Fig 2: ROCK1 is a target gene of miR-145-5p. (A) The predicted target sequences for miR-145-5p in the ROCK1 3' UTR. (B) Luciferase reporter assays were conducted to demonstrate that miR-145-5p directly binds to the WT target site of ROCK1. (C) ROCK1 protein expression following the exposure of H9c2 cells to H/R. (D) The mRNA levels of ROCK1 following exposure of H9c2 cells to H/R. ROCK1 (E) protein expression and (F) mRNA levels after transfection with miR-145-5p mimic. (G) ROCK1 mRNA levels following transfection with pcDNA3.1 ROCK1 overexpression vector. ***P<0.001 vs. the respective control (n=6). miR, microRNA; H/R, hypoxia/reoxygenation; NC, negative control; ROCK1, Rho-associated coiled-coil-containing kinase 1; WT, wild type; Mut, mutant; UTR, untranslated region.

Fig 3: Stratification of Endometrial Cell Subpopulations Based on SMAD4 and JAC Expression Levels and Patients’ BMIs(A–C) Expression profiles of SMAD4 and JAC based on CyTOF were classified into five categories: I, II, III, IV, and V. Violin plots show ordered SMAD4 expression level (low to high) and distribution in the individual subpopulations (A). Expression heatmaps of JAC were aligned according to SMAD4 expression levels (in descending order) in different cellular subpopulations (B). Area of circle denotes relative cell proportion of a subpopulation within each sample and ordered in the aforementioned five categories in cellular subpopulations of EME6/7t lines and primary tumors (C). Tumors were ordered from top to bottom based on increased patients’ BMIs and histology classes.(D) Balloon plots of subpopulation distributions of endometrial tumors in the five categories based on histology and BMI. Circle area indicates the size of each subpopulation. Categories IV and V were associated with relatively high expression levels of SMAD4 and JAC, whereas low levels of SMAD4 and JAC were noted in categories I, II, and III.(E) Violin plots showing the average expression levels of four JAC markers—ADAM15, ROCK1, Cx43, and Gas1—based on patients’ tumor histology: papillary serous and endometrioid.(F) Balloon plots of subpopulation distributions in the five categories stratified by BMI into three groups: normal weight (NW), obesity (OB), and morbid obesity (MOB).(G) Violin plots showing the average expression levels of ADAM15, ROCK1, Cx43, and Gas1, based on three BMI groups: NW, OB, and MOB. Lower JAC expressions corresponded with patients with higher BMIs. ****p < 0.0001.See also Figure S7.

Fig 4: Oestrogen up‐regulated the development of endometriosis and expression of RhoA、ROCK1/2 and phosphorylated ERK in endometriotic mouse models. A, Consistent treatment of oestrogen enhanced the size (upper) and weight of mouse endometriotic lesions (lower). EM: PBS‐injected endometriosis group, E2/EM: E2‐injected endometriosis group. B, The mRNA expression of RhoA and ROCK1/2 was increased in the E2/EM group. C, Oestrogen increased the expression of RhoA and phosphorylated ERK in mouse endometriotic lesions. Data are expressed as mean ± SEM. NS = no significant difference, *P < 0.05, ***P < 0.0001 vs control group. Figure S1 (A) Quantitative analysis of Rho/ROCK pathway referred to GEO database. B, The mRNA expression of RhoB and RhoC from different human endometria. C, The images of cell morphology and green fluorescent protein (GFP) in RhoAoe and siRhoA cells detected by fluorescence microscope. Optical: optical microscope. Data are expressed as mean ± SEM. NS = no significant difference, *P < 0.05, **P < 0.001, ***P < 0.0001 vs control group

Fig 5: The expression of RhoA/ROCK pathway was significantly increased in both human and mouse endometriotic lesions. A, The expression of Rho family including RhoA, RhoB, RhoC, ROCK1 and ROCK2 referring to GEO database. Norm: eutopic endometria from normal patients, Eut: eutopic endometria from endometriosis patients, Ect: ectopic endometriotic lesions B, The mRNA expression of RhoA (left), ROCK1 (middle) and ROCK2 (right) analysed by qRT‐PCR. C, The protein level of RhoA, ROCK1 and ROCK2 via Western blot assays in 5 groups including normal endometria, eutopic endometria and ectopic lesions. D, Quantitative analysis of RhoA, ROCK1 and ROCK2 proteins. E, Establishment of endometriotic mouse model (right) and characterized by HE staining (left). F, The expression of RhoA, ROCK1 and ROCK2 detected by IHC in eutopic endometria and ectopic lesions from mouse model of endometriosis after treating for 1 mo. Data are expressed as mean ± SEM. NS = no significant difference, *P < 0.05, **P < 0.001, ***P < 0.0001 vs normal group