Adipocyte alterations in endometriosis: reduced numbers of stem cells and microRNA induced alterations in adipocyte metabolic gene expression.

BACKGROUND: Endometriosis is an estrogen dependent, inflammatory disorder occurring in 5-10% of reproductive-aged women. Women with endometriosis have a lower body mass index (BMI) and decreased body fat compared to those without the disease, yet few studies have focused on the metabolic abnormalities in adipose tissue in women with endometriosis. Previously, we identified microRNAs that are differentially expressed in endometriosis and altered in the serum of women with the disease. Here we explore the effect of endometriosis on fat tissue and identified a role for endometriosis-related microRNAs in fat metabolism and a reduction in adipocyte stem cell number. METHODS: Primary adipocyte cells cultured from 20 patients with and without endometriosis were transfected with mimics and inhibitors of microRNAs 342-3p or Let 7b-5p to model the status of these microRNAs in endometriosis. RNA was extracted for gene expression analysis by qRT-PCR. PCNA expression was used as a marker of adipocyte proliferation. Endometriosis was induced experimentally in 9-week old female C57BL/6 mice and after 10 months fat tissue was harvested from both the subcutaneous (inguinal) and visceral (mesenteric) tissue. Adipose-derived mesenchymal stem cells in fat tissue were characterized in both endometriosis and non-endometriosis mice by FACS analysis. RESULTS: Gene expression analysis showed that endometriosis altered the expression of Cebpa, Cebpb, Ppar-γ, leptin, adiponectin, IL-6, and HSL, which are involved in driving brown adipocyte differentiation, appetite, insulin sensitivity and fat metabolism. Each gene was regulated by an alteration in microRNA expression known to occur in endometriosis. Analysis of the stem cell content of adipose tissue in a mouse model of endometriosis demonstrated a reduced number of adipocyte stem cells. CONCLUSIONS: We demonstrate that microRNAs Let-7b and miR-342-3p affected metabolic gene expression significantly in adipocytes of women with endometriosis. Similarly, there is a reduction in the adipose stem cell population in a mouse model of endometriosis. Taken together these data suggest that endometriosis alters BMI in part through an effect on adipocytes and fat metabolism.

Systemic Inflammation Induced by microRNAs: Endometriosis-Derived Alterations in Circulating microRNA 125b-5p and Let-7b-5p Regulate Macrophage Cytokine Production.

Context: Endometriosis is characterized by aberrant inflammation. We previously reported increased levels of microRNA (miRNA) 125b-5p and decreased levels of miRNA Let-7b-5p in serum of patients with endometriosis. Objective: Determine the regulatory function of miRNAs 125b-5p and Let-7b-5p on production of proinflammatory cytokines in endometriosis. Design: Case-control study. Setting: University hospital. Patients: Women with (20) and without (26) endometriosis; human U937 macrophage cell line. Intervention: Sera were collected from surgically diagnosed patients and differentiated U937 cells that were transfected with miRNAs 125b-5p and Let-7b-5p mimics and inhibitor. Main Outcome Measures: Enzyme-linked immunosorbent assay for tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-8, and IL-1ß levels and quantitative real-time polymerase chain reaction for expression of miRNAs 125b-5p and Let-7b-5p in sera of patients with and without endometriosis. Transfected macrophages were evaluated for expression of inflammatory cytokines, intracellular production, and secretion of these cytokines. Results: We noted substantial elevation of TNF-α, IL-1ß, and IL-6, marked upregulation of miRNA 125b, and considerable downregulation of Let-7b in sera of patients with endometriosis vs control. There was a positive correlation between miRNA 125b levels and TNF-α, IL-1ß, and IL-6 and a negative correlation between miRNA Let-7b levels and TNF-α in sera of patients with endometriosis. Transfection experiments showed a noteworthy upregulation of TNF-α, IL-1ß, IL-6, and IL-8 in macrophages transfected with miRNA 125b mimic or Let-7b inhibitor. The secreted cytokine protein levels and intracellular imaging studies closely correlate with the messenger RNA changes. Conclusions: Endometriosis-derived miRNAs regulate macrophage cytokine production that contributes to inflammation associated with this condition.