This aim of the present study was to investigate the expression and function of claudin 3 (CLDN3) in pregnancy-induced hypertension. The mRNA expression levels of CLDN3 in the placental tissue and peripheral blood of patients with pregnancy-induced hypertension were measured using reverse transcription-quantitative PCR. Human trophoblast HTR8/SVneo cells overexpressing CLDN3 were generated using a lentiviral vector. Cell Counting kit-8 (CCK-8) assay, flow cytometry, Transwell chamber assays, confocal laser scanning microscopy and western blot analysis were performed to detect cell proliferation, invasion, migration and apoptosis, in addition to matrix metalloproteinase (MMP) expression and ERK1/2 phosphorylation. The mRNA expression levels of CLDN3 were significantly reduced in the placental tissues and peripheral blood samples of patients with pregnancy-induced hypertension compared with healthy pregnant controls. CLDN3 overexpression significantly increased HTR8/SVneo cell proliferation, invasion and migration whilst reducing apoptosis. HTR8/SVneo cells overexpressing CLDN3 also exhibited increased myofiber levels, increased MMP-2 and MMP-9 expression and increased ERK1/2 signaling activity. CLDN3 downregulation may be associated with the pathogenesis of pregnancy-induced hypertension. In conclusion, CLDN3 promotes the proliferative and invasive capabilities of human trophoblast cells, with the underlying mechanisms possibly involving upregulation of MMP expression via the ERK1/2 signaling pathway.
Pregnancy-induced hypertension is a common disease observed during the gestational period. If not treated in time, pregnancy-induced hypertension may cause significant harm to the mother and fetus (
Trophoblast cells are one of the components of the maternal placental architecture, involved in the regulation of placental microenvironment remodeling, implantation of embryos and normal fetal development (
Tight junction proteins are important in the maintenance of cell-to-cell connections, which serve important roles in cell polarity and the formation of cellular barriers such as the intestinal epithelial barrier (
A total of 51 pregnant women with hypertension, including 25 patients diagnosed with pregnancy-induced hypertension, 11 patients with mild pre-eclampsia and 15 patients with severe pre-eclampsia, and 30 normal pregnant women were included in this study, all of whom were admitted to Laiwu Maternal and Child Health Hospital (Laiwu, China) for delivery from December 2016 to December 2017. In these patients, the pregnancy hypertension was defined as: i) BP ≥140/90 mmHg during pregnancy, which returned to normal within 12 weeks after delivery; ii) urine protein (-); and iii) cases that may be associated with upper abdominal discomfort or thrombocytopenia. Mild pre-eclampsia was defined as: i) BP ≥140/90 mmHg appeared during pregnancy, which returned to normal within 12 weeks after delivery; ii) urine protein (-); and iii) cases that may be associated with upper abdominal discomfort or thrombocytopenia. Severe pre-eclampsia was defined as maternal convulsions that cannot be explained by other reasons. None of the pregnant women had previous pregnancies or suffered from hypertension, diabetes or underlying diseases, including liver, kidney and autoimmune diseases, prior to the present pregnancy. All these patients received cesarean section due to pregnancy-induced hypertension, abnormal fetal position or other social factors. There were no significant differences in age, gestational period or neonatal weight between the pregnancy-induced hypertension and control group (mean age of patients with pregnancy-induced hypertension, 29.5±1.08 years; mean gestational period, 38±2.08 weeks; mean neonatal weight, 7.6±2.3 kg. Mean age of patients with mild-pre-eclampsia, 33.1±1.08 years; gestational period, 39±3.08 weeks; neonatal weight, 7.1±1.3 kg. Mean age of patients with severe pre-eclampsia, 31.45±1.08 years; gestational period, 40±2.60 weeks; neonatal weight, 7.7±1.7 kg). Prior written informed consent was obtained from every patient and the study was approved by the ethics review board of Laiwu Maternal and Child Health Hospital. In total 10 ml peripheral blood sample was obtained from each subject and the collection of placental tissues was performed 10 min after the placenta was delivered, with a 2x2x2 cm sample of tissue dissected from the central area (avoiding infarction and calcification) of the maternal placenta. The tissue was washed with saline immediately after collection, and then stored at -80˚C.
Total RNA was extracted from the placental tissue and peripheral blood samples using TRIzol® (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RNA concentrations were quantified using the NanoDrop method. Reverse transcription was performed with 0.5 µg RNA to obtain cDNA using BeyoRT™ cDNA First Chain Synthesis kit (cat. no. D7166; Beyotime Institute of Biotechnology). Subsequent qPCR was performed using BeyoFast™ SYBR Green qPCR Mix (cat. no. 7260; Beyotime Institute of Biotechnology) in a StepOnePlus™ Real-Time PCR instrument. The following primer sequences were used: CLDN3 forward, 5'-GCCACCAAGGTCGTCTACTC-3' and reverse, 5'-CCTGCGTCTGTCCCTTAGAC-3' and GAPDH forward, 5'-CGGAGTCAACGGATTTGGTCGTAT-3' and reverse, 5'-AGCCTTCTCCATGGTGGTGAAGAC-3'. The total 20 µl PCR mixture consisted of 10 µl RT-qPCR-Mix, 0.5 µl each primer, 2 µl cDNA and 7 µl ddH2O. The thermocycling conditions were as follows: Initial denaturation at 95˚C for 10 min; followed by 40 cycles of 95˚C for 1 min and 60˚C for 1 min. Target gene expression levels were calculated using the 2-ΔΔCq method (
Normal human trophoblast HTR8/SVneo cells were purchased from American Type Culture Collection. Cells were cultured using RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and supplemented with 100 U/ml penicillin and 100 U/ml streptomycin, in a humidified atmosphere at 37˚C containing 5% CO2. When cell confluence reached 90%, the cells were passaged using 0.5% trypsin.
The lentiviral Lv-GFP-CLDN3 vector was constructed by Hanbio Biotechnology Co., Ltd., with the titer of 1x108 pfu. HTR8/SVneo cells were first seeded into 24-well plates at a density of 1x105 cells/well and cultured with RPMI-1640 medium containing 10% FBS. When 70% confluence was achieved, the HTR8/SVneo cells were transfected with either empty vector or Lv-GFP-CLDN3 at a multiplicity of infectionof 20. The medium was changed to fresh RPMI-1640 medium containing 10% FBS after 6 h, and the cells were cultured at 37˚C under 5% CO2 for a further 48 h.
CCK-8 assay (Beyotime Institute of Biotechnology) was performed to measure the viability of the HTR8/SVneo cells. Briefly, at 48 h after transfection, the cells were seeded into 96-well plates at 2x103 cells/well. A total of 200 µl RPMI-1640 complete medium containing 10% FBS and 100 U/ml penicillin and streptomycin was added and the cells were then incubated at 37˚C under 5% CO2. Following cell adhesion, at 0, 24, 48 and 72 h, the cells were incubated with 20 µl CCK-8 reaction solution for 30 min. The optical density values at 490 nm were then obtained for each well using a microplate reader, which were used to produce a cell viability curve.
Flow cytometry was performed to measure HTR8/SVneo cell proliferation and apoptosis. For cell proliferation, the cells were collected and washed with PBS. Following fixation with 4% formaldehyde for 10 min at room temperature, the cells were centrifuged at 1,000 x g for 5 min at room temperature. The cells were treated with 0.5% Triton X-100 at room temperature for 5 min and then incubated with BD Cytofix/Cytoperm™ Plus reagent (cat. no. 555028; BD Biosciences) at room temperature in the dark for 15 min. Fluorescence was detected and the percentage of Ki-67 calculated from it using the BD FACSVerse™ flow cytometer (BD Biosciences) and the FlowJo™ VX10 software (FlowJo LLC).
For apoptosis detection, 48 h after transfection, 1x106 HTR8/SVneo cells were collected and cultured with a shaker for 2 h at a speed of 200 rpm. After washing twice with ice-cold PBS, the cells were labeled using the BD Pharmingen™ FITC Annexin V Apoptosis Detection Kit I (cat. no. 556547; BD Biosciences), according to the manufacturer's protocol. Flow cytometry was conducted to detect the fluorescence, from which apoptosis rate was calculated.
For Ki-67 detection, the cells were collected and treated with BD Cytofix/Cytoperm™ Plus reagent (cat. no. 555028; BD Biosciences) according to the manufacturer's protocols. The cells were then incubated with PE-Cy™7-conjugatedmouse anti-Ki-67 antibody (cat. no. 561283; BD Biosciences) in the dark at room temperature for 15 min, followed by detection of the fluorescence with flow cytometry.
The infiltration ability of the cells was detected using Transwell chamber assays. Matrigel® solution was diluted in serum-free RPMI-1640 medium (v:v, 1:3), evenly smeared onto the upper chamber and incubated at 37˚C for 60 min. The HTR8/SVneo cells were then seeded onto the upper chamber at a density of 1x105 cells/well and cultured in 200 µl serum-free RPMI-1640 medium while 500 µl RPMI-1640 medium containing 20% FBS was present in the lower chamber. After 24 h, the cells were fixed with 4% formaldehyde at room temperature for 10 min and subjected to Giemsa staining at room temperature for 2 min. Following rinsing for 2 min, the cells were air-dried. HTR8/SVneo cells that infiltrated to the lower chamber were observed and counted under a light microscope, from a total of five random fields of view under high magnification (x200). For the measurements of cell migration, identical procedures were performed as described aforementioned using Transwell chambers that were not coated with Matrigel.
The cytoskeleton was imaged using CLSM. After transfection, when 80% confluence was reached, HTR8/SVneo cells were fixed with 4% formaldehyde at room temperature for 10 min. After washing with PBS, the cells were permeabilized using 0.5% Triton X-100 at room temperature for 5 min and incubated with 200 µl 100 nmol/l Rhodamine Phalloidin at room temperature in the dark for 30 min. The cytoskeleton was then observed using a confocal microscope (no. of fields taken, 5; magnification, x400; model SP8; Leica Microsystems GmbH).
At 48 h after transfection, the cells were lysed using RIPA buffer (Beyotime Institute of Biotechnology) supplemented with PMSF (100 mM) on ice. The protein concentration was determined using Bicinchoninic Acid assay (Beyotime Institute of Biotechnology). Then, 20 µg sample was separated by 10% SDS-PAGE, and then transferred onto PVDF membranes. After blocking with 50 g/l fat-free milk diluted in 0.1% TBST at room temperature for 1 h, the membranes were incubated with rabbit anti-human anti-CLDN3 (1:1,000 dilution; cat. no. 83609), rabbit anti-human anti-matrix metalloprotease (MMP)-2 (1:1,000 dilution; cat no. 40994), rabbit anti-human anti-MMP-9 (1:1,000 dilution; cat no. 13667), rabbit anti-human anti-ERK1/2 (1:1,000 dilution; cat no. 5013), mouse anti-human anti-p-ERK1/2 (1:1,000 dilution; cat. no. 9106) or rabbit anti-human anti-GAPDH (1:5,000 dilution; cat no. 5174) primary antibodies (all from Cell Signaling Technology, Inc.) at 4˚C overnight. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit (cat. no. A0208) or goat anti-mouse (cat. no. A0216) secondary antibody (both 1:4,000 dilution; Beyotime Institute of Biotechnology) at room temperature for 1 h. Protein bands were visualized using the ECL method (Pierce; Thermo Fisher Scientific, Inc.) and analyzed using the Quantity one V4.6.7 software (Bio-Rad Laboratories, Inc.).
Data are presented as the mean ± SD. SPSS 20.0 software (IBM Corp.) was used for statistical analysis. Pairwise comparisons were performed using the Student's t-test, whereas one-way ANOVA followed by Dunnett's test was used for multiple group comparisons. P<0.05 was considered to indicate a statistically significant difference.
To investigate the expression of CLDN3 in the placenta of the pregnant patients with and without hypertension, RT-qPCR was performed. Compared with the healthy control group, the expression levels of CLDN3 mRNA in the placental tissue were significantly reduced in the pregnant patients with hypertension (P<0.01;
The effects of CLDN3 on the viability of HTR8/SVneo cells were next investigated. At 48 h after transfection with Lv-GFP-CLDN3, GFP fluorescence was distributed evenly in the cytoplasm of the HTR8/SVneo cells (
To investigate the effect of CLDN3 on the invasive and migratory abilities of HTR8/SVneo cells, Transwell chamber assays were performed. Compared with the control group, CLDN3 overexpression significantly promoted the migratory and invasive capabilities of HTR8/SVneo cells (P<0.05;
Apoptosis is also an important factor affecting the migratory and invasive abilities of HTR8/SVneo cells (
The ERK1/2/MMP pathway serves an important role in the cell migration and is regulated by tight junction proteins (
Pregnancy-induced hypertension is a common complication in obstetrics (
The placenta is the location where gas and nutrient exchange occurs and where metabolic products are eliminated between the mother and the fetus. As part of the main placental structure, invasion of trophoblast cells into the endometrium is an important physiological process in the formation of the placenta (
Tight junction proteins, of which the CLDN family of proteins is an important example, serve important roles in the maintenance of cell barriers and polarity. In recent years, a large number of studies have shown that CLDNs are closely associated with tumor cell invasion and metastasis (
It has previously been found that human trophoblast cells secrete MMPs to degrade the extracellular matrix during invasion (
However, in the present study, although the expression levels of CLDN3 in the placental tissue and the effects on the biological function of trophoblast cells were investigated, only a single trophoblast cell line was used, and further in-depth studies on primary trophoblast cells are required to confirm the regulatory effects of CLDN3.
In conclusion, the present study showed that CLDN3 can promote human trophoblast cell proliferation, migration and invasion, with the underlying mechanism possibly involving the upregulation of MMP-2 and MMP-9 expression levels via the ERK1/2 signaling pathway. These findings suggest that downregulation of CLDN3 may be associated with the pathogenesis of pregnancy-induced hypertension, which may serve to assist in the design of therapeutic interventions for the treatment of this disease in the clinic.
The authors thank Director Ailan Wang from the Laiwu Maternal and Child Health Hospital (Shandong, China) for kind assistance in the study design, experiment performance, data analysis and manuscript preparation.
No funding was received.
All data generated or analyzed during this study are included in this published article.
AZ, YQ and KL contributed to the study design, experiment performance, data collection and analysis, and manuscript preparation. All authors read and approved the final manuscript.
The study was approved by the ethics review board of Laiwu Maternal and Child Health Hospital (Shandong, China). Prior written informed consent was obtained from every patient.
Not applicable.
The authors declare that they have no competing interests.
Expression levels of CLDN3 mRNA in the placental tissues and peripheral blood of patients. The mRNA expression levels of CLDN3 in (A) placental tissues and (B) peripheral blood of normal control, pregnancy-induced hypertension, mild pre-eclampsia and severe mild pre-eclampsia groups were measured using reverse transcription quantitative PCR. Experiments were performed in triplicates. *P<0.05 vs. normal. CLDN3, claudin 3.
Effects of CLDN3 overexpression on the proliferation of HTR8/SVneo cells. (A) After transfection with Lv-GFP-CLDN3, GFP fluorescence was detected using confocal laser scanning microscopy (magnification, x200). (B) CLDN3 overexpression in HTR8/SVneo cells was verified using western blot analysis. (C and D) Following transfection, cell viability was determined using (C) Cell Counting kit-8 assay and (D) proliferation was assessed via the quantification of Ki-67 using flow cytometry. Experiments were performed in triplicates. *P<0.05 vs. NC. CLDN3, claudin 3; GFP, green fluorescent protein; NC, cells transfected with empty vector; OD, optical density; SSC, side scatter; PE, phycoerythrin.
Effects of CLDN3 overexpression on HTR8/SVneo cell invasion and migration. (A) The invasive and migratory capabilities of transfected HTR8/SVneo cells were measured using Transwell chamber assays. (B) The F-actin cytoskeleton (red fluorescence) was imaged using confocal laser scanning microscopy. Scale bar, 2 µm. *P<0.05 vs. NC. CLDN3, claudin 3; NC, cells transfected with empty vector.
Effects of CLDN3 overexpression on the apoptosis of HTR/SVneo cells. The apoptosis of transfected HTR/SVneo cells was detected using flow cytometry and the apoptotic rates were calculated accordingly. Experiments were performed in triplicates. *P<0.05 vs. NC. CLDN3, claudin 3; FITC, fluorescein isothiocyanate; PI, propidium iodide. NC, cells transfected with empty vector.
Effects of CLDN3 on MMP expression and ERK phosphorylation in HTR8/SVneo cells. Following transfection, the expression levels of MMP-2 and MMP-9 proteins, in addition to ERK1/2 phosphorylation were detected using western blot analysis. Experiments were performed in triplicates. *P<0.05 vs. NC. CLDN3, claudin 3; MMP, matrix metalloproteinase; NC, cells transfected with empty vector.