Serum lncRNAs in early pregnancy as potential biomarkers for the prediction of pregnancy-induced hypertension, including preeclampsia

Abstract

Long noncoding RNAs (lncRNAs) are key mediators of biological regulation with diagnostic value as disease biomarkers. We explored serum lncRNA levels in early pregnancy as potential biomarkers of pregnancy-induced hypertension (PIH), including gestational hypertension (GH) and preeclampsia (PE). We performed a two-phase nested case-control study in pregnant women before 20 weeks' gestation (before clinical diagnosis). The screening phase assessed lncRNA expression profiles with a human lncRNA microarray in 5 pairs of serum samples (5 PE patients and 5 matched controls). The second phase validated levels of 8 candidate lncRNAs selected via the random walk method by quantitative real-time polymerase chain reaction (qRT-PCR). Serum levels of the 8 lncRNAs were markedly increased in women with PIH compared with matched normotensive pregnant (NP) women (p < 0.001), consistent with the microarray results. In addition, 7 candidate lncRNAs were correlated with PIH severity. Logistic regression analysis revealed that serum levels of ENST00000527727 (odds ratio [OR], 1.113; 95% confidence interval [CI], 1.024-1.209; p = 0.0113) and ENST00000415029 (OR, 1.126; 95% CI, 1.000-1.267; p = 0.0496) were associated with adverse pregnancy outcomes, such as fetal growth restriction (FGR) and placenta accreta of PIH. Nine pathways associated with the candidate lncRNAs had confirmed associations with PIH.

Keywords: biomarker; gestational hypertension; lncRNA; preeclampsia; pregnancy-induced hypertension.

Graphical abstract

Figure 1

Differential expression analysis in PIH In total, 417 upregulated and 167 downregulated lncRNAs and 510 upregulated and 226 downregulated mRNAs were identified; the scatter points marked in red and blue correspond to up- and downregulated lncRNAs/mRNAs. (A) Volcano plot of lncRNAs. (B) Volcano plot of mRNAs. (C) Cluster heatmap of differentially expressed lncRNAs. (D) Cluster heatmap of differentially expressed mRNAs. (Statistics: unpaired t test, p < 0.05).

Figure 2

Expression of serum lncRNAs in the validation phase (A–H) Circulating expression levels of lncRNAs were determined by quantitative real-time PCR with serum samples from pregnant women at an early stage of gestation who later developed PIH (n = 97) compared to women with normotensive pregnancy (n = 97). (A) NR_002187 (p = 4.60E-05). (B) ENST00000415029 (p = 4.87E-06). (C) ENST00000398554 (p = 1.52E-04). (D) ENST00000586560 (p = 9.79E-04). (E) TCONS_00008014 (p = 9.79E-04). (F) ENST00000546789 (p = 8.74E-06). (G) ENST00000610270 (p = 5.90E-05). (H) ENST00000527727 (p = 6.28E-04). The expression of the 8 lncRNAs was significantly higher in pregnant women who later developed PE than in NP women (statistics: Wilcoxon rank sum test, ∗∗∗p < 0.001).

Figure 3

Expression of serum lncRNAs in PIH patients (A–E) 7 of the 8 lncRNAs were associated with the severity of PIH and showed higher expression levels in the PE group (n = 50) than in the GH group (n = 47), excluding ENST00000415029 (p = 0.0770). (A) NR_002187 (p = 0.0414). (B) ENST00000398554 (p = 0.0004). (C) ENST00000586560 (p = 0.0015). (D) TCONS_00008014 (p = 0.0060). (E) ENST00000546789 (p = 0.0262). (F) ENST00000610270 (p = 0.0093). (G) ENST00000527727 (p = 0.0162). (Statistics: Wilcoxon rank sum test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Figure 4

ROC analysis of early pregnancy serum lncRNAs for predicting PIH (A–H) NR_002187, AUC 0.6694 (95% CI = 0.5918–0.7471) (A); ENST00000415029, AUC 0.6900 (95% CI = 0.6138–0.7662) (B); ENST00000398554, AUC 0.6575 (95% CI = 0.5804–0.7345) (C); ENST00000586560, AUC 0.6370 (95% CI = 0.5577–0.7164) (D); TCONS_00008014, AUC 0.6490 (95% CI = 0.5706–0.7273) (E); ENST00000546789, AUC 0.6848 (95% CI = 0.6077–0.7619) (F); ENST00000610270, AUC 0.6670 (95% CI = 0.5883–0.7457) (G); and ENST00000527727, AUC 0.6422 (95% CI = 0.5634–0.7209) (H).

Figure 5

Functional annotation of the lncRNA biomarkers of hypertension in pregnancy (A) Bubble chart of significantly enriched KEGG pathways; the bubble size indicates the odds ratio, and the color represents the significance level. (B) The relationship of genes and pathways. The colors of each gene represent its participating pathways, and the background of each gene set indicates the corresponding pathway. (Statistics: Fisher’s exact test, p < 0.05.)

Figure 6

Study design