ACVR2A Gene Polymorphisms and Preeclampsia Risk Summary.

ACVR2A was downregulated in placental tissue associated with pre-eclampsia.

(A) Comparison of the RNA-seq volcano maps of all genes in the placenta of normal control (NC) and patients with pre-eclampsia (PE) evidently showed that the expression of the ACVR2A gene significantly decreased in patients with PE.

(B) Heat maps depicting the aberrant gene pathways in the NC and PE placentas revealed a decreased expression of the ACVR2A gene in patients with PE compared with NC.

(C) Western blot analysis demonstrated reduced levels of ACVR2A in preeclampsia placental tissue (n = 10) compared with control placentas.

(D) RT-qPCR was employed to assess the ACVR2A mRNA expression in placental tissues of normal control (NC, n = 10) and patients with pre-eclampsia (PE, n = 10).

(E) Immunohistochemical staining was conducted using rabbit IgG anti-human ACVR2A antibody on sections from normal control and pre-eclampsia (PE) placentas. Sections were counterstained with hematoxylin. The ACVR2A levels were markedly lower in patients with PE (n = 10) than in NC.

(F) Immunofluorescence co-localization of rabbit IgG anti-human ACVR2A antibodies and HLA-G antibodies (a marker of extrachorionic trophoblastic cells) was performed in normal control and pre-eclampsia maternal placenta. The expression pattern of the ACVR2A antibody closely resembles that of the HLA-G antibody, primarily expressed in EVT cells.

(*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with normal control group).

The ACVR2A gene in JAR and HTR8/SVneo cells was successfully knocked out using CRISPR/Cas9 gene editing technology.

(A) RT-qPCR was employed to assess the ACVR2A mRNA expression across various cell lines. The expression of ACVR2A in HTR8/SVneo and JAR was comparable to that in multiple cancer cell lines, with JAR exhibiting a higher ACVR2A expression.

(B) DNA sequence of ACVR2A and target sequence site information of sgRNA-1 and sgRNA-2.

(C) The knockout efficiency of the ACVR2A gene in HTR8/SVneo and JAR cell lines was assessed through polymeric primer PCR, confirmed by agarose gel electrophoresis, and reconfirmed through four rounds of monoclonal cell communities. The specific experimental results are shown in Figure S3.

(D) The ACVR2A mRNA levels were measured in HTR8/SVneo and JAR cell lines, and the normal control group was compared with the two ACVR2A-KO cell lines after three rounds of validation (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with normal control group).

(E) Western blot analysis showing ACVR2A protein levels in wild-type (WT) and knockout (KO) HTR8/SVneo and JAR cell lines. The ACVR2A protein is significantly absent in KO cell lines compared to WT, confirming successful knockout of the ACVR2A gene.

(F) Sanger sequencing confirmed the expression of ACVR2A in ACVR2A-KO monoclonal cell lines and successfully knocked out the DNA fragment between the ACVR2A gene sgRNA-1 and sgRNA-2. The specific experimental results are shown in Figure S4 and S5.

The invasion, migration, and proliferation in JAR and HTR8/SVneo cells were suppressed following ACVR2A knockout.

(A) Cell scratch assay was conducted in a six-well plate to assess alterations in the migration ability of HTR8/SVneo and JAR cells following ACVR2A gene knockout. Three visual fields were randomly selected under a 100× microscope for continuous observation, calculation, and difference analysis. Scale bar: 100 µm.

(B) CCK-8 method was employed to assess the proliferation of HTR8/SVneo and JAR cells after ACVR2A gene knockout.

(C) The alterations in the invasion ability of HTR8/SVneo and JAR cells after ACVR2A gene knockout were determined using Transwell invasion assay (100× magnification), with three fields randomly selected under a 300× microscope for counting and statistical analysis. Scale bar: 200µm.

(D) Colony formation was detected by single-cell clone assay. Colony formation assay conducted in a six-well plate to assess changes in the individual cell proliferation capacity of HTR8/SVneo and JAR cells following ACVR2A gene knockout (*P < 0.05,

**P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with normal control group).

Transcriptomic analysis revealed that ACVR2A may suppress cell biological behavior through the Wnt/TCF7 pathway in JAR cell lines.

(A) The Volcano plots of RNA-seq for all the genes compared JAR-ACVR2A-KO and WT.

(B) One representative hallmark pathway, the Wnt pathway, in the ACVR2A-KO group.

(C) The enriched biological process pathways of cell invasion and migration based on DEGs (p value cutoff = 0.05).

(D) The category netplot depicted the linkages of downregulated genes and three biological concepts that are related to ACVR2A as a network.

(E) The heatmap showed differentially expressed genes (DEGs) in the Wnt pathway between experimental and normal control groups. Red and blue represent significantly upregulated and downregulated genes, respectively.

Transcriptomic analysis revealed that ACVR2A may suppress cell biological behavior through the Wnt/TCF7 pathway in HTR8/SVneo cell lines.

(A) Volcano plots of RNA-seq for all the genes compared HTR8/SVneo-ACVR2A-KO and WT.

(B) One representative hallmark pathway, the Wnt pathway, in the ACVR2A-KO group.

(C) The enriched biological process pathways in cell invasion and migration based on DEGs (p value cutoff = 0.05).

(D) The category netplot depicted the linkages of downregulated genes and three biological concepts that are related to ACVR2A as a network.

(E) Heatmap of differentially expressed genes (DEGs) about Wnt pathway between experimental and normal control groups. Red and blue represent significantly upregulated and downregulated genes, respectively.

RT-PCR and immunohistochemistry validated that ACVR2A modulates cellular behavior via the TCF7/c-JUN pathway.

(A) RT-qPCR analysis of DEGs (Wnt3, Wnt4, TCF7, TCF7L1, CCND1, and c-JUN) expression enriched in Wnt/TCF pathway following HTR8/SVneo ACVR2A knockout.

(B) RT-qPCR analysis of DEGs (Wnt3, Wnt4, TCF7, TCF7L1, CCND1, and c-JUN) expression enriched in Wnt/TCF pathway following JAR ACVR2A knockout.

(C) RT-qPCR analysis of DEGs (Wnt3, Wnt4, TCF7, TCF7L1, CCND1, and c-JUN) expression enriched in Wnt/TCF pathway expression in the placenta of normal control pregnant women (NC) and patients with preeclampsia (PE).

(D) Immunohistochemical staining of normal control pregnant women and preeclampsia placentas using rabbit IgG anti-human Wnt3, Wnt4, TCF7L1, TCF7L2, CCND1, and c-JUN antibody. Sections were counterstained with hematoxylin, and positive cells were quantified using ImageJ software.

The inhibitory effect of ACVR2A on trophoblast cell invasion, migration, and proliferation through the TCF7/c-JUN pathway is illustrated.

(Colors: Green: Normal conditions and functional pathways. Red: Abnormalities or disrupted pathways in preeclampsia. Arrows: Solid Dark Brown Arrows: Normal signaling pathways and interactions. Bold Red Arrows: Disrupted or abnormal signaling pathways in preeclampsia.) The typical WNT pathways are intricately connected with ACVR2A and TGFβ. ACVR2A influences TCF7 transcriptional activation via the SMAD1/SMAD5-SMAD4 axis, subsequently affecting genes, such as JUN, CCND1, and others, and ultimately leading to the regulation of trophoblastic cell invasion, migration, and proliferation (draw by FigDraw).