Dysregulation of MYBL2 impairs extravillous trophoblast lineage development and function, contributing to recurrent spontaneous abortion

Abstract

Recurrent spontaneous abortion (RSA) is a pregnancy-related condition characterized by a complex etiology. While placental trophoblast dysfunction is strongly associated with the development and progression of RSA, the underlying molecular mechanisms remain poorly understood. In this study, we observed a significant decrease in the expression of MYB Proto-Oncogene Like 2 (MYBL2) in the villous tissue of patients with RSA and the placentas of abortion-prone (AP) mice. Utilizing human trophoblast stem cells (hTSCs), we identified MYBL2 as a critical regulator of hTSCs stemness maintenance, promoting the expression of the stemness-associated genes Tumor protein p63 (TP63) and TEA Domain Transcription Factor 4 (TEAD4). Furthermore, MYBL2 facilitates the differentiation of hTSCs into extravillous trophoblast (EVT) by positively regulating Ajuba LIM Protein (AJUBA) expression. Using HTR-8/SVneo cell line, an immortalized EVT-like model, we found that MYBL2 positively regulates AJUBA expression by binding to the distal region of the AJUBA promoter. Additionally, the MYBL2-AJUBA axis enhances the migration and invasion of HTR-8/SVneo cells by suppressing the Hippo signaling pathway. Our study indicates that the dysregulation of MYBL2 expression in placental trophoblasts is associated with the pathogenesis of RSA, highlighting its potential as a therapeutic target for this condition.

Keywords: Hippo; MYBL2; placenta; recurrent spontaneous abortion; trophoblast cell.

Fig. 1.

MYBL2 is decreased in the villous tissue of patients with RSA and placentas of AP mice. (A) Venn diagram shows the intersection of three sets of public databases, two of which represent downregulated genes in placental villous samples from patients with RSA, while the third set comprises essential and growth-restricting genes for hTSCs. (B) Representative immunofluorescence analysis of MYBL2 (red), Ki67 (green), CK7 (green), and HLA-G (green) in placental villous tissues of normal pregnancy and patients with RSA (n = 5 per group). Nucleus were stained with Hoechst (blue). (Scale bar, 100 μm.) (C and D) qRT-PCR (C) and western blot (D) analysis of MYBL2 in placental villous tissues of normal pregnancy and patients with RSA (n = 5 per group). (E) Representative immunohistochemical analysis of MYBL2 and CK7 expression in NP mice and AP mice (n = 6 per group). (Scale bars, 20 and 100 μm.) (F and G) qRT-PCR (F) and western blot (G) analysis of MYBL2 in NP mice and AP mice (n = 6 per group). Data are represented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2.

MYBL2 promotes the stemness maintenance of hTSCs. (A) qRT-PCR confirmed the mRNA level of hTSCs marker genes TP63 and TEAD4, EVT marker genes HLA-G and MMP2, and STB marker genes CGB and SDC1 in hTSCs, EVT, and STB (n = 3 per group), respectively. (B) Representative immunofluorescence analysis of MYBL2 (red), TP63 (green), HLA-G (green), CGB (green) in hTSCs, EVT, and STB (n = 3 per group). Nucleus were stained with Hoechst (blue). (Scale bar, 100 μm.) (C) qRT-PCR confirmed the mRNA level of MYBL2 in hTSCs, EVT, and STB (n = 3 per group). (D) Representative immunofluorescence analysis of MYBL2 (red), TEAD4 (green), CK7 (green) in MYBL2 knockdown or overexpressed hTSCs (n = 3 per group). Nucleus were stained with Hoechst (blue). (Scale bar, 100 μm.) (E) qRT-PCR confirmed the mRNA levels of MYBL2, TP63, TEAD4, and CK7 in MYBL2 knockdown or overexpressed hTSCs (n = 3 per group). (F–H) CCK8 (F) and EdU (G and H) analysis was used to detect the proliferation of hTSCs in MYBL2 knockdown or overexpression group (n = 3 per group). (Scale bar, 100 μm.) Data are represented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3.

MYBL2 negatively regulates the Hippo signaling pathway in hTSCs. (A and B) Volcano plot and Heatmap analysis of differentially expressed genes in control and MYBL2 knockdown hTSCs (n = 3 per group). (C and D) GO and KEGG analysis of differentially expressed genes in control and MYBL2 knockdown hTSCs (n = 3 per group). (E and F) Western blot analysis of p-YAP1, YAP1, and TEAD4 in MYBL2 knockdown or overexpressed hTSCs (n = 3 per group). β-actin (ACTB) was used as a loading control for whole-cell lysate (WCL); Histone3 (H3) was used as a loading control for nuclear extracts.

Fig. 4.

MYBL2 promotes the differentiation of hTSCs into EVT by positively regulating AJUBA. (A) Representative phase-contrast images depicting cell morphology of hTSCs and EVT in MYBL2 knockdown and MYBL2 overexpression group, respectively (n = 3 per group). (Scale bar, 200 μm.) (B) Representative immunofluorescence displaying MYBL2 (red), phalloidin (green) in MYBL2 knockdown or overexpressed hTSCs induced EVT (n = 3 per group). Nucleus were stained with Hoechst (blue). (Scale bar, 100 μm.) (C) Representative immunofluorescence analysis of TEAD4 (green) in MYBL2 knockdown and overexpressed hTSCs and HLA-G (green) in EVT differentiate from MYBL2 knockdown and overexpressed hTSCs, respectively (n = 3 per group). Nucleus were stained with Hoechst (blue). (Scale bar, 50 μm.) (D and E) qRT-PCR (D) and western blot analysis (E) of MYBL2, HLA-G, and MMP2 in MYBL2 knockdown or overexpressed hTSCs induced EVT (n = 3 per group). (F and G) qRT-PCR (F) and western blot (G) analysis confirmed the expression of AJUBA in MYBL2 knockdown or overexpressed hTSCs induced EVT (n = 3 per group). (H) AJUBA overexpression were performed in MYBL2 knockdown hTSCs and induced differentiation into EVT. Representative phase-contrast images depicting cell morphology of EVT with MYBL2 knockdown and AJUBA overexpression (n = 3 per group). (Scale bar, 50 and 200 μm.) Representative immunofluorescence displaying HLA-G (green) in EVT with MYBL2 knockdown and AJUBA overexpression (n = 3 per group). Nucleus were stained with Hoechst (blue). (Scale bar, 50 μm.) (I and J) qRT-PCR (I) and western blot (J) analysis confirmed the expression of MYBL2, AJUBA, HLA-G in EVT with MYBL2 knockdown and AJUBA overexpression (n = 3 per group). Data are represented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5.

AJUBA overexpression compensated the inhibitory effect of MYBL2 knockdown on trophoblast migration and invasion. (A and B) Representative wound-healing assay (A) and corresponding quantitative values (B) of HTR-8/SVneo cells with MYBL2 knockdown and AJUBA overexpression (n =3 per group). (Scale bar, 400 μm.) (C and D) Representative transwell analysis (C) and corresponding quantitative values (D) of HTR-8/SVneo cells with MYBL2 knockdown and AJUBA overexpression (n = 3 per group). (Scale bar, 300 μm.) (E and F) qRT-PCR (E) and western blot (F) analysis of AJUBA, Vimentin, ITGA5, MMP2, and MMP9 in AJUBA knockdown or overexpressed HTR-8/SVneo cells (n = 3 per group). Data are represented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.