RNA-seq Analysis of Changes in mRNA Expression for the hTERT-HMA/B Human Myometrial Cell Line treated with all Combinations of Progesterone, Forskolin, and Interleukin-1β

Parturition involves cellular signaling changes driven by the complex interplay between progesterone (P4), inflammation, and the cyclic adenosine monophosphate (cAMP) pathway. To characterize this interplay, comprehensive transcriptomic studies utilizing 8 treatment combinations were performed on the hTERT-HMA/B telomerase immortalized human myometrial cell line and myometrium obtained from term c-section deliveries. Genome-wide RNA-sequencing was performed on total RNA extracted from hTERT-HMA/B cells treated with all combinations of P4, forskolin (induces cAMP), and interleukin-1β (IL-1β). Gene set enrichment and regulatory network analyses was then used to identify pathways commonly, differentially, or synergistically regulated by each treatment. Tissue similarity index (TSI) was also applied to characterize the correspondence between cell lines and tissue phenotypes. In addition to their individual anti-inflammatory effects, P4 and cAMP synergistically blocked specific inflammatory pathways/regulators including STAT3/6, CEBPA/B, and OCT1/7, but not NFKB. TSI analysis indicated that forskolin+P4- and IL-1β-treated cells exhibit transcriptional signatures highly similar to non-laboring and laboring term myometrium, respectively. The data identify potential therapeutic targets to prevent preterm birth and show that the hTERT-HMA/B cell line provides is an accurate transcriptional model for term ± labor myometrium. Comparisons made between all single stimulus treated cells (P4, FSK, IL-1β) with untreated cells to identify signaling by progesterone, cAMP, and IL-1β and comparisons made between IL-1β conditions to identify anti-inflammatory actions of P4 and/or FSK in the presence of IL-1β.

分娩过程涉及由孕酮（progesterone, P4）、炎症反应与环磷酸腺苷（cyclic adenosine monophosphate, cAMP）通路之间的复杂互作所驱动的细胞信号转导改变。为解析这一互作机制，研究团队针对hTERT-HMA/B端粒酶永生化人子宫肌层细胞系，以及足月剖宫产分娩获取的子宫肌层组织，设置8种处理组合开展全面转录组学研究。研究人员对经孕酮（P4）、福司柯林（forskolin，可诱导cAMP通路）与白细胞介素-1β（interleukin-1β, IL-1β）的全部组合处理的hTERT-HMA/B细胞提取总RNA，进行全基因组RNA测序。随后通过基因集富集分析与调控网络分析，识别各处理方式共同调控、差异调控或协同调控的通路；同时应用组织相似性指数（tissue similarity index, TSI）表征细胞系与组织表型间的对应关系。实验结果表明，除各自独立的抗炎效应外，孕酮与cAMP可协同阻断特定炎症通路及调控因子，包括STAT3/6、CEBPA/B与OCT1/7，但不影响NFKB通路。TSI分析显示，经福司柯林+孕酮处理的细胞与未临产足月子宫肌层，经IL-1β处理的细胞与临产足月子宫肌层，分别具有高度相似的转录组特征。本数据集不仅明确了可用于预防早产的潜在治疗靶点，同时证实hTERT-HMA/B细胞系可作为临产/未临产足月子宫肌层的精准转录组学模型。此外，本研究还开展了两组对照比较：其一为分别比较单一刺激处理组（P4、FSK、IL-1β）与未处理组，以解析孕酮、cAMP与IL-1β各自的信号通路调控作用；其二为比较不同IL-1β处理条件下的样本，以明确IL-1β存在时孕酮与/或福司柯林的抗炎效应。