Are vascular endothelium and angiogenesis effective MicroRNA Biomarkers associated with the prediction of early-onset preeclampsia (EOPE) and adverse perinatal outcomes?

Purpose : We aimed to investigate serum MicroRNA (miRNA) levels in preeclampsia (PE) and to determine whether any changes in miRNA levels are useful in predicting early onset preeclampsia (EOPE) and adverse perinatal outcomes. Methods: A total of 89 pregnant patients were enrolled in this prospective case-control study (55 PE and 34 healthy controls). miR-17, miR-20a, miR-20b, miR126, miR155, miR-200, miR-222, and miR-210 levels were studied in maternal serum in preeclamptic pregnant women. The Real-time RT-PCR method was used to determine maternal serum miRNA levels. Results: Serum miR-17, miR-20a, miR-20b, miR126, and miR-210 levels were signi�cantly higher in PE than the control group . Increased miR-17, miR-20a, and miR-20b levels were independently associated with PE. Increased miR-17 and miR-126 levels were negatively correlated with serum EOPE in PE, and increased miR-210 levels were signi�cantly positively correlated with EOPE in PE. Conclusion: Increased expression of serum miR-17, miR-20a, miR-20b, miR126, and miR-210 were found to be associated with PE, also increased expression of miR-17, miR-20a and miR-20b were to be prediction with PE, also increased maternal serum miR-17 and miR-126 expressions were negatively correlated and increased miR-210 expression was positively correlated with EOPE in PE women.


Introduction
MicroRNAs (miRNAs) are small single-stranded and short non-coding RNA molecules that can negatively regulate gene expression.miRNAs play an important role in numerous cell biology, metabolism, placentation, implantation, invasion, migration, apoptosis, and differentiation of pregnancy pathological processes [1][2][3].miRNAs pro les have been described for the placenta, maternal plasma, and several pregnancy disorders [4].
Endovascular-trophoblastic (EVT) invasion and incomplete endovascular transformation impair spiral artery remodeling, resulting in reduced blood ow to the placental interface in the rst trimester of gestation.This aberrant placental development is involved in the pathogenesis of PE.Although PEs etiology and pathogenesis remain elusive.Multiple miRNAs may be involved in regulating the expression of various angiogenesis-related factors and endothelial cell function in PE [5,6].Recently, a member of this family, miR-20b, was found to be mainly expressed by EVTs and plays a speci c role in trophoblast invasion, additional expression of human umbilical vein endothelial cells, and plays role segregation of Vascular endothelial growth factor-A (VEGFA) in PE [7].Also, Wang et al. demonstrated that overexpression of miR -20a in the PE placenta could signi cantly inhibit proliferation, migration, and invasion of JEG -3 cells by direct regulation of Forkhead box protein A1 [8].Walter et al. reported that miR-17 is down-regulated in villous samples from early pregnancy loss [1].Liu et al. demonstrated that expression of miR-126 was higher and vascular cell adhesion molecule − 1 (VCAM-1) was lower in the placental tissues of the PE group [9].It has been determined that the increase of miR-210 at the placental level reduces the trophoblastic cell invasion of preeclampsia [10].
Besides, it's, angiogenesis and oxidative stress are already known to have an important role in regulatory molecular interaction at the maternal-fetal interface, so an imbalance in these regulatory factors may result in gestational-speci c diseases such as PE [11].In light of the above studies, we hypothesize that the differential expression levels of miRNAs play an important role in endothelial dysfunction, by inducing oxidative stress, membrane damage, and invasion of EVT in PE.The present study aimed to determine maternal serum miR-17, miR-19a, miR-20a, miR-20b, miR-126, miR-155, miR-200, miR-210 and miR-222 levels and also aimed to determine whether changes in these miRNA levels are associated with EOPE and adverse pregnancy outcomes.

Study population:
A prospective case-control study was carried out between February 2020 and February 2021.Fifty-ve patients diagnosed with preeclampsia and thirty-four patients with control (healthy pregnant) were recruited consecutively from the Perinatology Department of Selcuk University Faculty of Medicine.The local ethics committee approved the study and each patient gave written informed consent before participation.PE was de ned as systolic blood pressure of 140 mm Hg or diastolic blood pressure of 90 mm Hg that occurs after 20 weeks of gestation in a woman with previously normal blood pressure, with proteinuria as urinary excretion of 0.3 g protein in a 24-hour urine specimen.In the absence of proteinuria, preeclampsia was diagnosed as hypertension associated with at least one of the following: thrombocytopenia, new-onset cerebral, visual disturbances, doubling of serum creatinine in the absence of other renal disease or pulmonary edema.The preeclampsia group in our study was composed of patients with systolic blood pressure > 160 mm Hg or diastolic blood pressure > 110 mm Hg or accompanied by having any of the aforementioned criteria, before the 34th gestational week, which de nes EOPE [12].Adverse neonatal outcomes included preterm delivery, intrauterine fetal demise (IUFD), fetal growth restriction (FGR), and neonatal unit admission.Fetuses having estimated weight lower than 10% of their gestational ages according to the biometric measurements by sonographic assessment were FGR [13].
Patients were excluded if any of the following disorders were present: multiple pregnancies, allergic or dermatological diseases, type 1 or 2 diabetes mellitus, chronic hypertension, chronic liver or renal disease, cholelithiasis, and viral infections affecting the liver enzymes, and [3] fetal chromosomal aneuploidy and/or malformations.Thirty-four, age-and body mass index (BMI)-matched healthy, pregnant women were also recruited as a control group.
Adverse neonatal outcomes included IUFD, preterm delivery, FGR, and neonatal unit admission-.Gestational age was calculated according to the last menstrual date and was con rmed for all patients by routine examination with ultrasonography, performed during the 1st trimester of gestation.

Serum samples:
To determine miRNA levels, at least 5 ml of maternal venous blood sample was taken from the patients with preeclampsia and the control group at 28-34 weeks into a yellow-capped gel tube.The blood sample was gently inverted 5-6 times so that it came into good contact with the silica particles on the wall of the tube.It was waited for at least 30 minutes until the blood in the tube clotted spontaneously, and was centrifuged at 1500-2000xg for 10 minutes.The separated serum was transferred to a new tube, previously prepared and with patient information written.Since hemolyzed or turbid sera were not suitable for many examinations, samples were taken again.The serums were stored in cryotubes at -80°C until the study period.Also, routine laboratory automated techniques were used to determine serum biochemical markers and CBC parameters were measured by an automated blood counter Cell-Dyn 3700 automated hemocytometer (Abbott, IL).

a. Isolation of miRNA from Plasma
Plasmas were centrifuged at 12000 RPM for 5 minutes.The supernatant was discarded and 1 m triazole was placed on the pellet.(Invitrogen, USA).The cell-triol suspension was transferred to a 1.5 ml tube and slowly abundantly pipetted.200 µl of chloroform was added to it.After the tubes were inverted, they were vortexed and incubated on ice for 15 minutes.At the end of this period, the mixture was centrifuged at 12000×g for 15 minutes at 4°C and the clear top formed after phase separation was removed and transferred to a new 1.5 ml tube.500 µl of isopropanol was added to the RNA mixture and the mixture was vortexed and kept on ice for 10 minutes.It was centrifuged at 12000×g for 10 minutes at 4°C at the end of the period.After centrifugation, the supernatant was removed and 1 ml of 70% ethanol was added to the pellet and centrifuged again at 7500×g for 10 minutes at 4°C.The supernatant was completely removed and the RNA pellet was allowed to dry at the end of the time.After removing the ethanol, the miRNA pellet was dissolved in 50 µl of ddH2O.
b. Determination of RNA Quantity and Quality: After miRNA isolation, RNA quantity and quality were measured for each sample with a 260 and 280 nm UV Spectrophotometer (ACT Gene, USA).

c. Reverse Transcription (RT) Reaction (cDNA Synthesis):
For the production of single-stranded cDNA from 1 µg/10 µl total miRNA, a Poly A tail was added to the miRNA using the Poly(A) Polymerase, Yeast (ABM, Canada) kit.Performed using the commercial kit OneScript® Plus cDNA Synthesis Kit (ABM, Canada).For this, 2 ng RNA, 1 µl of 50 ng/µl random primer, 1 µl of 40µM oligo (dT) 18 primers, 10 mM dNTP mixture 1 µl and 10 µl of nuclease-free water was added into a tube at room temperature and incubated for 10 minutes at 65°C in a Thermal Cycler instrument (Bio-Rad-T100, USA) for denaturation.At the end of the period, 2 µl of 10x buffer M-MulV and 0.5 µl of 100 units of Reverse Transcriptase enzyme were added to the mixture in order, and the mixture was made up with nuclease-free water so that the total volume was 20 µl, and the tube was mixed slowly.For cDNA synthesis, the mixture was incubated at 42 ºC for 60 minutes, then incubated at 25 ºC for 10 minutes since random hexamer was used.The mixture was incubated at 85 ºC for 5 minutes for enzyme inactivation at the end of the period.The synthesized cDNA was diluted at 1:9 or 1:6 ratios and stored at -200C until use.

e. Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)
Selected miRNA primers to determine miRNA gene expression differences were ampli ed in RT-qPCR using cDNAs.BRYT Green, a new dye that can bind to double-stranded DNA and uoresce more than Sybr Green, was used as the intercalating agent.Bright green miRNA qPCR MasterMix (ABM, Canada) with reaction BRYT Green, 2X to 1X; 0.1µl of intermediate stock containing 5 µl mix, 10 pMol miRNA primer and 3' universal primer; 2 µl of 1/10 diluted cDNA was added and made up a total volume of 10 µl with sterile dH2O.The reaction was carried out in sterile 8-pack RT-qPCR strips (Axygen, USA).The temperature pro le of the reaction was set at + 95°C for 10 seconds, 45 cycles (15 seconds at 95°C, 30 seconds at 58°C, and 45 seconds at 72°C).Optical measurements were made automatically by the device during the elongation phase, just after the annealing temperature of 58°C of each cycle.Then, it was gradually heated from 63°C to 95°C at a rate of 0.1°C per second, and the melting curve analysis was performed by making optical measurements at every 1°C increase in these temperatures.The data obtained as a result of optical analysis in RT qPCR were recorded as Cq.A mixture containing the same amount of double distilled water (ddH2O) was used as a negative control, instead of cDNA as a non-template control (NTC).The obtained data were analyzed using the 2-ΔΔCt method.

Statistical Analysis
Data analysis was performed by using SPSS for Windows, version 22.0 (SPSS Inc., Chicago, IL, USA).miRNA analysis was performed using GraphPad Prism® V.6.00 (GraphPad Software Inc.).Whether the distribution of continuous variables was normal or was not determined by the Kolmogorov-SmiRnov test, the homogeneity of variances was evaluated by the Levene test.Continuous variables were shown as mean ± standard deviation (SD) or median (min-max), where applicable.While the mean differences between preeclampsia and control groups were compared by the independent Sample t-test.The optimal cutoff points of the clinical measurements which discriminate groups from each other were evaluated by ROC analysis calculating the area under the curve (AUC) as giving the maximum sum of sensitivity and speci city for the signi cant test.Sensitivity and speci city values for miR-17, miR-20a, miR-20b, miR-126, and miR-210 have also been calculated at the best cutoff point for preeclampsia; sensitivity and speci city values of miR-17, miR-20a, miR-20b, miR-126, and miR-210 were also calculated at the best cutoff point for preeclampsia.The best predictors of miR-17, miR-20a, miR-20b, miR-126, and miR-210 which discriminated groups from each other were determined by multiple logistic regression analysis, where applicable.Any variable, whose univariable test had a p-value < 0.05, was accepted as a candidate for the multivariable model along with all variables of known clinical importance.Adjusted odds ratios and 95% con dence intervals were calculated for each variable, for preeclampsia and adverse perinatal outcome.A p-value less than < .05 was considered statistically signi cant.

Results
A total of 89 participants (55 preeclampsia patients and 34 healthy controls) were enrolled in the study.

Discussion
In the present prospective case-control study in pregnant patients, increased expression of serum miR-17, miR-20a, miR-20b, miR-126, and miR-210 were found to be associated with preeclampsia, also increased expression of miR-17, miR-20a and miR-20b were to be prediction with preeclampsia.
miRNA are important regulators for angiogenesis [5] and thus play an important role in the pathogenesis of pregnancy-related diseases [14].It has been shown that miR-15, miR-16, miR-20a, and miR-20b play a role in apoptosis by targeting VEGF, and Bcl2, especially in vascular diseases and in ammation [15].Proangiogenetic miRNAs of the miR-17-92 family [16] and over-expression of miR-210 have been reported to be involved in tubulogenesis and migration in oncological patients [17].miR-210 is induced by hypoxia in endothelial cells, also MiR-210 overexpression enhanced the formation of capillary-like structures and VEGF-driven migration of normoxic endothelial cells, whereas inhibition of miR-210 decreased tube formation and migration [18].miR-126 inhibited the expression of VCAM-1.Thus, decreasing miR-126 in endothelial cells increases TNFα-stimulated VCAM-1 expression and enhances leukocyte adherence to endothelial cells [19].In addition, miR-155 is co-expressed in endothelial cells and vascular smooth muscle cells and miR-155 translationally represses the expression of angiotensin II type 1 receptor (AT1R) [17].miR-155 is induced in macrophages by cytokines such as TNFα and IFN-beta and thus contributes to in ammation [20,21].
Although the pathogenesis of preeclampsia is not known, many factors are accused: abnormal placental function, immune-system alterations, implantation aşamasında placental oxidative stress, increased in ammatory activation, the abnormal balance of angiogenic and antiangiogenic factors, and metabolic changes [22].The fundamental pathological changes of PE include systemic endothelium damage, which can cause renal tubular and vascular dysfunction.These pathological processes may lead to multiorgan dysfunction ve EOPE [23].Several studies have shown differential expression of miRNA in pregnant patients with PE.Additionally, deregulated miRNAs have also been reported in human villous trophoblasts during hypoxic stress.miR-17 was upregulated and altered in PE compared with healthy pregnancies, also miR-17 was elevated in severe versus mild PE [24].
Abdelazim et al. [24] miR-17, miR-363, and MALAT-1 showed diagnostic potential and discriminated severe PE.In additional miR-17 and MALAT-1 were signi cantly correlated with gestational age and albuminuria in PE.Kum et al. [25] showed, that members of the miR-17 ~ 92 cluster caused a signi cant reduction in syncytiotrophoblast differentiation and resulted in defective differentiation using microarray analysis.Also, Chen et al. [26] showed that angiogenesis-related miR-17-family miRNAs are increased in preeclamptic placentas compared to normotensive placentas and play an important role in trophoblast invasion into the maternal spiral artery and vascular model during early human placental development.
Wang et al. [7] showed that miR-17, miR-20a, and miR-20b, which are important in angiogenesis, are signi cantly increased in PE placenta compared to healthy term placenta.They showed that miR-20b is expressed primarily in villous syncytiotrophoblasts and regulates differentiation in endothelial and trophoblast cells.It has been shown that miR-210 is upregulated in PE by HIF-1a and NF-kBp50 induced by hypoxia and/or immune-mediated processes and high miR-210 may contribute to PE by inhibiting antiin ammatory Th2-cytokines [27].In addition, miR-210 has been shown to inhibit the invasion of trophoblast cells in the placentas of preeclamptic pregnant women [11].It has been shown that increased miR-20a can inhibit the proliferative and invasive activities of trophoblast cells by suppressing FOXA1 expression in human preeclamptic placental tissue [8].Hu et al. [28] reported that VEGF, miR-200c, -20a, and − 20b, which are the main factors of angiogenesis and vasculogenesis in the preeclamptic placenta, are decreased, and they reported that it may be responsible for angiogenic factor disorders in preeclampsia.MiR-17-5p levels were increased in maternal serum in FGR pregnancies [29].It has been shown that miR-17-5p, miR-20b-5p, miR-29a-3p, and miR-126-3p are increased in combination in gestational hypertension and severe preeclampsia.It has been reported that increased expression of miR-19a, and miR-210, and decreased expression of miR-126 is associated with the prediction of severe preeclampsia [30].The combination of seven microRNA biomarkers (miR-16-5p, miR-20a-5p, miR-145-5p, miR-146a-5p, miR-181a-5p, miR-342-3p, and miR-574-3p) was able to identify 42.68% FGR pregnancies at 10.0% FPR in early stages of gestation [31][32].MiR-126-3p and some microRNAs tend to decrease in preeclamptic pregnancies with FGR requiring delivery before 34 weeks of gestation [33].In the MADRES study, it was shown that miR-126 was negatively correlated with gestational week and birth weight, while miR-120 was positively correlated [34].Although it is associated with many adverse perinatal outcomes as the gestational week progresses [35], changes in maternal serum blood miR expression were not associated with the prediction of adverse perinatal outcomes in our study.
MiR-17, miR-126, and miR-210 are known to play a role in vascular in ammation and angiogenesis [35].It has been shown that there is a negative correlation between miR-126 and VCAM-1 expression in EOPE, thus contributing to the formation and development of EOPE by affecting the invasion ability of trophoblast cells [10].In our study, the increase in miR-17 and miR-126 was negatively correlated and the increase in miR-210 was positively correlated with EOPE in preeclamptic pregnant women.

Conclusion
In light of the above studies, it has been shown that miR-17, miR-20a, miR-20b, miR-126, and miR-210 have an important role in vascular remodeling and endothelial dysfunction.The changes in serum miR-17, miR-20a, miR-20b, miR-126, and miR-210 expression in our study suggest that there may be a relationship between vascular complications in preeclampsia.We also showed that EOPE was negatively correlated with the increase in miR-17 and miR-126 expression, and the increase in miR-210 expression was positively correlated.As a result of our study, we think that changes in miR-17, miR-20a, miR-20b, miR-126, and miR-210 expression may be markers associated with vascular endothelial damage and placental insu ciency in preeclampsia and EOPE.
We think that our study should be expanded by increasing the number of patients and that more miRNA expression should be investigated so that we can use it in the predictivity of preeclampsia, and fetal and maternal complications.

Table 1
Baseline characteristics and anthropometric preeclampsia and control groups

Table 4
Regression analysis for prediction of preeclampsia, EOPE and adverse perinatal outcome risk in the patient group