背景:我们之前确定了Il17RB,IL17超家族的一员,作为子宫内膜衰老的候选标记基因。虽然IL17RB与几个器官系统的炎症和恶性肿瘤有关,其在子宫内膜中的功能尚未被研究,因此知之甚少。在本研究中,我们对该受体进行了功能分析,目的是确定其与年龄相关的过表达对子宫环境的影响.
方法:我们分析了通过慢病毒转导(“IL17RB-hEM”)被迫表达受体的永生化人子宫内膜腺上皮细胞系(“hEM”)中IL17RB相关信号通路和下游基因表达。我们还从子宫切除术患者的子宫内膜组织中制备了子宫内膜类器官(“患者来源的EO”),并将其暴露于由IL17RB表达上调的细胞因子中,以研究类器官形成能力和衰老标志物的变化。我们分析了来自我们先前研究的RNA-seq数据(GEO登录号GSE132886)以鉴定与改变的IL17RB表达相关的信号通路。我们还分析了JNK途径对类器官形成能力的影响。
结果:用白细胞介素17B刺激可增强IL17RB-hEM中的NF-κB通路,导致编码衰老相关分泌表型(SASP)因子IL6,IL8和IL1β的基因表达显着升高。在这些细胞因子中,IL1β抑制子宫内膜类器官生长。生物信息学分析表明,JNK信号通路与IL17RB表达的年龄相关变异有关。当IL17RB阳性细胞在存在IL17B的情况下培养时,它们的类器官形成能力略低于未暴露的IL17RB阳性细胞,但是当IL17B与JNK抑制剂(SP600125)配对时,恢复到控制水平。Further,相对于未暴露(对照),IL1β暴露显着降低了子宫内膜类器官的形成能力和p21表达增加,但当IL1β与SP600125配对时,两项指标均恢复至与对照条件相当的水平.
结论:我们揭示了IL17RB之间的关联,其在子宫内膜腺上皮中的表达随着年龄的增长而增加,和细胞衰老。使用人子宫内膜类器官作为体外模型,我们发现IL1β通过JNK途径抑制细胞增殖并导致子宫内膜衰老。
BACKGROUND: We previously identified Il17RB, a member of the IL17 superfamily, as a candidate marker gene for endometrial aging. While IL17RB has been linked to inflammation and malignancies in several organ systems, its function in the endometrium has not been investigated and is thus poorly understood. In the present study, we performed a functional analysis of this receptor with the aim of determining the effects of its age-associated overexpression on the uterine environment.
METHODS: We analyzed IL17RB-related signaling pathways and downstream gene expression in an immortalized human endometrial glandular epithelial cell line (\"hEM\") forced to express the receptor via lentiviral transduction (\"IL17RB-hEM\"). We also prepared endometrial organoids from human endometrial tissue sourced from hysterectomy patients (\"patient-derived EOs\") and exposed them to cytokines that are upregulated by IL17RB expression to investigate changes in organoid-forming capacity and senescence markers. We analyzed RNA-seq data (GEO accession number GSE132886) from our previous study to identify the signaling pathways associated with altered IL17RB expression. We also analyzed the effects of the JNK pathway on organoid-forming capacity.
RESULTS: Stimulation with interleukin 17B enhanced the NF-κB pathway in IL17RB-hEM, resulting in significantly elevated expression of the genes encoding the senescence associated secretory phenotype (SASP) factors IL6, IL8, and IL1β. Of these cytokines, IL1β inhibited endometrial organoid growth. Bioinformatics analysis showed that the JNK signaling pathway was associated with age-related variation in IL17RB expression. When IL17RB-positive cells were cultured in the presence of IL17B, their organoid-forming capacity was slightly but non-significantly lower than in unexposed IL17RB-positive cells, but when IL17B was paired with a JNK inhibitor (SP600125), it was restored to control levels. Further, IL1β exposure significantly reduced organoid-forming capacity and increased p21 expression in endometrial organoids relative to non-exposure (control), but when IL1β was paired with SP600125, both indicators were restored to levels comparable to the control condition.
CONCLUSIONS: We have revealed an association between IL17RB, whose expression increases in the endometrial glandular epithelium with advancing age, and cellular senescence. Using human endometrial organoids as in vitro model, we found that IL1β inhibits cell proliferation and leads to endometrial senescence via the JNK pathway.