Potential regulatory network in the PSG10P/miR-19a-3p/IL1RAP pathway is possibly involved in preeclampsia pathogenesis

Abstract

Preeclampsia (PE), a pregnancy-specific disorder, is a leading cause of perinatal maternal-fetal mortality and morbidity. Impaired cell migration and invasion of trophoblastic cells and an imbalanced systemic maternal inflammatory response have been proposed as potential mechanisms of PE pathogenesis. Comparative analysis between PE placentas and normal placentas profiled differentially expressed miRNAs, lncRNAs, and mRNAs, including miR-19a-3p (miRNA), PSG10P (lncRNA), and IL1RAP (mRNA). This study was conducted to investigate their potential roles in PE pathogenesis. The expression of miR-19a-3p, PSG10P, and IL1RAP was examined in PE and normal placentas using RT-qPCR. An in vitro experiment was performed in human trophoblast HET8/SVneo and TEV-1 cells cultured in normoxic and hypoxic conditions. MiR-19a-3p targets were identified using Targetscan, miRanda, and PicTar analysis as well as luciferase reporter assays. The mouse model of PE was conducted using sFlt-1 for in vivo tests. Lower levels of miR-19a-3p, but higher levels of PSG10P and IL1RAP were observed in PE placentas and the trophoblast cells in hypoxia. Luciferase reporter assays confirmed that PSG10P and IL1RAP were both direct targets of miR-19a-3p. Exposure to hypoxia inhibited cell viability, migration, and invasion of HET8/SVneo and TEV-1 cells. Knocking out PSG10P and IL1RAP or overexpressing miR-19a-3p rescued the inhibition caused by hypoxia. In vivo experiments showed that IL1RAP promoted the expression of caspase-3, a key apoptosis enzyme, but inhibited MMP9, which is responsible for degrading the extracellular matrix, suggesting a significant role of IL1RAP in cell proliferation, migration, and invasion. miR-19a-3p, PSG10P, and IL1RAP were all found to be involved in PE pathogenesis. With a common targeting region in their sequences, a regulatory network in the PSG10P/miR-19a-3p/IL1RAP pathway may contribute to PE pathogenesis during pregnancy.

Keywords: IL1RAP; PSG10P; miR-19a-3p; preeclampsia pathogenesis.

Figure 1

Expression profiles of PSG10P, miR‐19a‐3p, and IL1RAP in placentas of patients with pre‐eclampsia (PE) and controls and their association with mean arterial pressure (MAP). Expression profiles of PSG10P (A1) and IL1RAP (D1) in GSE50783 datasets were analysed. RT‐PCR was performed to detect the expression of PSG10P (A2), miR‐19a‐3p (B1), and a series of mRNAs (C) in placentas of patients with PE and controls. miR‐19a‐3p expression was also detected in the serum samples using RT‐PCR (B2). Western blotting was performed to detect the expression of IL1RAP in placentas of patients with PE and controls (D2). Association of the relationship between MAP and relative RNA expression of PSG10P, miR‐19a‐3p, and IL1RAP was analysed by Pearson correlation analysis in patient with PE (F) and normal controls (E). Nor and N:Normal group; Pre and P: Pre‐eclampsia group; MAP: Mean arterial pressure. *P < 0.05, **P < 0.01, ***P < 0.001 vs Control

Figure 2

Expression profiles of PSG10P, miR‐19a‐3p, and IL1RAP in trophoblast cells under hypoxia and normoxia. HTR‐8/SVneo and TEV‐1 cells were cultured under hypoxia or normoxia. Expression profiles of PSG10P (A), miR‐19a‐3p (B), and IL1RAP (C) in trophoblast cells were assessed by RT‐PCR and western blot assays. Hyp: hypoxia; Nor: normoxia. *P < 0.05, **P < 0.01 vs HTR‐8/SVneo cells under normoxia; ^# P < 0.05, ^## P < 0.01 vs TEV‐1 cells under normoxia

Figure 3

Changes of PSG10P, miR‐19a‐3P, and IL1RAP expression in trophoblast cells after transfection. HTR‐8/SVneo and TEV‐1 cells were transfected with shRNA‐PSG10P (A), hsa‐miR‐19a‐3p mimics (B), shRNA‐IL1RAP (C), and their negative controls. The transfection rate was determined by light microscopy and fluorescent images. PSG10P, miR‐19a‐3P, and IL1RAP expression were assessed by PT‐PCR. Con: control; Scr: scramble; Kno: knockdown of PSG10P or IL1RAP. *P < 0.05, **P < 0.01 vs HTR‐8/SVneo cells in control; ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 vs TEV‐1 vs TEV‐1 cells in control

Figure 4

Negative correlation between PSG10P and miR‐19a‐3p expression in trophoblast cells. miR‐19a‐3p expression in trophoblast cells was analysed by RT‐PCR after PSG10P knockdown (A); PSG10P expression in the trophoblast cells was analysed by RT‐PCR after transfection with miR‐19a‐3p mimics (B). *P < 0.05, **P < 0.01 vs HTR?8/SVneo cells in control; ^# P < 0.05, ^## P < 0.01 vs TEV?1 cells in control. In a luciferase reporter assay, transfection with miR‐19a‐3p mimics significantly reduced the luciferase activity of the wild‐type of PSG10P constructs, but not that of the mutant PSG10P constructs (C). RNA pull‐down analysis indicated that PSG10P interacted with WT‐hsa‐miR‐19a‐3p, but not with MT‐hsa‐miR‐19a‐3p (D). *P < 0.05, **P < 0.01, and ***P < 0.001

Figure 5

IL1RAP is a direct target of miR‐19a‐3p. miR‐19a‐3p and IL1RAP expression in trophoblast cells was assessed after transfection with miR‐19a‐3p mimics (Mim) and inhibitors (Inh) (A). *P < 0.05, **P < 0.01 vs HTR‐8/SVneo cells in control; ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 vs TEV‐1 cells in the control. In the luciferase reporter assay, transfection with miR‐19a‐3p mimics significantly reduced the luciferase activity of the wild‐type of IL1RAP constructs, but not that of the mutant IL1RAP constructs (B). **P < 0.01

Figure 6

PSG10P, miR‐19a‐3P, and IL1RAP changed the growth and invasion properties of trophoblast cells under hypoxia. HTR‐8/SVneo and TEV‐1 cells were transfected with shRNA‐PSG10P, hsa‐miR‐19a‐3p mimics, or shRNA‐IL1RAP under hypoxia. After 48 h, cell viability (A), migration rate (B), and invasion rate (C) of both cell lines were evaluated. PSG10 (−) and P (−): PSG10P knockdown; miR19 (+) and M (+): miR‐19a‐3p overexpression; I (−) and IL1RAP (−): IL1RAP knockdown. *P < 0.05, **P < 0.01 vs HTR‐8/SVneo cells in control; ^$ P < 0.05, ^$$ P < 0.01 vs HTR‐8/SVneo cells under hypoxia. ^# P < 0.05, ^## P < 0.01 vs TEV‐1 cells in control; ^& P < 0.05, ^&& P < 0.01 vs TEV‐1 cells under hypoxia

Figure 7

Increased miR‐19a‐3p expression rescued pre‐eclampsia (PE) symptoms in a mouse model. A mouse model of PE was established using sFlt‐1. Among 36 PE mice, 12 mice were randomly selected for mmu‐miR‐agomir treatment as a negative control (PE+NC), 12 for mmu‐miR‐19a‐3p agomir treatment (PE+Agomir), and 12 as a PE control (PE). In addition, another 12 normal pregnant mice were orally treated with saline solution as a control (Nor). (A) miR‐19a‐3p expression in placentas was assessed via RT‐PCR. (B) Protein levels of IL1RAP, caspase‐3, and MMP‐9 in placentas were assessed via western blot assay. (C) Ki67 expression in placentas was assessed via immunohistochemical staining. (D) The blood pressure, (E) urine protein concentrations, and (F) fetal survival number of the mice were recorded. *P < 0.05, **P < 0.01 vs Normal group; ^# P < 0.05, ^## P < 0.01 vs PE group