Differentially expressed mRNAs, lncRNAs, and circRNAs, and cluster analysis
Because of the central role of the placenta in PE pathogenesis, we first performed microarray analysis of six placenta samples from SPE patients and six placenta samples from matched normal controls (N). After applying a stringent filtering approach that compared SPE and N groups (adjusted p value, < 0.05; fold change, > 2 or < 0.5), we identified 731 upregulated and 1617 downregulated mRNAs, 5127 upregulated and 3813 downregulated lncRNAs, and 4569 upregulated and 3984 downregulated circRNAs. The top 10 upregulated and downregulated circRNAs are shown in Additional file 1: Table S3. Moreover, based on these differentially expressed mRNAs, lncRNAs, and circRNAs, a tree with a clear distinction between SPE and N groups was generated by MeV_4_9_0 cluster analysis (Fig. 1). The variability between controls in microarray vs variability between PE samples was shown in BOX plot (Additional file 1: Figure S2). These results suggested that ceRNA expression in the SPE group can be robustly separated from that in the N group.
GO enrichment of differentially expressed mRNAs, lncRNAs, and circRNAs
We identified the functional categories of these differentially expressed mRNAs, lncRNAs, and circRNAs by performing GO analysis to gain further insights into the biological processes that were potentially mediated by these differentially expressed molecules in PE. For differentially expressed mRNAs, 217 GO terms in the category of biological processes were significantly enriched at a false discovery rate (FDR) threshold of < 0.05. As shown in Fig. 2a, the top 10 significantly enriched biological processes were enzyme-linked receptor protein signaling pathway, tetraspanin-enriched microdomain, female pregnancy, immune system process, leukocyte migration, defense response, integral component of plasma membrane, plasma membrane, mating, and apoptotic process. Many biological processes were associated with the pathogenesis of PE, such as immune system and apoptotic processes.
There were 72 significantly enriched GO terms (FDR threshold of < 0.05) for differentially expressed lncRNAs. The top 10 significantly enriched biological processes are shown in Fig. 2b. They were CTPase activity, selenocysteine-tRNA ligase activity, vascular smooth muscle cell differentiation, response to other organism, l-aspartate transmembrane transporter activity, response to hydrostatic pressure, cellular response to osmotic stress, cell differentiation involved in embryonic placenta development, metalloendopeptidase activity, and positive regulation of apoptotic processes. Many biological processes were related to the mechanisms of PE, such as cell differentiation involved in embryonic placenta development and metalloendopeptidase activity. Four hundred and forty-four GO terms were significantly enriched (FDR threshold of < 0.05) for host genes of differentially expressed circRNAs. As shown in Fig. 2c, the top 10 significantly enriched biological processes were protein binding, cytosol, ATP binding, membrane, cytoplasm, nucleoplasm, actin binding, extracellular exosome, positive regulation of GTPase activity, and nuclear membrane. The most abundant categories were those associated with pathological processes that may induce damage in the membrane, cytoplasm, nucleoplasm, and nuclear membrane of placental cells.
KEGG enrichment of differentially expressed mRNAs, lncRNAs, and circRNAs
Ninety-four KEGG pathways (FDR p value < 0.05) were associated with differentially expressed mRNAs. As shown in Fig. 3a, the top 10 enriched pathways were ECM-receptor interaction, alanine, aspartate and glutamate metabolism, sphingolipid metabolism, metabolic pathways, protein digestion and absorption, PI3K-Akt signaling pathway, proteoglycans in cancer, signaling pathways regulating pluripotency of stem cells, focal adhesion, and peroxisome. In particular, a study has shown that the phosphatidylinositide 3-kinase (PI3K)-Akt signaling pathway is associated with PE [22]. Therefore, the roles of the PI3K-Akt signaling pathway in human PE placentas were investigated further.
There were 14 KEGG pathways (FDR p value < 0.05) associated with differentially expressed lncRNAs. As shown in Fig. 3b, the top 10 enriched pathways were microRNAs in cancer, thiamine metabolism, terpenoid backbone biosynthesis, pentose phosphate pathway, cysteine and methionine metabolism, ether lipid metabolism, aminoacyl-tRNA biosynthesis, hepatitis B, RIG-I-like receptor signaling pathway, and protein digestion and absorption. These results indicated that differentially expressed lncRNAs in PE control gene expression on post-transcription level. One hundred and fifty-three KEGG pathways (FDR p value < 0.05) were associated with host genes of differentially expressed circRNAs. As shown in Fig. 3c, the top 10 enriched pathways were focal adhesion, ECM-receptor interaction, regulation of actin cytoskeleton, metabolic pathways, DNA replication, phosphatidylinositol signaling system, Epstein-Barr virus infection, propanoate metabolism, cell cycle, and the mammalian target of rapamycin (mTOR) signaling pathway. These results indicated that host genes of differentially expressed circRNAs in PE were involved in the mTOR signaling pathway that is related to angiogenic factor expression in the placenta of rats [23].
Validation of mRNA, lncRNA, and circRNA expression
Some mRNAs (Chd5 and Furin), lncRNAs (lnc-ELAVL4-9:1 and lnc-RAP1GAP2-5:2), and circRNAs (hsa_circ_0036877, hsa_circ_0036878, hsa_circ_0055724, hsa_circ_0049730, and hsa_circ_0036474) identified as differentially expressed by the microarray analysis were selected for validation (Fig. 4). qRT-PCR was performed to detect the expression of these mRNAs, lncRNAs, and circRNAs in PE (n = 10) and N (n = 10) placenta samples. As shown in Fig. 4, the qRT-PCR results were highly consistent with the microarray data. Melting curve analysis and agarose gel electrophoresis were used to check for the specificity of the qRT-PCR products.
Global ceRNA network integration in PE
Previous studies show that miRNAs (miR-210 [24], miR-181a [25], miR-126-3p and miR-126-5p [26], miR-223 [27], miR-515 [28], and miR-519d [29]) are involved in the pathogenesis of PE. Based on our microarray data and significant expression of miRNAs associated with PE in previous reports, a global ceRNA network was predicted using Cytoscape_V2_8_3. As shown in Fig. 5a, a portion of the global ceRNA network was observed and GO analysis was performed to gain further insights into the biological processes in this module. Remarkably, we observed that the target genes of this module were implicated in biological processes and molecular functions in the pathogenesis of PE, such as blood vessel maturation and angiogenesis (Fig. 5b).
Hsa_circ_0036877 can function as a ceRNA
The proprotein convertase Furin, which is required for the development of the syncytiotrophoblast structure in the labyrinth layer and normal embryonic development [30, 31], is the host gene of hsa_circ_0036877. In view of this, we explored whether has_circ_0036877 can function as a ceRNA in htra-8 cells (the htra-8 cell derived from first trimester human placenta was cell line of trophoblasts).
Firstly, we investigated the expression of hsa_circ_0036877 in placenta of patients with PE and htra-8 cells by RNA FISH. As shown in Additional file 1: Figure S3A and B, hsa_circ_0036877 was high expressed in cytoplasm of syncytial trophoblasts (arrows show) and was higher expressed in normal placenta than PE. Next, to detect the ability of hsa_circ_0036877 to sponge miRNAs, endogenous hsa_circ_0036877 binding of Ago2 was determined by RNA-binding protein immunoprecipitation (RIP) in htra-8 cells. RIP assay showed that the amount of endogenous hsa_circ_0036877 pulled-down using AGO2 antibody was detected by qRT-PCR and significantly higher than control using IgG antibody (Additional file 1: Figure S3C). This experiment can identify that hsa_circ_0036877 might recruit AGO2 to sponge miRNAs, because the “minimal RNA-induced silencing complex (RISC)” appears to include AGO2 bound to a short guide RNA such as a miRNA or short interfering RNA (siRNA), which direct RISC to complementary mRNAs and silence gene expression [32].
Furthermore, as predicted by Cytoscape_V2_8_3, hsa_circ_0036877 may function as a ceRNA to bind to miR-519d-3p and miR-15b-5p, thus affecting gene expression (Additional file 1: Figure S3D). GO analysis in this module was performed to gain further insights into the biological processes (Additional file 1: Figure S3E). It is worth noting that biological processes and molecular functions in the pathogenesis of PE, such as regulation of blood vessel remodeling and female pregnancy, were enriched in this module. These results suggested that hsa_circ_0036877 may function as a ceRNA and sponge miRNAs, thus implicating it in the pathogenesis of PE.
Hsa_circ_0036877 can serve as a potential novel blood biomarker for early PE
Hsa_circ_0036877 expression was lower in placenta samples of PE patients (Fig. 4c). To confirm that differentially expressed circRNAs in microarray data can serve as blood biomarkers for early PE, we selected hsa_circ_0036877 whose expression in maternal whole peripheral blood from 110 matched normal participants and 34 patients with PE at 24 weeks of gestation was detected by qRT-PCR. The qRT-PCR results showed that expression of hsa_circ_0036877 was significantly higher in blood samples of 34 patients with PE (ΔCt mean ± s.e.m., 1.779 ± 0.6888) than in those of 110 normal participants (ΔCt mean ± s.e.m., 6.651 ± 0.2192), which were diagnosed at the end of gestation based on ACOG (Fig. 6a). Higher ΔCt value indicated lower expression. Furthermore, Additional file 1: Figure S4 shows the correlation between the expression in placenta and maternal blood from the same individuals in the PE (n = 40) and non-PE groups (n = 33). These results indicated that hsa_circ_0036877 expression was lower in placenta samples of PE, but higher in whole peripheral blood samples of patients with PE than normal controls.
Leading hypotheses suggest that necrotic breaks occur in the syncytiotrophoblast as a direct consequence of an imbalance in the regulation of apoptosis, leading to increased release of trophoblast particles (TPs) into maternal circulation in PE [33]. Extracellular nucleic acids of both fetal and placental origin, packed into either trophoblast-derived apoptotic bodies or shedding syncytiotrophoblast microparticles, which are significantly increased in PE, may be detected in maternal circulation [34, 35]. In our study, hsa_circ_0036877 may be one of the extracellular nucleic acids originating from the placenta. As shown in Fig. 6b, the significantly increased apoptosis of syncytial trophoblasts (arrows) can explain the phenomenon that lower expression of hsa_circ_0036877 in PE placentas, but higher expression in blood circulation.
Furthermore, the area under the receiver operating characteristic (ROC) curve (AUC) of hsa_circ_0036877 was 0.846 [95% confidence interval (CI) 0.754–0.938]. The cutoff value (the optimal cutoff point was determined at the maximum of Youden index (YI)) was 5.13 (ΔCt value) (Fig. 6c). The sensitivity and specificity were 85.3 and 72.7%, respectively. The positive predictive value (PPV) of an ΔCt value of 5.13 or lower for a diagnosis of preeclampsia was 49.1%. An ΔCt value above 5.13 had a high negative predictive value (NPV) of 94.1%. These results suggested that hsa_circ_0036877 may have some predictive value for early PE.