Estradiol promotes EMT in endometriosis via MALAT1/miR200s sponge function

in Reproduction
Correspondence should be addressed to Y Liu or L Zhang; Email: liqun1994@hust.edu.cn or zhanglingxh@hust.edu.cn
Restricted access

Endometriosis is an estrogen-dependent benign gynecological disease that shares some common features of malignancy. Epithelial–mesenchymal transition (EMT) has been recognized as a core mechanism of endometriosis. MALAT1 is widely known as EMT promoter, while miR200 family members (miR200s) are considered as EMT inhibitors. Previous studies have reported that MALAT1 upregulation and miR200s downregulation are observed in endometriosis. MiR200c has been regarded as the strongest member of miR200s to interact with MALAT1. However, whether MALAT1/miR200c regulates EMT remains largely unclear. In this study, the roles of miR200s and MALAT1 in ectopic endometrium were investigated. Additionally, the effects of E2 on EMT and MALAT1/miR200s were examined in both EECs and Ishikawa cells. Notably, E2 could upregulate MALAT1 and downregulate miR200s expression levels and induce EMT in EECs and Ishikawa cells. PHTPP, an ERβ antagonist, could reverse the effect of E2. Overexpression of miR200c and knockdown of MALAT1 significantly inhibited E2-mediated EMT, suggesting that both miR200c and MALAT1 are involved in the E2-induced EMT process in endometriosis. In addition, a reciprocal inhibition was found between miR200s and MALAT1. Therefore, the role of MALAT1/miR200c in EMT is influenced by the presence of estrogen during endometriosis development.

Downloadable materials

  • Supplementary Figure 1: The prediction of the upstream transcription factors of MALAT1 by JASPAR.
  • Supplementary Figure 2: Expression levels of E-cadherin and Vimentin in EECs were identified by immunofluorescence.
  • Supplementary Figure 3: Protein expression levels of EMT-associated markers with the treatment of E2 and/or ICI in EECs for 48h analyzed by western blot.
  • Supplementary Figure 4: A, Protein expression levels of EMT-associated markers with the treatment of E2 and/or PHTPP in Ishikawa cells for 48h analyzed by Western blot; B~D, RNA expression of EMT-associated markers, MALAT1 and miR200s treated with E2 and/or PHTPP in Ishikawa cells for 24h analyzed by qRT-PCR; E,E2 promoted migration and invasion of Ishikawa cells and PHTPP reversed the effect. Data were evaluated by one-way ANOVA analysis (*P<0.05, **P<0.01, ***P<0.001). Photographs were taken at magnifications of 200×.
  • Supplementary Figure 5: The results of Western blot statistical chart of Figure1-3 and Supplementary Figure4.
  • Supplementary Figure 6: The results of Western blot statistical chart of Figure4-6.

 

An official journal of

Society for Reproduction and Fertility

Sections

Figures

  • View in gallery

    (A) Expression level of miR200s in normal (N), ectopic (ovarian) (EC) and eutopic (EU) endometriosis. (B) Expression levels of MALAT1, Zeb1, Zeb2, E-cadherin and Vimentin in normal, ectopic (ovarian) and eutopic endometriosis examined by qRT-PCR. (*: P < 0.05; **: P < 0.01; ***P < 0.001 by the Kruskal–Wallis test.)

  • View in gallery

    (A and B) Expression levels of EMT-associated markers (Zeb1, Zeb2, E-cadherin and Vimentin) with E2 treatment for different doses (0, 10−12, 10−10, 10−8, 10−6 mol/L) and time (0, 24, 48 or 72 h) in EECs detected by Western blot.(M: mol/L); (C and D) Expression levels of MALAT1 and miR200s under E2 for various doses (0, 10−12, 10−10, 10−8, 10−6 mol/L) for 24 h in EECs measured by qRT-PCR. Data were evaluated by one-way ANOVA analysis (*P < 0.05, **P < 0.01, ***P < 0.001 compared with untreated group).

  • View in gallery

    (A) Protein expression levels of EMT-associated markers in EECs with treatment of E2 and/or PHTPP for 48 h analyzed by Western blot; (B, C and D) RNA expression levels of EMT-associated markers, MALAT1 and miR200s treated with E2 and/or PHTPP in EECs for 24 h analyzed by qRT-PCR; (E) E2 promoted migration and invasion of EECs and PHTPP reversed the effect. Data were evaluated by one-way ANOVA analysis (*P < 0.05, **P < 0.01, ***P < 0.001). Photographs were taken at magnifications of 200×.

  • View in gallery

    (A) The predicted results of the interaction between MALAT1 and miR200s by RNAhybrid (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid). (Red: MALAT1. Green: each member of miR200s) (MALAT1 and miR200a, mfe: −22.6 kcal/mol; MALAT1 and miR200b, mfe: −26.8 kcal/mol; MALAT1 and miR200c, mfe: −30.1 kcal/mol; MALAT1 and miR141, mfe: −24.2 kcal/mol; MALAT1 and miR429, mfe: -21.0 kcal/mol.); (B and C) Ishikawa cells were transfected with miR200c mimic or miRNA Negative Control (miRNA NC) and were treated with or without E2 (10−8 mol/L) for 24 h. The expression levels of miR200c and MALAT1 were analyzed by qRT-PCR. (D) Expression levels of EMT-associated markers detected by Western blot after the same treatment for 48 h. (E) The function of miR200c mimic and/or E2 affects the ability of migration (24 h) and invasion (48 h) in Ishikawa cells. Data were evaluated by one-way ANOVA analysis (*P < 0.05, **P < 0.01, ***P < 0.001). Photographs were taken at magnifications of 200×.

  • View in gallery

    (A) Ishikawa cells were transfected with MALAT1 siRNA (si-MALAT1-1 and si-MALAT1-2), expression level of MALAT1 was quantified by qRT-PCR. (B) Ishikawa cells were transfected with si-MALAT1(-2) with or without addition of E2 (10−8 mol/L) for 24 h, expression of MALAT1 was quantified by qRT-PCR; (C) Ishikawa cells were transfected with si-MALAT1-2 with or without the addition of E2 (10−8 mol/L) for 24 h, expression levels of miR200s were quantified by qRT-PCR; (D) The altered expression levels of EMT-associated markers after the same treatment for 48 h were analyzed by Western blot. (E) the function of si-MALAT1(-2) and/or E2 affects the ability of migration (24 h) and invasion (48 h) in Ishikawa cells. Data were evaluated by one-way ANOVA analysis and Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001). Photographs were taken at magnifications of 200×.

  • View in gallery

    (A) The effect of miR200c inhibitor on protein expression levels of down-stream EMT-associated markers for 48 h analyzed by Western blot. (B, C and D) Ishikawa cells were co-transfected with si-MALAT1(-2) or si-NC, and miR200c inhibitor or inhibitor Negative Control (inhibitor NC); (B) Expression levels of Zeb1, Zeb2, E-cadherin and Vimentin after treatment for 48 h analyzed by Western blot; (C) Expression level of MALAT1 after treatment for 24 h analyzed by qRT-PCR; (D) The treatment influences the ability of migration and invasion; (E) Estradiol promotes epithelial–mesenchymal transition in endometriosis via MALAT1/miR200s sponge function. E2-activated ERβ may promote the expression of MALAT1 and mediate EMT in endometriosis. Data were evaluated by one-way ANOVA analysis (*P < 0.05, **P < 0.01, ***P < 0.001). Photographs were taken at magnifications of 200×.

Index Card

PubMed

Google Scholar

Related Articles

Altmetrics

Metrics

All Time Past Year Past 30 Days
Abstract Views 160 160 160
Full Text Views 45 45 45
PDF Downloads 14 14 14