Fig 2: MALAT1 acts as a ceRNA for miR-145-5p.A qRT-PCR analysis was performed to detect the mRNA expression of ACSL4, GPX4, SLC7A11, and MUC1 in EESCs transfected with siMALAT1 (50 nM) or siNC (50 nM) for 24 h. B Western blot analysis was performed to detect the protein expression of ACSL4, SLC7A11, and MUC1 in EESCs transfected with siMALAT1 (50 nM) or siNC (50 nM) for 48 h. C Venn graph of MALAT1-targeted miRNAs predicted by starBase and miRNAs predicted by TargetScan and miRTarBase to target MUC1. D The predicted binding sites between miR-145-5p and MALAT1 are shown. E Luciferase activities were assessed in 293T cells cotransfected with the MALAT1 mutant (MALAT1 mut) and miR-145-5p mimics or cotransfected with MALAT1 wild type (MALAT1 wt) and miR-145-5p mimics. F qRT-PCR analysis was performed to detect the expression of miR-145-5p in 17 EC tissues and 11 EN tissues. G The correlation between the expression of MALAT1 and miR-145-5p was analyzed using the Pearson’s test (r < 0 denotes a negative correlation). H miR-145-5p expression after MALAT1 silencing was determined using qRT-PCR. All data are shown as the mean ± SD of three independent experiments (NS is nonsignificant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Statistical significance was calculated using Student’s t test. EC ectopic endometrial, EN normal endometrial, EESC ectopic endometrial stromal cell, MALAT1 metastasis-associated lung adenocarcinoma transcript 1, siNC negative control siRNA, siMALAT1 siRNA targeting MALAT1.

Fig 3: Erastin-induced ferroptosis shrinks endometriotic lesions by regulating the MALAT1/miR-145-5p/MUC1 axis in vivo.A Flowchart for the establishment of the mouse endometriosis therapeutic model. B The successful establishment of the endometriosis model was verified by H&E staining of ectopic lesions. The scale bar = 100 µm for ×200, 50 µm for ×400. C Representative visible lesions in the peritoneal cavity of a mouse model of endometriosis after treatment with erastin (20 mg/kg) or DMSO for 7 days are shown. D The volume of ectopic lesions was reduced after treatment with 20 mg/kg erastin for 7 days. E Body weights of the DMSO group and erastin group measured every day. Body weight did not change significantly after erastin treatment compared with the control group. F H&E staining showed that the glandular and stromal structures of ectopic lesions completely disappeared after erastin treatment. G The mRNA expression of MALAT1, miR-145-5p, and MUC1 in the DMSO group and erastin group was analyzed by qRT-PCR. H Correlations between the expression of MALAT1 and miR-145-5p, miR-145-5p and MUC1, MALAT1 and MUC1 in the erastin group were analyzed using the Pearson’s test. I The protein expression of MUC1 was detected by immunohistochemical staining. The scale bar =50 µm for ×400. All data are shown as the mean ± SD of three independent experiments (**P < 0.01, ****P < 0.0001, Student’s t test). MALAT1 metastasis-associated lung adenocarcinoma transcript 1, MUC1 mucin 1, i.p. intraperitoneal injection, i.m. muscle injection, E2 17-ß-estradiol-3-benzoate, qod every other day, qd once a day.

Fig 4: MiR-145-5p promoted erastin-induced ferroptosis in EESCs by regulating MUC1.A Cell viability was evaluated in miR-145-5p-overexpressing cells after cotransfection with the MUC1 plasmid. B, C Intracellular Fe2+ and Liperfluo levels were evaluated in miR-145-5p-overexpressing cells after cotransfection with the MUC1 plasmid (magnification, ×200). D Cell viability was evaluated in cells with miR-145-5p inhibition after cotransfection with siMUC1. E, F Intracellular Fe2+ and Liperfluo levels were evaluated in miR-145-5p-inhibited cells after cotransfection with siMUC1 (magnification, ×200). All data are shown as the mean ± SD of three independent experiments (NS is nonsignificant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Statistical significance was calculated using Student’s t test. EESC ectopic endometrial stromal cell, MUC1 mucin 1, siNC negative control siRNA, siMUC1 siRNA targeting MUC1, mimic NC negative control miRNA mimic, inhibitor NC negative control miRNA inhibitor.

Fig 5: Ferroptosis resistance occurs in endometriosis.A Representative images of ferroptosis-related protein (ACSL4, GPX4, SLC7A11, and MUC1) expression in EN and EC tissues were analyzed by immunohistochemical staining. The scale bar = 100 µm for ×200, 50 µm for ×400. B Semiquantitative analysis software (ImageJ) was used to measure the mean IOD of the sections. C The mRNA levels of ACSL4, GPX4, SLC7A11, and MUC1 in NESCs and EESCs were determined using quantitative RT-PCR (qRT-PCR) analysis. D Protein expression of ACSL4, GPX4 SLC7A11, and MUC1 in NESCs and EESCs was determined using western blot analysis. ImageJ was used to calculate the integrated density of the protein bands. All data are shown as the mean ± SD of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Statistical significance was calculated using Student’s t test. EC ectopic endometrial, EN normal endometrial, NESC normal endometrial stromal cell, EESC ectopic endometrial stromal cell, ACSL4 acyl-CoA synthetase long-chain family member 4, GPX4 glutathione peroxidase 4, SLC7A11 solute carrier family 7 member 11, MUC1 mucin 1, IOD integrated optical density.