Preeclampsia-Associated lncRNA INHBA-AS1 Regulates the Proliferation, Invasion, and Migration of Placental Trophoblast Cells

Abstract

Preeclampsia is believed to be caused by impaired placentation with insufficient trophoblast invasion, leading to impaired uterine spiral artery remodeling and angiogenesis. However, the underlying molecular mechanism remains unknown. We recently carried out transcriptome profiling of placental long noncoding RNAs (lncRNAs) and identified 383 differentially expressed lncRNAs in early-onset severe preeclampsia. Here, we are reporting our identification of lncRNA INHBA-AS1 as a potential causal factor of preeclampsia and its downstream pathways that may be involved in placentation. We found that INHBA-AS1 was upregulated in patients and positively correlated with clinical severity. We systematically searched for potential INHBA-AS1-binding transcription factors and their targets in databases and found that the targets were enriched with differentially expressed genes in the placentae of patients. We further demonstrated that the lncRNA INHBA-AS1 inhibited the invasion and migration of trophoblast cells through restraining the transcription factor CENPB from binding to the promoter of TNF receptor-associated factor 1 (TRAF1). Therefore, we have identified the dysregulated pathway "INHBA-AS1-CENPB-TRAF1" as a contributor to the pathogenesis of preeclampsia through prohibiting the proliferation, invasion, and migration of trophoblasts during placentation.

Keywords: cell invasion; cell migration; long noncoding RNA; placenta; preeclampsia; transcriptome; trophoblast.

Graphical abstract

Figure 1

Expression of INHBA-AS1 in Placentae of Patients with Preeclampsia (PE) (A) Boxplot of INHBA-AS1 expression levels in EOSPE placentae (n = 9) compared with normal placentae (n = 32). (B) The relative expression level of INHBA-AS1 measured by qRT-PCR. y axis, the fold change; red bar, placentae of patients with PE, n = 20; blue bar, normal placentae, n = 20. The values are shown as the mean ± SD; ∗∗p < 0.01. (C) The relative expression level of INHBA-AS1 in PE samples measured by qRT-PCR. Average expression level in normal samples was set at 0; red bars represent relative expression levels in placentae of PE patients. (D) Positive correlation (r = 0.5504, p < 0.001) between systolic blood pressure and the expression levels of lncRNA INHBA-AS1 (Pearson one-tail correlation analysis). (E) Positive correlation (r = 0.6475, p < 0.001) between diastolic blood pressure and the expression of lncRNA INHBA-AS1 (Pearson one-tail correlation analysis). (F) Hierarchical “INHBA-AS1-transcription factor (TF)-Target” interaction network. (G) Gene-coding proteins interacting with INHBA-AS1 are enriched with coding differentially expressed genes (DEGs) in EOSPE. (H) The targets in the INHBA-AS1-TF-Target interaction network are enriched with DEGs in EOSPE. (I) The enriched pathways with DEGs as targets of the INHBA-AS1-TF-Target interaction network. The values are shown as the mean ± SD of three independent experiments; ∗∗p < 0.01, ∗p < 0.05. See also Table S1.

Figure 2

The Effect of INHBA-AS1 on Invasion, Migration, and Proliferation in Trophoblast Cell Line (A and B) Invasion and migration of HTR-8/SVneo cells were detected by a Transwell assay after INHBA-AS1 overexpression (A) or knockout (B). Upper panel: pictures of cells stained with 0.1% crystal violet solution; lower panel: statistics of cells. Scale bars, 100 μm. (C and D) The proliferation of HTR-8/SVneo cells was detected by an EdU assay after INHBA-AS1 overexpression (C) or knockout (D). Upper panel: pictures of cells stained with 4′,6-diamidino-2-phenylindole (DAPI) and EdU; lower panel: statistics of EdU-positive cells. (E and F) Cell Counting Kit-8 was used to evaluate the cell proliferation of HTR-8/SVneo cells after INHBA-AS1 overexpression (E) or knockout (F). LV, lentivirus; NC, normal control. The values are shown as the mean ± SD of three independent experiments; ∗∗p < 0.01, ∗p < 0.05. See also Figure S1.

Figure 3

INHBA-AS1 Prohibits Cell Cycle and Promotes Apoptosis in HTR8/SVneo Cells (A) Cell-cycle analysis by flow cytometry on cells transfected by LV harboring human full-length INHBA-AS1 or empty vector. Upper panels: frequency histograms of cell-cycle phases; lower panels: statistics of cell-cycle phases. (B) Cell-cycle analysis by flow cytometry on cells with INHBA-AS1 knockout. Upper panels: frequency histograms of cell-cycle phases; lower panels: statistics of cell-cycle phases. (C and D) Apoptosis analysis using Annexin-V assay coped with flow cytometry on cells with INHBA-AS1 overexpression (C) or knockout (D). Upper panels: histograms of apoptotic cells; lower panels: statistics of apoptotic cells. The apoptotic cells include cells in quad 2 (Q2) and Q4. Cells in Q2 are dead apoptotic cells (PI positive and Annexin-V positive), and cells in Q4 are ongoing apoptotic cells (PI negative but Annexin-V positive). The values are shown as the mean ± SD of three independent experiments; ∗∗p < 0.01, ∗p < 0.05. See also Figure S1.

Figure 4

INHBA-AS1 Interacts with CENPB and Inhibits TRAF1 Expression (A) The subcellular localization of INHBA-AS1 in HTR-8/SVneo detected by FISH assays. Red, INHBA-AS1; blue, nucleus. Scale bar, 20 μm. (B) The subcellular localization of INHBA-AS1 in the HTR-8/SVneo cell detected by cell fractionation assays. U6, nucleus marker; GAPDH, cytoplasm marker. (C) INHBA-AS1-binding proteins detected by pulldown and mass spectrometry. (D) Enrichment analysis of GO term cellular component (GO-CC) on INHBA-AS1-binding proteins. (E) TF-target network (TFTNet) for EOSPE DEGs. Red triangles, TFs; blue nodes, targets of the TFs. (F) Overlaps between CENPB target genes (188) and DEGs (3,116) in EOSPE detected in transcriptome sequencing. (G) Binding of INHBA-AS1 with CENPB detected by RIP assays and qRT-PCR. (H) Transcription level of CENPB target genes in cells with INHBA-AS1 overexpression. Left panel shows qPCR results; blue bars, cells transfected with empty vector; red bars, cells transfected with INHBA-AS1; right panel shows western blotting results for TRAF1. (I) Transcription level detected of CENPB target genes in cells with INHBA-AS1 knockout. Left panel shows qPCR results; blue bars, cells without knockout; red bars, cells with INHBA-AS1 knockout; right panel shows western blotting result of TRAF1. The values are shown as the mean ± SD of three independent experiments; ∗∗p < 0.01, ∗p < 0.05. See also Figure S2 and Tables S2, S3, and S4.

Figure 5

CENPB Binds the Promoter Region of TRAF1 and Promotes Its Transcription (A) The expression of CENPB in HTR8/SVneo cells transfected with full-length CENPB. Left panel: results of qPCR; right panel: results of western blot. (B) Increased expression of TRAF1 in HTR8/SVneo cells after CENPB overexpression. Left panel: results of qPCR; right panel: results of western blot. (C) The expression of CENPB in HTR8/SVneo cells transfected with shRNA of CENPB. Left panel: results of qPCR; right panel: results of western blot. (D) Decreased expression of TRAF1 in HTR8/SVneo cells after CENPB overexpression. Left panel: results of qPCR; right panel: results of western blot. (E) Binding of CENPB to the promoter of TRAF1 detected using ChIP assays and qPCR in HTR8/SVneo cells. (F) Luciferase reporter assay on the transcription activity of the TRAF1 promoter in HTR8/SVneo cells after INHBA-AS1 overexpression or knockout. The values are shown as the mean ± SD of three independent experiments; ∗∗p < 0.01, ∗p < 0.05. See also Table S5.

Figure 6

TRAF1 Promoted the Invasion and Migration of Trophoblast Cells and Weakened the Effect of INHBA-AS1 (A) Overexpression of TRAF1 partially rescued the invasion and migration of HTR-8/SVneo cells inhibited by INHBA-AS1. Scale bars, 100 μm. (B) INHBA-AS1 knockout partially rescued invasion and migration caused by knockdown of TRAF1. Scale bars, 100 μm. (C) Overexpression of TRAF1 upregulated the expression of MMP2, MMP3, and vimentin at mRNA level and protein level. (D) Knockdown of TRAF1 in HTR-8/SVneo cells downregulated the expression of MMP2, MMP3, and vimentin at the mRNA level and protein level. The values are shown as the mean ± SD of three independent experiments; ∗∗p < 0.01, ∗p < 0.05. See also Figure S3 and Table S5.