Fig 1: Inhibition of miR-218-5p prevents the epithelial-mesenchymal transition. A Immunofluorescent analysis of Vimentin (Red) and LASP1 (Green) in the control eutopic endometrium (CEu), adenomyosis eutopic endometrium(AEu) and adenomyosis ectopic endometrium(AEc) respectively. DAPI staining was included to visualize the cell nucleus (Blue), Scale bar = 100 μm. B and C Statistic analysis of Immunofluorescent staining of LASP1 and VEGF-α in the gland epithelial cell of three groups. D and E Statistic analysis of Immunofluorescent staining of LASP1 and VEGF-α in the stromal cell of three groups: CEu (n = 17), AEu (n = 16) and AEc (n = 16). P-values were determined by Student’s t-test, ***p < 0.001. F Western blot analysis showing effects of miR-218-5p on protein levels of Vimentin. GAPDH was used as the loading control
Fig 2: Lasp1 enhanced cyclin and EMT proteins via activating FAK-AKT pathwayTreatment of FAK inhibitor (PF-562271) markedly prevented the phosphorylation of FAK and AKT and subsequently counteracted increasing expression of CyclinA2, CyclinB1, Snail and decreasing expression of E-cadherin caused by Lasp1 overexpression (A). LY294002, an inhibitor of AKT, attenuated the levels of p-AKT but not p-FAK, and thereby reduced the upregulating expression of CyclinA2, CyclinB1, Snail and reversed the inhibition of E-cadherin expression induced by Lasp1 cDNA transfection (B). Tumor proliferation (C) and invasion (D); Magnification, 400×; scale bar=50µm) caused by Lasp1 overexpression were also reversed by FAK inhibitor incorporation. Immunoprecipitation assay showed that Lasp1 directly interacted with FAK (E). Immunofluorescence assay showed that Lasp1 co-localized with FAK in the cytoplasm of H460 cells. Magnification; 600× (F).
Fig 3: The diagram of miR-218 regulating EMT of endometrial cells. In CEu, the highly expressed miR-218 can inhibit LASP1 in stromalcells, thereby reducing the expression of Vimentin, and ultimately hinder the process of EMT. On the contrary, in AEu/AEc, the low expression of miR-218 promotes the overexpression of LASP1, Which in turn caused the rise of Vimentin and the acceleration of the EMT process
Fig 4: miR-218-5p inhibits LASP1 mRNA level in ESCs. A and B qPCR analysis of miR-218-5p and LASP1 mRNA levels in different ESCs, respectively. ThE: Eutopic stromal cell line of the control endometrium. Q-Z1: Primary eutopic stromal cell of the endometriosis endometrium. Q-Y19: Primary ectopic stromal cell of the endometriosis endometrium. X-Y19: Primary ectopic stromal cell of the adenomyosis endometrium. C and D qPCR analysis of miR-218-5p and LASP1 mRNA levels in ThESC transfected with the control inhibitor, 20 nM and 50 nM miR-218-5p inhibitor respectively. E and F qPCR analysis of miR-218-5p and LASP1 mRNA levels in Q-Z1 transfected with the control inhibitor, 20 nM and 50 nM miR-218-5p inhibitor respectively. G and H qPCR analysis of miR-218-5p and LASP1 mRNA levels in Q-Y19 transfected with the control inhibitor, 20 nM and 50 nM miR-218-5p inhibitor respectively. P-values were determined by Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001
Fig 5: miR-218-5p has no effect on the proliferation of ESCs but can inhibit the migration of ESCs. A Western blot analysis and quantification of LASP1 protein level in lysates of different ESCs. B Western blot analysis and quantification of LASP1 protein level in lysates of Q-Z1 with or without 50 nM miR-218-5p mimic. C Western blot analysis and quantification of LASP1 protein level in lysates of ThESc with or without 50 nM miR-218-5p inhibitor. GAPDH was used as the loading control. P-values were determined by Student’s t-test, *p < 0.05, ***p < 0.001. D Predicted miR-218-5p target recognition sites in the 3’-UTR of human LASP1. Mutation in the miR-218-5p target site is red. 3’UTR of LASP1 luciferase constructs, as indicated, were transfected into 293 T cells together with indicated plasmid for 48 h and subjected to a luciferase reporter assay. The results were normalized to the Renilla luciferase activity and are expressed as the fold change in relative luciferase activity compared with the control. E ThE cells were transfected with control inhibitor or miR-218-5p inhibitor. F Q-Z1 cells were transfected with control mimic or miR-218-5p mimic. After 24 h of transfection, cells were starved for 24 h before cell migration assay was performed without matrigel transwell filters, Scale bar = 200 µm. The migrated cells were stained and counted. Quantification was done in and is shown with counting six nonoverlaping fields. G and H EdU incorporation assays of ThE or Q-Z1 cells were transiently transfected with control inhibitor/miR-218-5p inhibitor or control mimic/miR-218-5p mimic, DAPI staining was included to visualize the cell nucleus (Blue), Scale bar = 100 µm. Each bar indicates mean ± SEM. of a representative experiment performed in triplicate. P-values were determined by Student’s t-test. *** p < 0.001
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