Effect of TNFa and RELA inhibition on gene expression in human PDAC cells

The canonical NF-?B transcription factor RELA is a master regulator of immune and stress responses and is upregulated in PDAC tumours. Here, we characterised previously unknown endogenous RELA-GFP dynamics in PDAC cell lines by live single cell imaging, which revealed rapid, sustained and non-oscillatory nuclear RELA following TNFa stimulation. Using Bayesian analysis of single cell datasets with variation in nuclear RELA, we predicted that RELA heterogeneity in PDAC cell lines is dependent on F-actin dynamics. Using RNA-seq, we identified the actin regulators NUAK2 and ARHGAP31 as transcriptionally regulated by RELA. In turn, NUAK2 or ARHGAP31 siRNA depletion downregulates TNFa-stimulated RELA nuclear localisation in PDAC cells, establishing a novel negative feedback loop regulating RELA activation by TNFa. We identify an additional actin-independent feedback loop involving RELB, which suppresses TNFa-mediated RELA nuclear localisation following RELA mediated upregulation of RELB. Taken together, we provide computational and experimental support for interdependence between the F-actin network and RELA translocation dynamics in PDAC. Overall design: To study the role of TNFa-activated RELA on gene expression in PDAC cells, we introduced doxycycline-inducible lentiviral I?B-SR (RELA inhibitor) into the human PDAC cell lines MIA PaCa2 and PANC1. We performed RNA-seq analysis on cells treated with TNFa at 0, 0.01, 0.1 or 10 ng/ml in the presence or absence of I?B-SR induction.

经典核因子κB（canonical NF-κB）转录因子RELA是免疫与应激反应的核心调控因子，且在胰腺导管腺癌（Pancreatic Ductal Adenocarcinoma, PDAC）肿瘤中表达上调。本研究通过活单细胞成像技术，对胰腺导管腺癌细胞系中此前尚未被揭示的内源性RELA-GFP动态变化进行了表征，结果揭示了肿瘤坏死因子α（Tumor Necrosis Factor α, TNFα）刺激后，RELA快速、持续且非振荡性地入核的现象。本研究通过对存在核RELA表达差异的单细胞数据集进行贝叶斯分析，预测胰腺导管腺癌细胞系中RELA的异质性依赖于纤维肌动蛋白（F-actin）的动态变化。通过RNA测序（RNA-sequencing, RNA-seq），本研究鉴定出肌动蛋白调控因子NUAK2与ARHGAP31为RELA的转录调控靶点。反过来，通过小干扰RNA（small interfering RNA, siRNA）敲低NUAK2或ARHGAP31的表达，可抑制胰腺导管腺癌细胞中TNFα刺激诱导的RELA入核过程，从而确立了一条由TNFα调控RELA激活的新型负反馈环路。本研究还发现了一条不依赖于肌动蛋白的额外反馈环路：RELB可在RELA介导的RELB表达上调后，抑制TNFα介导的RELA入核过程。综上，本研究为胰腺导管腺癌中纤维肌动蛋白网络与RELA核转位动态之间的相互依存关系提供了计算与实验层面的双重证据支持。

总体实验设计：为探究TNFα激活的RELA在胰腺导管腺癌细胞基因表达中的调控作用，本研究将多西环素诱导型慢病毒包装的IκB超抑制因子（IκB-SR，RELA抑制剂）转入人胰腺导管腺癌细胞系MIA PaCa2与PANC1。本研究分别在存在或不存在IκB-SR诱导的条件下，对经浓度为0、0.01、0.1或10 ng/ml的TNFα处理的细胞进行了RNA测序分析。