RBM25 induces trophoblast epithelial-mesenchymal transition and preeclampsia disorder by enhancing the positive feedback loop between Grhl2 and RBM25

Abstract

Defects in migration and invasion caused by dysregulation of trophoblast epithelial-mesenchymal transformation (EMT) are one of the key factors in the pathogenesis of preeclampsia (PE). RNA-binding motif protein 25 (RBM25) is an RNA-binding protein involved in a variety of cellular processes, including cell proliferation, apoptosis, cell migration and invasion, and EMT. However, the expression and function of RBM25 in placental of PE remain unclear. In this study, we reveal that the expression of RBM25 is significantly elevated in PE placental tissue. RBM25 depletion and over-expression in trophoblast cells increase and decrease, respectively, cell migration and invasion by regulating EMT marker E-cadherin and Vimentin expression. Mechanistically, Grhl2 is involved in RBM25-regulated trophoblast cell migration, invasion, and EMT through RBM25-facilitated mRNA stabilization. Furthermore, the upregulation of Grhl2 enhances the expression of RBM25 through transcription and forms a positive feedback regulation in the progression of PE. These findings suggest that upregulation of RBM25 induces dysregulation of trophoblast EMT by enhancing positive feedback regulation of Grhl2 and RBM25, leading to defects in cell migration and invasion. Targeting this newly identified regulatory axis may provide benefits in the prevention and treatment of PE.

Keywords: Grhl2; Preeclampsia; RBM25; epithelial-mesenchymal transition; migration and invasion; trophoblast.

Figure 1.

The expression of RBM25 was upregulated in PE tissues. (A) Hematoxylin and eosin (HE) staining was used to identify normal and preeclampsia (PE) placental tissues. Scar bar = 100 μm. (B) Placental tissues from three normal and three PE cases were randomly selected for Western blot analysis of RBM25 protein expression. (C) Quantitative analysis of Western blot results in (B). (D) RT-qPCR was used to examine RBM25 mRNA expression in normal (n = 30) and PE (n = 30) placental tissues. (E) RBM25 expression was measured in cell lines derived from placental tissue by using RT-qPCR. All data were from three independent experiments and were presented as a mean ± standard error. **P < 0.01 compared with the corresponding controls.

Figure 2.

Knockdown of RBM25 promoted trophoblast cells migration and invasion. (A) HTR-8/SVneo trophoblast cells were transfected with RBM25 shRNAs (shRBM25-1# and shRBM25-2#) or control vector (pLKO) and then RT-qPCR was used to detect RBM25 mRNA expression. (B) HTR-8/SVneo cells were transfected as in (A) and Western blot was used to examine RBM25 protein level. (C) Quantitative analysis of Western blot results in (B). (D) HTR-8/SVneo cells were transfected as in (A), and then a transwell assay was used to explore cells migration. Scar bar = 50 μm. (E) Quantitative analysis of relative cell number of migration in (D). (F) A 3D Matrigel droplet assay was used to explore the invasion of HTR-8/SVneo cells after transfected as in (A). Scar bar = 100 μm. (G) Quantitative analysis of fold change of cell invasion in (F). All data were collected from three independent experiments and were presented as a mean ± standard error. **P < 0.01, ***P < 0.001 vs corresponding controls.

Figure 3.

Over-expression of RBM25 inhibited trophoblast cells migration and invasion. (A) HTR-8/SVneo cells were transfected with RBM25-over-expressed vector (oeRBM25) or control vector (pWPI), and then RT-qPCR was used to measure RBM25 mRNA level. (B) Western blot was used to examine RBM25 protein level in oeRBM25 or pWPI vector transfected-HTR-8/SVneo cells. (C) Quantitative analysis of Western blot results in (B). (D) HTR-8/SVneo cells were treated as in (A), and then a transwell assay was used to examine cells migration. Scar bar = 50 μm. (E) Quantitative analysis of relative cell number of migration in (D). (F) The 3D Matrigel droplet assay was used to detect the invasion of HTR-8/SVneo cells after oeRBM25 or pWPI vector transfection. Scar bar = 100 μm. (G) Quantitative analysis of fold change of cell invasion in (F). All data were collected from three independent experiments and were presented as a mean ± standard error. **P < 0.01, ***P < 0.001 vs corresponding controls.

Figure 4.

RBM25 is involved in EMT of trophoblast cells by regulating EMT marker gene expression. (A) HTR-8/SVneo cells were transfected with shRBM25-1# or shRBM25-2# or pLKO and then Western blotting was used to detect E-cadherin and Vimentin protein expression. (B) Quantitative analysis of Western blot results in (A). (C) Western blotting was used to examine E-cadherin and Vimentin protein expression in oeRBM25 or pWPI-transfected HTR-8/SVneo cells. (D) Quantitative analysis of Western blot results in (C). (E) HTR-8/SVneo cells were transfected with shRBM25-2# or oeRBM25 or their corresponding control vectors, and then double immunofluorescence staining was used to explore E-cadherin and Vimentin protein expression. DAPI (blue), Vimentin (green), and E-cadherin (red). Scar bar = 25 μm. (F) Quantitative analysis of relative fluorescence intensity in (E). (G and H) Immunohistochemical staining detects E-cadherin or Vimentin expression in normal (n = 30) and PE (n = 30) placental tissues. All data were collected from three independent experiments and were presented as a mean ± standard error. **P < 0.01, ***P < 0.001 vs corresponding controls.

Figure 5.

RBM25 promotes Grhl2 expression by stabilizing mRNA. (A) HTR-8/SVneo cells were transfected with oeRBM25 or shRBM25 or their control vectors, and then RT-qPCR was used to detect ZEB1 and Grhl2 mRNAs expression. (B) HTR-8/SVneo cells were transfected as in (A), and Western blot was performed to measure Grhl2 and ZEB1 protein level. (C) Quantitative analysis of Western blot results in (B). (D) HTR-8/SVneo cells were transfected as in (A) and treated with actinomycin D (Act D) for 0, 2, 4 and 8 h. RT-qPCR was used to examine Grhl2 mRNA. (E) HTR-8/SVneo cells were transfected with oeRBM25 or pWPI vector, and the CoIP-Western blot was used to confirm RBM25 protein precipitation by a RBM25 antibody. (F) RNA binding protein immunoprecipitation assay (RIP) was used to examine proteins and Grhl2 mRNA interaction. RT-qPCR was used to detect the Grhl2 mRNA enrichment by using RBM25, RBM5, and IgG antibodies. (G) ZEB1 and Grhl2 mRNA probes were designed and synthesized, then a Pulldown-Western blot assay was used to explore RNA interaction with RBM25. All data were collected from three independent experiments and were presented as a mean ± standard error. *P < 0.05, **P < 0.01 vs corresponding controls.

Figure 6.

Grhl2 is involved in RBM25-regulated trophoblast cells migration, invasion, and EMT. (A) HTR-8/SVneo cells were transfected with shRBM25 or oeGrhl2 alone or together, and then Western blotting was used to measure E-cadherin, Vimentin, and RBM25 expression. (B) Quantitative analysis of Western blot results in (A). (C) HTR-8/SVneo cells were transfected as in (A), and double immunofluorescence staining was used to explore E-cadherin and Vimentin protein expression. DAPI (blue), Vimentin (green), and E-cadherin (red). Scar bar = 25 μm. (D) Quantitative analysis of relative fluorescence intensity in (C). (E) Transwell assay was used to explore cell migration in HTR-8/SVneo cells after transfection with shRBM25 or oeGrhl2 alone or together. Scar bar = 50 μm. (F) Quantitative analysis of relative cell number of migration in (D). (G) The 3D Matrigel droplet assay was used to explore the invasion of HTR-8/SVneo cells after transfected as in (A). Scar bar = 100 μm. (H) Quantitative analysis of fold change of cell invasion in (G). All data were collected from three independent experiments and were presented as a mean ± standard error. **P < 0.01, ***P < 0.001 vs corresponding controls.

Figure 7.

Grhl2 promotes RBM25 expression by transcription. (A) HTR-8/SVneo cells were transfected with oeGrhl2 or shGrhl2 or their control vectors, and then RT-qPCR was used to detect ZEB1 and RBM25 mRNAs expression. (B) HTR-8/SVneo cells were transfected with oeGrhl2 or pwpI, and then treated with Act D. Western blot was used to examine RBM25 and ZEB1 expression. (C) Quantitative analysis of Western blot results in (B). (D) The potential Grhl2-binding motif on the promoter of RBM25 gene. (E) ChIP-PCR was used to identify the binding site of Grhl2 on the RBM25 promoter. (F) Dual luciferase reporter gene examined the role of Grhl2 and RBM25 in regulating RBM25 promoter activity by co-transfection with indicated constructs. All data were collected from three independent experiments and were presented as a mean ± standard error. *P < 0.05, **P < 0.01, ***P < 0.001 vs corresponding controls.