A novel regulated network mediated by downregulation HIF1A-AS2 lncRNA impairs placental angiogenesis by promoting ANGPTL4 expression in preeclampsia

Figures

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  FIGURE 1. Modulation of HIF1A-AS2 expression in trophoblasts. qPCR analysis of HIF1A-AS2 expression in HTR-8/SVneo (A) and HUVECs (B) after transfected with HIF1A-AS2–specific siRNAs, respectively. HIF1A-AS2 expression levels were tested by qPCR after treating with empty vector and pcDNA3.1-HIF1A-AS2 plasmid in HTR-8/SVneo (C) and HUVECs (D). Error bars are mean with SEM of technical replicates (n = 3). *p < 0.05, **p < 0.01; significance by Student’s t test.

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  FIGURE 2. HIF1A-AS2 promotes trophoblast proliferation, migration and invasion in vitro. Transwell assays showed the migratory and invasive abilities of siHIF1A-AS2-treated in HTR-8/SVneo (A) and HUVECs (C). The quantity of cells was markedly decreased compared to siCon control. With the treatment of overexpressed HIF1A-AS2, the 2 cell lines showed significant increase than that in pcDNA control (B,D). Colony formation assay to assess the ability of proliferation in HTR-8/SVneo and HUVEC cells, transfected with siRNAs or plasmid. Silenced HIF1A-AS2 expression in HTR-8/SVneo and HUVEC cells (E) and overexpressed-HIF1A-AS2 (F). The statistics of the relevant data is on the right side of the corresponding graph. Error bars are mean with SEM of technical replicates (n = 3), *p < 0.05, **p < 0.01; significance by Student’s t test.

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  FIGURE 3. HIF1A-AS2 affects angiogenesis of HTR-8/SVneo trophoblast and HUVEC cells in vitro and in vivo. Consequent to network formation, the number of segments and total segments length of the network were decreased in HIF1A-AS2 with siRNAs (A) and increased in overexpressed with pcDNA3.1-HIF1A-AS2 (B) in HTR-8/SVneo and HUVECs, respectively. The quantity of cells is shown below the corresponding graph. The Matrigel plug assay showed that HIF1A-AS2-related siRNA inhibited angiogenesis in nude mice (C), and overexpression of HIF1A-AS2 promoted angiogenesis (D). Error bars are mean with SEM of technical replicates (n = 3). *p < 0.05, **p < 0.01; significance by Student’s t test.

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  FIGURE 4. HIF1A-AS2 interacts with the transcription factor FOXP1. FOXP1 expression level was detected at RNA and protein level by qPCR (A,C) and Western blotting (B), respectively, after FOXP1-specific siRNAs were transfected into HTR-8/SVneo. After the treatment with siRNAs against HIF1A-AS2, the lncRNA expression was tested by qPCR (C). The putative binding sites of FOXP1 on HIF1A-AS2 promoter region, and four potentially relevant binding sequences were acquired by using the JASPAR website (D). ChIP assay confirmed the binding of FOXP1to the promoter of HIF1A-AS2 in HTR-8/SVneo. qPCR data were compared with IgG negative control (E). Error bars are mean with SEM of technical replicates (n = 3). *p < 0.05, **p < 0.01; significance by Student’s t test. Performing network formation, cells transfected with FOXP1 siRNAs and HIF1A-AS2 plasmid showed an increase in node numbers as compared with silencing of FOXP1 in HTR-8/SVneo (F). Error bars are mean with SEM of technical replicates (n = 3). *p < 0.05 and **p < 0.01; significance by one-way ANOVA with Tukey post-test. All data are representative of at least two independent experiments.

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  FIGURE 5. HIF1A-AS2 binds to LMNA to inhibit ANGPTL4 expression. qPCR analysis of HTR-8/SVneo cells uncovered the relative mRNA expression of vascularized genes, with (A) HIF1A-AS2 overexpression or (B) knockdown by siHIF1A-AS2. RNA pull down, silver staining and mass spectrometry analysis were combined to analyze several potential binding proteins (C). After transfected with siHIF1A-AS2, the changes in the mRNA expression of several possible binding proteins were analyzed by qPCR (D). RIP results showed that HIF1A-AS2 could bind LMNA in HTR-8/SVneo cells (E). After LMNA knockdown, the expression of ANGPTL4 was increased at RNA levels (F,G). Error bars are mean with SEM of technical replicates (n = 3). *p < 0.05, **p < 0.01; significance by Student’s t test. ChIP assays revealed the enrichment of LMNA in ANGPTL4 promoter region (H). Consequent to tube formation, the number of segments and total segments length of the tube were decreased in the inhibition of HIF1A-AS2 and LMNA (I); ANGPTL4 and LMNA expression levels were detected at protein level by WB in HTR-8/SVneo (J). Error bars are mean with SEM of technical replicates (n = 3). *p < 0.05 and **p < 0.01; significance by one-way ANOVA…

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  FIGURE 6. Expression of ANGPTL4 in preeclampsia placental tissues. qPCR results showed the fold change of placental tissue in PE patients compared to healthy pregnant women (n = 34) (A). The abundance of ANGPTL4 protein was detected by immunohistochemistry (B), quantification was performed by positive areas (C). All data are representative of at least three independent experiments. *p < 0.05, **p < 0.01; significance by Student’s t test.

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  FIGURE 7. Schematic of the regulatory pathway.