Inactivation of CFTR by CRISPR/Cas9 alters transcriptional regulation of inflammatory pathways and other networks
Inactivation of CFTR by CRISPR/Cas9 alters transcriptional regulation of inflammatory pathways and other networks
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DOI：
10.1016/j.jcf.2019.05.003
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发表时间：
2020-01-01
影响因子：
5.2
通讯作者：
Drumm, Mitchell L.
中科院分区：
文献类型：
--
作者：
Hao, Shuyu;Roesch, Erica A.;Drumm, Mitchell L.
背景资料：患有囊性纤维化（CF）的个体在多个器官中经历升高的炎症，但这是否反映CF细胞的固有特征或促炎环境的结果尚不清楚。克隆线与功能CFTR（CFTR+）相比，那些没有（CFTR-），直接解决CFTR在炎症基因regulation.Results的作用：所有线路保持CFTR mRNA的生产和紧密连接的形成。CFTR+线显示短路电流响应毛喉素，而CFTR-线没有。细胞因子IL 6和CXCL 8（IL 8）的基线表达在细胞系之间没有差异，无论CFTR基因型如何。所有细胞系都通过增加IL 6和CXCL 8 mRNA水平来响应TNF α和IL 1 β，但CFTR-细胞系比CFTR+细胞系产生更多的CXCL 8 mRNA。在TNF α刺激之前和之后，比较6个CFTR-和6个CFTR+系的转录组的差异表达作为CFTR基因型的函数。虽然有些基因出现差异表达，只是因为CFTR的情况下，其他人需要刺激的差异是apparent.Conclusion：总之，这些数据表明细胞响应CFTR的情况下，通过调节转录网络，其中一些是明显的，只有当细胞暴露于不同的环境背景下，如炎症。关于炎症，这些数据表明了一种模型，其中CFTR的缺失导致细胞的稳定的促炎状态，这种状态仅通过刺激来揭示。(C)2019年欧洲囊性纤维化协会。Elsevier B. V.出版，保留所有权利。
Background: Individuals with cystic fibrosis (CF) experience elevated inflammation in multiple organs, but whether this reflects an inherent feature of CF cells or is a consequence of a pro-inflammatory environment is not clear.Method: Using CRISPR/Cas9-mediated mutagenesis of CFTR, 17 subclonal cell lines were generated from Caco-2 cells. Clonal lines with functional CFTR (CFTR+) were compared to those without (CFTR-) to directly address the role of CFTR in inflammatory gene regulation.Results: All lines maintained CFTR mRNA production and formation of tight junctions. CFTR+ lines displayed short circuit currents in response to forskolin, while the CFTR- lines did not. Baseline expression of cytokines IL6 and CXCL8 (IL8) was not different between the lines regardless of CFTR genotype. All lines responded to TNF alpha and IL1 beta by increasing IL6 and CXCL8 mRNA levels, but the CFTR- lines produced more CXCL8 mRNA than the CFTR+ lines. Transcriptomes of 6 CFTR- and 6 CFTR+ lines, before and after stimulation by TNF alpha, were compared for differential expression as a function of CFTR genotype. While some genes appeared to be differentially expressed simply because of CFTR's absence, others required stimulation for differences to be apparent.Conclusion: Together, these data suggest cells respond to CFTR's absence by modulating transcriptional networks, some of which are only apparent when cells are exposed to different environmental contexts, such as inflammation. With regards to inflammation, these data suggest a model in which CFTR's absence leads to a poised, pro-inflammatory state of cells that is only revealed by stimulation. (C) 2019 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.