circCRAMP1L is a novel biomarker of preeclampsia risk and may play a role in preeclampsia pathogenesis via regulation of the MSP/RON axis in trophoblasts

Abstract

Background: Preeclampsia is a severe disease in pregnant women, which is primarily managed by early screening and prevention. Circular RNAs (circRNAs) have increasingly been shown to be important biological regulators involved in numerous diseases. Further, increasing evidence has demonstrated that circRNAs can be used as diagnostic biomarkers. This study was conducted to evaluate the potential of circCRAMP1L, previously identified to be downregulated in preeclampsia, as a novel biomarker for predicting the development of preeclampsia.

Methods: We measured the expression of circCRAMP1L, which is reportedly relevant to trophoblast physiology, in plasma samples from 64 patients with preeclampsia and 64 age-, gestational age-, and body mass index-matched healthy pregnant women by qRT-PCR. MTT proliferation and transwell invasion assays revealed the biological role of circCRAMP1L in preeclampsia pathogenesis. RNA immunoprecipitation and dual-luciferase reporter assays clarified the mechanism underlying the biological function of circCRAMP1L in TEV-1 cells.

Results: circCRAMP1L circulating levels were significantly lower in patients with preeclampsia (2.66 ± 0.82, △Ct value) than in healthy pregnant women (3.95 ± 0.67, △Ct value, p < 0.001). The area under the receiver operating characteristic curve for circCRAMP1L was 0.813. Univariate and multivariate analyses identified circCRAMP1L as an independent predictor of preeclampsia. Furthermore, when circCRAMP1L was utilised in combination with its target protein macrophage stimulating protein (MSP), the predictive performance increased, with an area under the receiver operating characteristic curve of 0.928 (95% CI 0.882-0.974), 80.0% sensitivity, and 80.0% specificity. The in vitro results indicated that circCRAMP1L regulates cell proliferation, and invasion via MSP and RON proteins. We investigated the molecular mechanisms of these effects. In vitro, relative to the control group, circCRAMP1L overexpression significantly enhanced cell proliferation; furthermore, trophoblast cell invasion increased proportionally with circCRAMP1L expression. RNA immunoprecipitation and luciferase reporter gene illustrated that circCRAMP1L participated in regulation of trophoblast cell by regulating MSP.

Conclusion: Reduced plasma levels of circCRAMP1L may be associated with an increased risk of preeclampsia, and circCRAMP1L may be a novel biomarker of preeclampsia risk.

Keywords: MSP/RON; Preeclampsia; circCRAMP1L.

Fig. 1

a Fluorescent in situ hybridisation results showing the localisation of circCRAMP1L in placenta tissue and b immunohistochemistry analysis showing MSP and RON expression in villous tissue of placenta

Fig. 2

Expression of MSP and RON protein levels determined by western blotting. MSP protein levels in PE placentas were substantially increased compared to in PTL pregnancy placentas, whereas RON protein levels showed the opposite expression patterns (*P < 0.05)

Fig. 3

circCRAMP1L expression in circulating plasma of patients with preeclampsia compared to normal pregnant women (2.66 ± 0.82 vs. 3.95 ± 0.67, P = 0.001)

Fig. 4

Plasma MSP of PE group were significantly elevated than the control (170.25 ± 42.11 vs. 106.44 ± 38.31, ng/mL, P < 0.001). In contrast, the plasma RON in PE group were significantly reduced than the control (55.36 ± 31.25 vs. 87.41 ± 27.48 ng/mL, P < 0.001)

Fig. 5

Regulatory effects circCRAMP1L on trophoblast cell proliferation. Compared to the control group, cell proliferation was highly facilitated by circCRAMP1L overexpression (* compared with the control and P < 0.01)

Fig. 6

Regulatory effects circCRAMP1L on trophoblast cell invasion. Invasion of trophoblast cells, which were TEV-1 cells treated with siRNA-RON, proportionally increased with the expression level of circCRAMP1L

Fig. 7

Cell proliferation in siRNA-RON and siRNA-MSP groups was slower than in the siRNA-NC groups (* compared with the si-NC and P < 0.01)

Fig. 8

TEV-1 Cell invasion capacities variation when siRNA-RON or siRNA-MSP. Cell invasion in the siRNA-RON group (41.30 ± 4.83 cells) was inhibited compared to in the siRNA-NC group (155.10 ± 6.72 cells, *P < 0.01). Similarly, cell invasion in the siRNA-MSP group (140.21 ± 5.55) significantly differed from that in the siRNA-NC group (27.62 ± 3.48) (*P < 0.01)

Fig. 9

RNA immunoprecipitation assay of the interaction of circCRAMP1L with MSP /RON in TEV-1 cells

Fig. 10

Dual-luciferase assay of the interaction of circCRAMP1L with MSP. Compared to the negative control, circCRAMP1L significantly inhibited the activity of the wild-type, but not the mutant, human MSP 3′UTR reporter gene (*P < 0.05)

Fig. 11

Receiver operating characteristic curve analysis. As a single predictor, MSP or circCRAMP1L showed low efficiency. When MSP was utilised in combination with circCRAMP1L, the predictive performance increased [AUC = 0.928 (95%CI 0.882–0.974)], with 80.0% sensitivity and 80.0% specificity