DIRAS3 regulates autophagy in an endometriosis epithelial cell line.

RESEARCH QUESTION: What is the role of DIRAS3 in endometriosis pathogenesis? DESIGN: Prospective patient cohort study combined with experiments in the 12Z human endometriosis epithelial cell line model to determine the role of DIRAS3 in endometriosis. Endometrium and endometriosis lesion samples were collected from premenopausal women from 24 control and 40 endometriosis patients by laparoscopic surgery. The role of DIRAS3 in endometriosis was assessed by siRNA knockdown in 12Z cells followed by proliferation, apoptosis, invasion and autophagy assays. Autophagy was induced by serum starvation and the levels of autophagy determined by assessing changes in the expression levels and localization of autophagy marker proteins, such as LC3. RESULTS: DIRAS3 mRNA showed a large increase in expression in ectopic endometriosis lesions compared with endometrium from control patients, with expression largely localized to the epithelium. DIRAS3 knockdown in 12Z endometriosis epithelial cells caused a significant reduction in the number of proliferating cells (1.6-fold, adjusted P = 0.0007) and increased apoptosis (AnnexinV/7AAD double-positive cells +48%, P = 0.01), indicating an effect on cell proliferation. Induction of autophagy by serum starvation caused significant upregulation in DIRAS3 expression after 24 h (mRNA +2.4-fold [adjusted P = 0.017], protein +8.1-fold (adjusted P = 0.029), reduced LC3I/LC3II ratio (-2.2-fold, adjusted P = 0.044) and an increase in the number of double positive LC3/DIRAS3 puncta (+2.3-fold, P = 0.02). Knockdown of DIRAS3 in serum-starved cells led to a reduction in autophagy, indicated by an overall decrease in LC3 expression and significant increase in LC3I/LC3II ratio. CONCLUSIONS: DIRAS3 is highly upregulated in endometriosis lesions. Studies in an endometriosis epithelial cell line indicate that DIRAS3 facilitates cell survival in this context by inducing autophagy.

MLLT11 Regulates Endometrial Stroma Cell Adhesion, Proliferation and Survival in Ectopic Lesions of Women with Advanced Endometriosis.

MLLT11 is a gene implicated in cell differentiation and the development and progression of human cancers, but whose role in the pathogenesis of endometriosis is still unknown. Using quantitative RT-PCR and immunohistochemistry, we analyzed 37 women with and 33 women without endometriosis for differences in MLLT11 expression. We found that MLLT11 is reduced in the ectopic stroma cells of women with advanced stage endometriosis compared to women without endometriosis. MLLT11 knockdown in control stroma cells resulted in the downregulation of their proliferation accompanied by G1 cell arrest and an increase in the expression of p21 and p27. Furthermore, the knockdown of MLLT11 was associated with increased apoptosis resistance to camptothecin associated with changes in BCL2/BAX signaling. Finally, MLLT11 siRNA knockdown in the control primary stroma cells led to an increase in cell adhesion associated with the transcriptional activation of ACTA2 and TGFB2. We found that the cellular phenotype of MLLT11 knockdown cells resembled the phenotype of the primary endometriosis stroma cells of the lesion, where the levels of MLLT11 are significantly reduced compared to the eutopic stroma cells of women without the disease. Overall, our results indicate that MLLT11 may be a new clinically relevant player in the pathogenesis of endometriosis.