Alleviation of preeclampsia-like symptoms through PlGF and eNOS regulation by hypoxia- and NF-κB-responsive miR-214-3p deletion

Preeclampsia is caused by placental hypoxia and systemic inflammation and is associated with reduced placental growth factor (PlGF) and endothelial nitric oxide synthase (eNOS) levels. The molecular signaling axes involved in this process may play a role in the pathogenesis of preeclampsia. Here, we found that hypoxic exposure increased hypoxia-inducible factor-1α (HIF-1α)/Twist1-mediated miR-214-3p biogenesis in trophoblasts, suppressing PlGF production and trophoblast invasion. TNF-α stimulation increased NF-κB-dependent miR-214-3p expression in endothelial cells, impairing eNOS expression and causing endothelial dysfunction. Synthetic miR-214-3p administration to pregnant mice decreased PlGF and eNOS expression, resulting in preeclampsia-like symptoms, including hypertension, proteinuria, and fetal growth restriction. Conversely, miR-214-3p deletion maintained the PlGF and eNOS levels in hypoxic pregnant mice, alleviating preeclampsia-like symptoms and signs. These findings provide new insights into the role of HIF-1/Twist1- and NF-κB-responsive miR-214-3p-dependent PlGF and eNOS downregulation in the pathogenesis of preeclampsia and establish miR-214-3p as a therapeutic or preventive target for preeclampsia and its complications.


Generation of miR-214-3p KO mice
MiR-214-3p KO mice were generated using the CRISPR/Cas9 system.Briefly, single-guide RNAs (sgRNAs; Supplementary Table 1) were cloned into the pT7-Guide-IVT vector (GE100025, Origene, Rockville, MD, USA).The constructs were linearized using the restriction enzyme BsmBI (ER0451, Thermo Fisher Scientific, Waltham, MA, USA) and used as templates for in vitro transcription to yield sgRNAs using the MEGAshortscript T7 kit (AM1334, Thermo Fisher Scientific).Cas9 mRNA (50 ng/l final concentration) and sgRNAs (50 ng/l final concentration) were mixed in TE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.5) and injected into the cytoplasm of C57BL/6 zygotes using micromanipulators.The zygotes were then transferred into the uteri of pseudopregnant recipient females at 2.5 days post-coitum.After birth, DNA was extracted from the tails of the pups, and the deletion of the miR-214 precursor sequence was analyzed using PCR with its specific primers, followed by further confirmation by sequencing analysis of the amplicons.The founder lines were expanded and maintained under a 12-h light:12-h dark cycle and provided free access to a standard chow diet (JA Bio, Republic of Korea) and water in a pathogen-free animal facility.All animal experiments were approved by the Institutional Animal Care and Use Committee of the Kangwon National University (KW-200305-3) and followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication, 8th edition, 2011).

Isolation of placental trophoblasts and aortic endothelial cells
Mouse primary trophoblasts were isolated using a protocol modified from previous methods 1,2 .
Briefly, mouse placentas were separated from the underlying endometrium in a sterile dish containing RPMI 1640 medium using dissecting forceps and incubated in dissociation medium (serum-free RPMI 1640 supplemented with 1 mg/ml collagenase IV [C5138, Sigma-Aldrich, St. Louis, MO] and 20 g/ml DNase I [DN25, Sigma-Aldrich]) (20 ml/g tissue) for 1 h at 37C, with periodic pipetting to separate cells.Cells were washed to remove dissociation medium and filtered through 40-µm cell strainers.After washing, cell pellets were resuspended in 5 ml RPMI 1640, layered on the top of a preformed Percoll gradient (65 %25) and centrifuged at 730 ×g at 4°C without braking for 30 min.Trophoblasts were collected from the layer between the 45% and 35% (density 1.050-1.060g/ml) Percoll aliquots.Mouse aortic endothelial cells were isolated as described previously 3 .In brief, abdominal aorta was harvested, and its lumen was washed with serum-free medium.A 24-gauge cannula was inserted into the proximal site of the aorta and ligated with a silk thread.The other side was bound, filled with 2 mg/ml collagenase II (NC9870009, Worthington Biochemical Co. Lakewood, NJ, USA), and incubated for 40 min at 37C.Endothelial cells were collected from aorta by flushing with 5 ml of RPMI 1640 supplemented with 20% FBS and washed by centrifugation at 1,000 ×g for 5 min.The precipitate was gently suspended in an appropriate volume of RPMI 1640.The trophoblasts and endothelial cells were used to analyze miRNA and mRNA expression.

MiRNA profiling and quantitative real time PCR (qRTPCR) analysis
Expression of miRNAs in HTR-8/SVneo cells exposed to normoxia or hypoxia for 24 h was analyzed at Macrogen (Seoul, Korea) using the Affymetrix miRNA expression microarray version 3.0 (902017).Total RNA was extracted from tissues, cells, and sera using the miRNeasy Mini kit (217084, QIAGEN) or miRNeasy serum/plasma kit (217184, QIAGEN), and cDNA was then synthesized using 1 g RNA and an miScript II RT Kit (218161, QIAGEN).Thereafter, miRNA levels were quantified via qPCR using the miScript SYBR Green PCR kit (218073, QIAGEN), miR-214-3p primers (MS00031605 for human; MS00032571 for mouse), miR-199a-3p primer (MS00007602 for human), SNORD95 miScript miRNA primer (H/M/R:MS00033726, QIAGEN), and human pre-miR-214-3p primers (Supplementary Table 1) according to methods described previously 4,5 .MiRNA levels were normalized to that of the housekeeping gene SNORD95.For mRNA quantification, total RNA was extracted from cultured cells and placental tissues using the TRIzol reagent (Invitrogen) and used to synthesize first strand cDNA using M-MLV Reverse Transcriptase (Promega, Madison, WI, USA), followed by quantification of PlGF and eNOS mRNA levels using qPCR and target-specific primers (Supplementary Table 1).mRNA levels were normalized to that of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene.

In vitro endothelial cell angiogenesis and trophoblast migration/invasion assays
HUVEC migration and tube formation were assessed as described previously 5 .For the migration assay using Transwell plates with polycarbonate filters (8-m pore size; Corning Inc.), CM were placed in the lower wells, and HUVECs (2 × 10 4 cells) were loaded in the upper wells, followed by incubation at 37C for 4 h.The cells that migrated to the lower side of the filter were quantified by counting those in all fields in each assay, as described above for the trophoblast invasion assay.HUVEC tube formation was determined after treatment with CM on a layer of growth factor-reduced Matrigel in a humidified CO 2 incubator.After culture for 24 h, tube formation was observed under an Olympus IX71 microscope, and images were captured using a video graphic system.Tube formation was then quantified using ImageJ software (NIH, Bethesda, MD, USA).Trophoblast migration and invasion were assessed by scratch-wound assay and Boyden chamber assay, respectively.For the scratch-wound assay, a linear scratch was gently made at the center of the HTR-8/SVneo cell monolayer using the SPLScar scratcher (201907, SPL Life Sciences, Pocheon, Republic of Korea), and cells were incubated with trophoblastderived CM for 24 h.Cell migration was recorded using an Olympus IX71 microscope (Tokyo, Japan) equipped with a digital camera (Canon Inc., Tokyo, Japan).The wound width was calculated as the average distance between the edges of the scratch using ImageJ software.For the Boyden chamber assay to determine trophoblast invasion, CM were placed in the lower chambers of Transwell plates with 6.5 mm diameter polycarbonate filters (8-m pore size; Corning Inc., Corning, NY, USA), and HTR-8/SVneo cells (5 × 10 3 cells) were placed into the upper wells, which were pre-coated with 100 l of growth factor-reduced Matrigel (354230, Corning Inc.).After incubation for 4 h in a humidified CO 2 incubator, cells were carefully fixed with cold methanol (4C) and stained with crystal violet, and those remaining on the upper surface of each filter were wiped off using a cotton swab.The cells found on the lower side of the filter were observed under an Olympus IX71 microscope, and images were captured using a video graphic system.Cell invasion was quantified by counting the cells in all fields in each assay.

Histology and immunohistochemistry
Paraffin-embedded kidney and placental tissues were sectioned at a thickness of 5 m using a cryostat microtome (CM1850 UV, Leica Biosystems, Wetzlar, Germany).Sections of renal tissue were stained with H&E, followed by analysis under an Olympus IX71 microscope.The mean glomerular diameter was calculated from five glomeruli that were randomly selected from the renal tissue of each mouse.Bowman's capsule space was calculated by subtracting the glomerular tuft area from the renal corpuscle area using ImageJ.The sections of placental tissues were stained with H&E as well as an anti- g, h Computational analysis of the putative transcription factor-binding sites, E-box and κB, in the promoter regions of human (g) and mouse miR-199a/214 (Dnm3os) (h).i Schematic plasmid (pGL3) constructs containing WT, truncated, or deleted promoter of human miR-199a/214 (Dnm3os).j HTR-8/SVneo cells were transfected with WT or mutant pGL3-Dnm3os promoter and exposed to normoxia or hypoxia for 24 h.Luciferase reporter activities were determined in cell lysates using an enzyme assay kit (n = 4).k HTR-8/SVneo cells were transfected with siRNA for control (C), HIF1A (H), or Twist1 (T), followed by exposure to normoxia or hypoxia for 24 h.The levels of target proteins were determined using western blotting.l Schematic illustration of psiCHECK-2 luciferase constructs containing WT or mutants at site 1 (7177 nt, Mut S1), site 2 (111118 nt, Mut S1), and site 3 (415421 nt, Mut S3) of human PlGF 3-UTR (876 bp).Data are presented as the mean ± SEM.Statistical significance was determined using one-way ANOVA (af, j), followed by the Holm-Sidak′s multiple comparisons test.