Multi-omic study of genome-edited human colonoids reveal patterns of microRNA regulation specific to different colorectal cancer mutation profiles. Multi-omic study of genome-edited human colonoids reveal patterns of microRNA regulation specific to different colorectal cancer mutation profiles

Colorectal cancer (CRC) is the third most diagnosed cancer and the second leading cause of cancer-related death worldwide Unique combinations of mutations can affect responsiveness to specific therapeutics. This can be, at least in part, due to mutation-specific alterations in gene regulatory mechanisms. To better understand how unique combinations of mutations affect gene regulation, we generated small RNA-seq, length extension chromatin run-on-seq (leChRO-seq), and RNA-seq data from APC (A-mutant), APC/KRAS (AK-mutant), APC/KRAS/TP53 mutant (AKP-mutant), and iGFP control human colonic organoids. From these analyses, we found that our colonic organoid models demonstrate mutation-specific patterns of miRNA transcription, miRNA expression and transcriptional regulatory element actvitiy. Furthermore, we defined 10 patterns of miRNA expression across organoid models. We highlighted one group of miRNAs that exhibited a unique downregulation of expression in AKP-mutant organoids (including miR-34a-5p and miR-10a-5p). Analysis of miRNA transcription revealed that most changes in miRNA expression are correlated with changes in miRNA transcription. Analysis of leChRO-seq data revealed that transcriptional regulatory elements upregulated in AKP-mutant colonic organoids have an enrichment of predicted binding sites for oncogenic transcription factors. Overall design: Genetically modfieid colonic organoid (termed colonoid) models were generated using a dox-inducible CRISPR system. smRNA-seq data was generated for APC+dox mutant (A-mutant; n=3), APC/KRAS+dox mutant (AK-mutant; n=4), APC/KRAS/TP53+dox mutant (AKP-mutant; n=2), iGFP+dox (iGFP_dox; n=4), iGFP-dox (iGFP_noDox; n=4). Length extension chromatin run-on sequencing (leChRO-seq) data was generated for A-mutant (n=2), AK-mutant (n=2), AKP-mutant (n=2), iGFP_dox (n=2), and iGFP_noDox (n=2). RNA-seq data was generated for iGFP_dox (n=4) and AKP-mutant (n=3).

结直肠癌（Colorectal cancer, CRC）是全球第三大确诊癌症，亦是第二大癌症相关死亡病因。突变的独特组合可影响肿瘤对特定疗法的响应性，这一现象至少部分源于基因调控机制中的突变特异性改变。为深入阐明突变的独特组合如何调控基因表达，我们分别对APC突变型（A-mutant）、APC/KRAS双突变型（AK-mutant）、APC/KRAS/TP53三突变型（AKP-mutant）以及iGFP对照人结肠类器官，开展了小RNA测序（small RNA-seq）、长度延伸染色质延续测序（leChRO-seq）与RNA测序（RNA-seq）实验。通过上述分析，我们发现本研究构建的结肠类器官模型，呈现出突变特异性的miRNA转录模式、miRNA表达模式与转录调控元件活性模式。此外，我们在所有类器官模型中鉴定出10种miRNA表达模式，并重点关注了一组在AKP突变型类器官中呈现独特表达下调的miRNA，其中包括miR-34a-5p与miR-10a-5p。对miRNA转录的分析显示，绝大多数miRNA的表达变化与其转录变化呈显著相关。对leChRO-seq数据的分析表明，在AKP突变型结肠类器官中上调的转录调控元件，显著富集了致癌转录因子的预测结合位点。整体实验设计如下：本研究采用多西环素（dox）诱导型CRISPR系统，构建了基因修饰结肠类器官（colonoid）模型。小RNA测序（smRNA-seq）的样本包括：APC+dox突变型（A-mutant，n=3）、APC/KRAS+dox突变型（AK-mutant，n=4）、APC/KRAS/TP53+dox突变型（AKP-mutant，n=2）、iGFP+dox组（iGFP_dox，n=4）以及iGFP无dox处理组（iGFP_noDox，n=4）。长度延伸染色质延续测序（leChRO-seq）的样本包括：A-mutant（n=2）、AK-mutant（n=2）、AKP-mutant（n=2）、iGFP_dox（n=2）以及iGFP_noDox（n=2）。RNA测序（RNA-seq）的样本包括：iGFP_dox（n=4）与AKP-mutant（n=3）。