MON-153 Bacterial endotoxin lipopolysaccharide dependent TNFα-signaling dysregulates proinflammatory and anti-inflammatory miRNAs in endometrial stromal cell pathogenesis

Abstract Disclosure: I. Chowdhury: None. S. Banerjee: None. S. Mcallister: None. L.P. Villacorta: None. N. Sidell: None. W.E. Thompson: None. Endometriosis is an estrogen-dependent chronic inflammatory gynecological disorder that is predominant mostly in reproductive-age women. Our previous studies demonstrated that besides hormonal regulation, the secondary cytokine, tumor necrosis factor α (TNFα), triggers the inflammatory process in endometriosis. TNFα is a non-glycosylated cytokine protein with potent inflammatory, cytotoxic, and angiogenic potential. Other studies established that increased levels of bacterial endotoxin lipopolysaccharide (LPS), the major outer membrane protein of gram-negative bacteria, present in the menstrual blood and peritoneal fluid induces an inflammatory response to endometriotic cells by contributing lesion proliferation in patients. Therefore, in the current study, we investigate the effects of LPS on TNFα-signaling in the dysregulation of inflammatory, anti-inflammatory, and proangiogenic microRNAs (miRNAs) in the normal human endometrial stromal cells (NESCs) compared with the untreated cells derived from eutopic endometrium of endometriosis subjects (EESC). miRNAs are short, 18- to 22-nucleotide- size, non-coding RNAs that act as post-transcriptional modulators of gene expression. The quantitative RT-QPCR analysis showed that TNFα and receptor TNFR1, interleukins (IL1β, IL6, IL8), and chemotactic CCl5 were significantly upregulated by LPS in a dose and time-dependent manner in NESCs, similar to EESCs. In contrast, anti-inflammatory miRNAs were substantially lower in LPS-treated NESCs similar to EESCs. In addition, the analysis showed that LPS treatments increased the phosphorylation of the NF-κB signaling pathways in a dose and time-dependent manner in NESCs along with the higher expression of TNFα, TNFR1, IL1β, IL6, IL8, and CCl5 at the protein levels. These results suggest that bacterial endotoxin, LPS-dependent TNFα-signaling through activation of NF-κB-pathway dysregulates the expression of pro-inflammatory, anti-inflammatory, and proangiogenic miRNA in NESCs, which may contribute to the pathophysiology of endometriotic cells. Sources of Research Support: This work was supported in part by the National Institutes of Health (Grant Nos. 1SC1 GM130544-01A1, 1SC3GM113751, G12RR03034, HD66439, 1R01HD057235, U54 CA118948, HD41749, S21MD000101, and G12-MD007602); and The Endometriosis Foundation of America (EndoFound). This research was conducted in a facility constructed with support from the Research Centers in Minority Institutions (RCMI) Grant Nos. C06RR018386 and U54MD007602 from the National Institute of Minority Health and Health Disparities (NIMHD). Presentation: Monday, July 14, 2025