Nuclear Factor-Kappa B-induced miRNA-518a-5p represses trophoblast cell migration and invasion by the Nuclear Factor-Kappa B pathway

Abstract Preeclampsia is associated with the insufficient invasion of trophoblasts. NF-κB is a transcription factor in almost all mammalian cells and has been validated to be upregulated in the maternal circulation and placenta of women with preeclampsia. MiR-518a-5p is also overexpressed in pre-eclamptic placenta. The present study was designed to explore whether NF-κB can transcriptionally activate miR-518a-5p and investigate the influences of miR-518a-5p on the viability, apoptosis, migration, and invasion of HTR8/SVneo trophoblast. In situ hybridization and real time polymerase chain reaction were used to reveal miR-518a-5p expression in placenta tissues and HTR8/SVneo cells, respectively. Cell migration and invasion were detected using Transwell inserts. Our findings indicated that NF-κB p52, p50, and p65 can bind to miR-518a-5p gene promoter. MiR-518a-5p further influences the levels of p50 and p65 but not p52. HTR8/SVneo cell viability and apoptosis were not influenced by miR-518a-5p. However, miR-518a-5p represses the migratory/invasive capacities of HTR8/SVneo cell and decreased gelatinolytic activity of MMP2 and MMP9, which was reversed by an NF-κB inhibitor. To sum up, miR-518a-5p is induced by NF-κB and represses trophoblast cell migration and invasion by the NF-κB pathway.


INTRODUCTION
Preeclampsia is a de-novo hypertension after gestation for 20 weeks combined with proteinuria, haematological complications, uteroplacental dysfunction, or fetal growth retardation (Kintiraki et al. 2015).According to the American College of Obstetrics and Gynecology, preeclampsia diagnosis is based on gestational hypertension, regardless of other diagnostic criteria that are complementary (ACOG 2019).Preeclampsia is associated with higher risk of disseminated intravascular coagulation, pulmonary oedema, hemolysis, elevated liver enzymes, and low platelets syndrome, and placental abruption in pregnant women (Kintiraki et al. 2015, Ahmed et al. 2017).There is no cure for preeclampsia at present, and the main treatment is placental and fetal delivery.Preeclampsia causes preterm birth, posing immediate and long-term health burdens to the fetus and mother in severe cases (Armistead et al. 2020).During preeclampsia, the placenta is exposed to excessive inflammation and oxidative stress, trophoblast differentiation is disrupted, and secretion of anti-angiogenic proteins is enhanced (Chiarello et al. 2020, Michalczyk et al. 2020).Impaired trophoblast migration/invasiveness leads to poor placental perfusion during early pregnancy and causes fetal injury and growth retardation, causing pre-eclampsia clinical manifestations (Lala & Chakraborty 2003).
MicroRNAs (miRNAs) are noncoding RNAs at the length of around 18-22 nucleotides.D i f fe re n t i al ly ex p re ss e d m i R N A s a re characteristic of preeclampsia.MiRNAs target signaling pathway-related genes, altering the preeclampsia-involved biological processes in many conditions.Dysregulated miRNAs control trophoblast proliferation and invasion, angiogenesis, regulates the immunome system as well as other essential aspects of placentation, which makes them serve as promising diagnostic tool and therapeutic target for preeclampsia (Skalis et al. 2019).A previous study revealed that late-onset mild preeclampsia may have no placenta-specific causal factors but associated maternal factors with distinct regulators driving the different molecular pathways (Ren et al. 2021).Whereas other studies revealed that miRNAs are putative preeclampsia-specific biomarkers and can differentiate early onset preeclampsia and late onset preeclampsia from uncomplicated placentas (Lykoudi et al. 2018, Kolkova et al. 2021, Demirer et al. 2020).MiR-518a-5p is overexpressed in 11 placentas with early onset preeclampsia complicated compared with 8 healthy controls (Lykoudi et al. 2018).We made a hypothesis that miR-518a-5p can be activated by NF-κB in preeclampsia and leads to the dysregulated functions of trophoblast.

Placental tissue collection
Term placentas were collected from healthy (n = 4) and pre-eclamptic women (n = 4) after cesarean birth in the Affiliated Huaian No.1 People's Hospital of Nanjing Medical University.The placentas are collected from decidua region far from the umbilical cord insertion.Preeclamptic women were at the age of 27-33 years old and delivered at 38-40 weeks.No participants had chronic hypertension, obesity, gestational diabetes, or eclampsia.The basic clinical characteristics of the participants in this study are provided in Table I.All participants had signed the written consents to donate placenta for the present study.All placental tissues were immediately kept in liquid nitrogen and then transferred to a laboratory refrigerator (−80°C).This study was granted by the Ethic Committee of the Affiliated Huaian No.1 People's Hospital of Nanjing Medical University.

In situ hybridization
MiR-518a-5p expression in pre-eclamptic placentas was assessed by in situ hybridization.Four placentas from each group were used for in situ hybridization.Each placenta was cut into 3-6 fragments, and about a third of the tissue is used for pathological analysis.Tissues were fixed on formalin, embedded in paraffin, cut into 5 μm sections, and deparaffinized with gradient concentration of ethanol (100%, 95%, 75%, 50%).miRCURY LNA miRNA Detection Probe specific to miR-518a-5p and the miRCURY LNA Optimization Kit (Qiagen, Germany) were used according to the manufacturer's instructions.The stained tissues were observed under a computer-connected light microscope.

Chromatin immunoprecipitation (ChIP)
A ChIP assay kit (#P2078, Beyotime, Shanghai, China) was used according to the manufacturer's protocols.HTR8/SVneo cells were cross-linked with 1% formaldehyde for 12 min and sonicated into DNA fragments of 200 and 1000 bp.Cell lysates were incubated with the NF-κB antibodies (Abcam) that were coated with protein A/G beads at 4°C overnight.The goat-anti-rabbit IgG served as a negative control.The immunocomplexes that were bound to protein A/G beads were then eluted with elution buffer to remove the nonspecific binding.Samples were treated with 5 M NaCl and heated at 65°C overnight to eliminate histone-DNA crosslinks.Next, proteinase K was added followed by incubation at 45°C for 2 h.A DNA Extraction Kit (BIO-RAD) was used to purify the bound DNA fragments.Products were finally analyzed by real-time PCR using the primers specific to miR-518a-5p promoter.

Quantitative real-time PCR
Quantitative real-time PCR (qRT-PCR) was used to detect miR-518a-5p expression in HTR8/ SVneo cell line after QNZ treatment.Primers for miR-518a-5p were purchased from Invitrogen (Carlsbad, CA).A TaqMan MicroRNA Reverse Transcription Kit and a TaqMan Universal Master Mix II (Applied Biosystems, CA, USA) were used for reverse transcription and miRNA amplification, respectively.qRT-PCR was conducted with an Applied Biosystems 7900HT Fast Real-Time PCR System.Expression of snRNA U6 was also assessed to serve as a loading control.Relative  (2023) 95(1) e20220596 4 | 11 miR-518a-5p expression was calculated using the 2 −ΔΔCT method (Livak & Schmittgen 2001).CT refers to the number of fractional cycle when the signal passes a fixed threshold.

Cell apoptosis assay
HTR8/SVneo cells were stained with Annexin V/ Prodium Iodide (PI) to measure cell apoptosis using a commercial kit (#40302ES20, YEASEN, Shanghai, China) according to the manufacturer's instructions.In brief, 5 × 10 5 cells were resuspended in 100 μL room temperature in the dark and added with 400 μL binding buffer on ice.
Cell apoptosis was analyzed by flow cytometry using a BD FACSCalibur TM flow cytometer (BD Biosciences, Switzerland).Apoptotic cells (%) were defined as the percentage of cells in the third quadrant (late apoptosis) of total cells.

Cell viability assay
Cells were plated in a 96-well plate at the concentration of 5000 cells/well.After transfection of miR-518a-5p inhibitor/mimics for 12 h, cell viability was detected using a Cell Counting Kit-8 (Dojindo, Tokyo, Japan) according to the manufacturer's instructions.Optical density was detected by assessing cell absorbance of 450 nm using a microplate reader (SpectraMax i3x, Molecular Devices).
The inserts were pre-coated with Matrigel matrix (1 mg/ml; 50 µL) at 37°C for 4 h.Approximate 1 × 10 5 HTR8/SVneo cells in serum-free medium (200 µL) were transfected with miR-518a-5p inhibitor or mimics for 24 h and then placed in the upper chamber.The bottom chamber was added with medium containing 10% FBS.Cells on the Matrigel side of the Transwell insert were wiped by a cotton swab after 24 h of incubation.After fixation with methanol for 10 min, the remaining cells were stained with crystal violet (Beyotime, China) at room temperature.An Olympus IX51 light microscope was used to observe the cells in five random fields.To assess cell migration, similar methods were used except that Matrigel was not used.

Gelatin zymography
HTR8/SVneo was cultured in serum-free media and underwent transfection for 12 h.Culture media were collected for measuring the activities of gelatinases matrix metalloproteinase (MMP)-2 and MMP-9 using gelatin zymography.The 10% SDS-PAGE containing 0.1% gelatin (BIO-RAD) was used.After being diluted in NuPAGE™ LDS sample buffer (4×) containing LDS (pH 8.5), SERVA Blue G250, and phenolic red, the conditioned medium was incubated at 37°C for half an hour.
The gel was washed with elution buffer twice, 40 min per washing, at room temperature after electrophoresis.Next, samples were incubated in calcium assay buffer (ab182458, Abcam) at 37°C for one day.Coomassie Brilliant Blue R250 (#20278, Thermo Scientific) was used to stain the gel for 3 h.Finally, gels were treated with the destaining solution 10% acetic acid (#984303, Thermo Scientific) for 1 h.

Data analysis
GraphPad software v7.0 was used for statistical analysis and graph drawing.The significance of intergroup differences was calculated using the student's t test, or analysis of variance (ANOVA) as appropriate.All results are exhibited as the mean ± standard deviation from three independent biological and technical experiments.A probability level of < 0.05 indicates statistical significance.

DISCUSSION
The miR-518 family is a special biomarker of the placenta (Yang et al. 2019).Hromadnikova et al. (2015) detected the decreased expression of miR-518f-5p in placentas of 36 fetal growth restriction pregnancies.A study revealed the downregulation of miR-518b in 30 fetal growth restriction placentas (Wang et al. 2014) while another study found the elevated miR-518b expression during early gestation in 7 pregnancies with later onset of preeclampsia (Hromadnikova et al. 2012).MiR-518a-5p is upregulated in preeclamptic placenta tissues (Inno et al. 2021, Lykoudi et al. 2018) or plasma (Yang et al. 2015), while its functions on trophoblasts were not studied.MiR-518a-5p has the potential to suppress diffuse large B cell lymphoma cell line proliferation and invasion (Huang et al. 2021), while miR-518a-5p induces the migration and invasion of cancer cancer cells (Qian et al. 2019).In the present study, we identified the negative influences of miR-518a-5p on the migration and invasion of HTR8/SVneo cells and revealed that miR-518a-5p has no significant effects on the apoptosis and viability of HTR8/SVneo cells.Degradation of extra-cellular matrix promotes EVT invasion in human placenta.MMPs are secreted from the cell and can degrade the extra-cellular matrix (Hiden et al. 2018).TIMPs inhibit MMPs activities in the extracellular space (Librach et al. 1991).Migratory trophoblasts express MMPs (Lala & Chakraborty 2003), while decidua produces TIMPs (Schatz & Lockwood 1993) to restrict invasiveness.In this study, miR-518a-5p decreased the gelatinolytic activities of MMP-2 and MMP-9 in the culture medium of HTR8/SVneo.Secretion of TIMP-1/2 was increased in HTR8/SVneo cells by overexpressing miR-518a-5p.However, whether miR-518a-5p directly targets these MMPs and TIMPs remains unknown, and the underling mechanisms need further investigation.
In conclusion, this study confirms the upregulation of miR-518a-5p in human preeclamptic placentas, reveals a vital role for miR-518a-5p in suppressing the migration and invasion of HTR8/SVneo trophoblast, and supports the NF-κB/miR-518a-5p feedback as a possible mechanism of preeclampsia.

Figure 3 .
Figure 3. MiR-518a-5p is a negative regulator of HTR8/ SVneo cell migration and invasion.a) and b) Migration and invasion of HTR8/SVneo cells after treatment of miR-518a-5p mimics or inhibitor were revealed using Transwell inserts that were pre-coated Matrigel or not.One way ANOVA followed by Tukey's post hoc test was performed.**p<0.01,***p<0.001.N = 3 for each assay.

Table I .
Clinical characteristics of the selected pregnant women.

weeks) Systolic blood pressure (mm Hg) Newborn weight (g)
P indicates pregnant women with preeclampsia; N indicates normal pregnant women.XING PENG et al.MiR-518a-5p REPRESSES TROPHOBLAST INVASIONAn Acad Bras Cienc

Table II .
Binding of NFKB1 on miR-518a-5p promoter.Data were derived from Jaspar database.Relative profile score threshold is set as 80%.

Table III .
Binding of NFKB2 on miR-518a-5p promoter.Data were derived from Jaspar database.Relative profile score threshold is set as 80%.

Table IV .
Binding of RELA on miR-518a-5p promoter.Data were derived from Jaspar database.Relative profile score threshold is set as 80%.