gene promoters leads to gene silencing and is the therapeutic target of methylation inhibitors such as 5-Aza-2'-deoxycytidine (5-Aza-CdR). By analyzing the time series RNA-seq data (days 5, 9, 13, 17) obtained from human bladder cells exposed to 5-Aza-CdR with 0.1 uM concentration, we showed that 5-Aza-CdR can affect isoform switching and differential exon usage (i.e., exon-skipping), in addition to its effects on gene expression. We identified more than 2,000 genes with significant expression changes after 5-Aza-CdR treatment. Interestingly, 29 exon-skipping events induced by treatment were identified and validated experimentally. Particularly, exon-skipping event in Enhancer of Zeste Homologue 2 (EZH2) along with expression changes showed significant down regulation on Day 5 and Day 9 but returned to normal level on Day 13 and Day 17. EZH2 is a component of the multi-subunit polycomb repressive complex PRC2, and the down-regulation of exon-skipping event may lead to the regain of functional EZH2 which was consistent with our previous finding that demethylation may cause regain of PRC2 in demethylated regions. In summary, our study identified pervasive transcriptome changes of bladder cancer cells after treatment with 5-Aza-CdR, and provided valuable insights into the therapeutic effects of 5-Aza-CdR in current clinical trials.
基因启动子中的DNA甲基化会导致基因沉默，是5-氮杂-2'-脱氧胞苷（5-Aza-CdR）等甲基化抑制剂的治疗靶点。通过分析从暴露于浓度为0.1μM的5-Aza-CdR的人膀胱细胞中获得的时间序列RNA-seq数据（第5、9、13、17天），我们发现5-Aza-CdR除了影响基因表达外，还能影响异构体转换和外显子使用差异（即外显子跳跃）。我们鉴定出2000多个在5-Aza-CdR处理后有显著表达变化的基因。有趣的是，经实验鉴定并验证了处理诱导的29个外显子跳跃事件。特别是，zeste同源物2（EZH2）增强子中的外显子跳跃事件以及表达变化在第5天和第9天显示出显著下调，但在第13天和第17天恢复到正常水平。EZH2是多亚基多梳抑制复合物PRC2的一个组成部分，外显子跳跃事件的下调可能导致功能性EZH2的恢复，这与我们之前的发现一致，即去甲基化可能导致去甲基化区域中PRC2的恢复。总之，我们的研究确定了5-Aza-CdR处理后膀胱癌细胞普遍存在的转录组变化，并为当前临床试验中5-Aza-CdR的治疗效果提供了有价值的见解。
