ABSTRACT
Objective:To investigate the changes of ROS/TXNIP/NLRP3 signaling pathway in pyroptosis of human embryonic trophoblast cells induced by high glucose.Methods:Human embryonic trophoblast cells were cultured in vitro to establish high glucose injury model, and they were randomly divided into control group, high glucose (HG) group and HG + ROS inhibitor N-acetyl-L-cysteine (HG + NAC) group. MTT assay was used to detect the cell survival rate. The level of ROS in each group was detected by dihydroethidine ROS fluorescence probe. Expression of TXNIP and NLRP3 mRNA was detected by real-time quantitative PCR (RT-qPCR). Western blot analysis was used to detect the expression levels of TXNIP, NLRP3, Caspase-1, interleukin (IL) -1β, tumor necrosis factor-α (TNF-α) and GSDMD proteins. In addition, pyroptosis was detected by flow cytometry.Results:The optimal glucose concentration for high glucose-induced injury of human embryonic trophoblast cells was 30 mmol/L. Compared with the control group (96.27±3.10) %, the survival rate of human embryonic trophoblast cells in HG group (55.44±2.15) % was significantly lower ( P<0.05), while the fluorescence intensity (ROS level) of 7 'dichlorofluorescein (DCF), the expression levels of TXNIP and NLRP3 proteins, the number of pyroptosis, expression levels of Caspase-1, GSDMD, IL-1β and TNF-α proteins were significantly higher ( P<0.05) ; Compared with HG group, the survival rate of human embryonic trophoblast cells in HG+NAC group (84.75±2.33) % was significantly higher ( P<0.05), the fluorescence intensity (ROS level) of DCF, the expression levels of TXNIP and NLRP3 proteins, the number of pyroptosis, and expression levels of Caspase-1, GSDMD, IL-1β and TNF-α proteins were significantly lower ( P<0.05) . Conclusion:Inhibition of ROS level in human embryonic trophoblast cells induced by high glucose may promote cell proliferation and reduce the occurrence of pyroptosis by inhibiting TXNIP/NLRP3 signaling pathway.
ABSTRACT
OBJECTIVE: This study aims to identify the effect of miR-146a-5p on trophoblast cell invasion as well as the mechanism in preeclampsia (PE). METHODS: Expression levels of miR-146a-5p and Wnt2 in preeclamptic and normal placentae were quantified. Trophoblast cells (HTR-8) were separately transfected with miR-146a-5p mimic, miR-146a-5p inhibitor, pcDNA3.1-Wnt2 or sh-Wnt2, and then the expression levels of miR-146a-5p, Wnt2, and epithelial-mesenchymal transition (EMT)-related proteins (Vimentin, N-cadherin and E-cadherin) were measured. Moreover, the proliferative, migratory and invasive capacities of trophoblast cells were detected, respectively. Dual luciferase reporter assay determined the binding of miR-146a-5p and Wnt2. RESULTS: Compared with normal placental tissues, the placentae from PE patients showed higher miR-146a-5p expression and lower Wnt2 expression. Transfection of miR-146a-5p inhibitor or pcDNA3.1-Wnt2 exerted pro-migratory and pro-invasive effects on HTR-8 cells and encouraged EMT in HTR-8 cells; transfection with miR-146a-5p mimic or sh-Wnt2 weakened the proliferative, migratory and invasive capacities as well as reduced EMT process of HTR-8 cells. Moreover, Wnt2 overexpression could partially counteract the suppressive effects of miR-146a-5p overexpression on the progression and EMT of HTR-8 cells. CONCLUSION: miR-146a-5p mediates trophoblast cell proliferation and invasion through regulating Wnt2 expression.
Subject(s)
Humans , Female , Pregnancy , Pre-Eclampsia , Trophoblasts/cytology , MicroRNAs/genetics , Epithelial-Mesenchymal Transition , Placenta , Cell Movement , Cell ProliferationABSTRACT
OBJECTIVE@#To investigate the effect of vitamin D on microRNA-21(miR-21) expression and migration and invasion of human placental trophoblast cells.@*METHODS@#The changes in the expression of miR-21 were detected using RT-qPCR in HTR-8/SVneo cells following stimulation by vitamin D at different doses for 24, 48 and 72 h.HTR-8/SVneo cells transfected with miR-21 mimic or inhibitor with or without vitamin D treatment were examined for changes in cell migration and invasion abilities using Transwell assay, and Western blotting was used to detect protein expressions of E-cadherin, fibronectin, and MMP9.@*RESULTS@#Vitamin D obviously inhibited the expression of micoRNA-21 in HTR-8/SVneo cells in a concentration-and time-dependent manner.Transfection with the miR-21 mimic significantly inhibited the migration and invasion of HTR-8/SVneo cells, and this inhibitory effect was abolished by treatment with vitamin D; transfection with miR-21 inhibitor obviously promoted the migration and invasion of HTR-8/SVneo cells, and these effects were not significantly affected by vitamin D treatment.@*CONCLUSIONS@#Vitamin D may promote trophoblast cell migration and invasion to accelerate the development of preeclampsia by down-regulating the expression of miR-21.
Subject(s)
Female , Humans , Pregnancy , Cell Movement , MicroRNAs , Genetics , Placenta , Pre-Eclampsia , Trophoblasts , Vitamin DABSTRACT
Aim: To explore the role of apoptosis stimulating protein 2 of p53 (ASPP2) on L-NAME induced apoptosis of placental trophoblast cells by regulating glucose-regulated protein78(GRP78), and provide a theoretical basis for the study of clinical pregnancy-induced hypertension. Methods: The HTR-8/SVneo human placental trophoblast cells were cultured in vitro, and in the absence (control group) or presence of 100 μmol · L-1 L-NAME (L-NAME group) for 48 h. The effects of L-NAME on placental trophoblast cell apoptosis were tested using flow cytometry and AO/EB assay. The expressions of caspase-12, GRP78 and ASPP2 were detected by Western blot. The ASPP2 interference with adenovirus was used to transfect the cells, and the mRNA expression level of ASPP2 and the protein expression level of GRP78 were detected by qRT-PCR and Western blot, respectively. After treated with 100 (xrnol · L-1 L-NAME for 48 h, the protein expression of caspase-12 and GRP78 was detected by Western blot and immunofluorescence. Results: Compared with control group, the placental trophoblast cell apoptosis significantly increased in L-NAME group (P < 0. 05). AO/EB staining showed that compared with control group, the majority of cells in L-NAME group showed bright orange and the number of late apoptotic cells increased significantly. At the same time, caspase-12, GRP78 and ASPP2 protein expression increased (P < 0. 05, P < 0. 01). After interfering with ASPP2, caspase-12 and GRP78 protein expressions decreased (P < 0. 05). Conclusions: Down-regulation of ASPP2 could decrease GRP78 expression and inhibit L-NAME-induced apoptosis in placental trophoblast cells.
ABSTRACT
Objective: To investigate the effect of activation of SHH signaling pathway on the apoptosis and invasion of the trophoblast cells in the preeclampsia (PE) patients, and to clarify its mechanism. Methods: The HTR8/SVneo cells were divided into normal control group, hypoxia/reoxygenation (H/R) treatment group and purmorphamine+H/R treatment group. The expression levels of SHH signaling pathway related proteins SHH, Ptchl, Smo, Glil, and Gli3 in the placenta tissues of PE and late trimester of normal pregnancy were detected by Western blotting method; the apoptotic rates of HTR8/SVneo cells in various groups were detected by flow cytometry (FCM); Transwell assay was used to detect the number of transmembrane cells; the expression levels of matrix metalloproteinase-2 (MMP-2) and metalloproteinase-9 (MMP-9) proteins in the HTR8/SVneo cells in various groups were detected by Western blotting method. Results: The expression levels of SHH, Ptchl, Smo, Glil and Gli3 proteins in the placenta tissue of PE were significantly lower than those in normal pregnancy placenta tissue (P<0. 05); the expression levels of SHH, Ptchl, Smo, Glil, Gli3, MMP-2, and MMP-9 proteins and the number of transmembrane cells in H/R treatment group were significantly lower than those in normal control group (P
ABSTRACT
Objective · To investigate the effects of growth arrest and DNA damage 45 alpha (Gadd45α) on the migration and invasion function of human extravillous trophoblast cells under hypoxia/re-oxygenation (H/R). Methods · Human extravillous trophoblast cells were infected by shRNA lentivirus targeting Gadd45α gene, to knock down Gadd45α gene expression. Then the oxidative stress model of preeclampsia was used in vitro to observe the changes of cell biological functions. The experiments were divided into 4 groups, nontreated group, hypoxia/re-oxygenation group, shRNA Gadd45α+H/R group and shRNA negative control+H/R group. Human villous explant experiments were used to determine the effects of silencing Gadd45α on human extravillous trophoblast cell under oxidative stress. Protein expression of Gadd45α was identified by Western blotting. Changes of cell migration and invasion were detected by transwell migration and Matrigel invasion assay. Gelatin zymography was used to detect the expression of matrix metalloproteinase (MMP) -2/9 in culture medium. Results · Hypoxia/re-oxygenation can increase the expression of Gadd45α in HTR8/SVneo cells and damage the trophoblast cell migration and invasion. Knocking down Gadd45α can increase the activities of MMP2/9, which can increase the cell migration and invasion. Conclusion · Knockdown of Gadd45α gene has promoted cell migration and invasion function of human extravillous trophobalst cells under oxidative stress.
ABSTRACT
Objective:To investigate the effect on the CXCR4/PI3K/AKT pathway after the transfection of miR-155 mimics and miR-155 inhibitor combined with the research on the ability of invasion and migration of human chorionic JEG-3 trophoblast cells. Methods:Chemically synthesized miR-155 mimics and miR-155 inhibitor were transfected into JEG-3 cells. The effect on the ability of invasion and migration were analyzed by Transwell migration assay and Wound healing assay. The expression of CXCR4 mRNA was detected by Real-time PCR. The expression of CXCR4 and p-AKT protein were detected by Western blot. Results: Transfection with miR-155 mimics significantly down-regulated the expression of CXCR4 as compared with the control group(P<0. 05);JEG-3 cells transfected miR-155 mimics had lower levels of migration and invasion capacity than cells in the control group(P<0. 05). However, transfection with miR-155 inhibitor significantly up-regulated the expression of CXCR4 as compared with the control group(P<0. 05);JEG-3 cells transfected miR-155 inhibitor had higher levels of migration and invasion capacity than cells in the control group ( P<0.05).Addition,the expression of p-AKT protein of JEG-3 cells was down-regulated after transfected miR-155 mimics,and the expression of p-AKT protein of JEG-3 cells was up-regulated after transfected miR-155 inhibitor. Conclusion:miR-155 may inhibits the invasion and migration of trophoblast cells by regulating CXCR4/PI3K/AKT pathway contributing to the development of preeclampsia.
ABSTRACT
Objective · To investigate the effects of a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 on biological function changes of human extravillous trophoblast cells induced by hypoxia/re-oxygenation (H/R). Methods · In-vitro cultured early pregnancy villus explants and human extravillous trophoblast cell line HTR8/SVneo were assigned to 4 groups according to different interventions, i.e. control group, SB203580 group (p38 MAPK inhibition), H/R group (simulation of preeclampsia by the oxidative stress model), and SB203580+H/R group. The effects of SB203580 on biological functions of human extravillous trophoblast cells under oxidative stress in early pregnancy villus explants were observed. Protein expression and phosphorylation of p38 MAPK in HTR8/SVneo cells were measured with Western blotting. Cell migration and invasion were observed with Transwell migration and Matrigel invasion assay, respectively. Gelatin zymography was used to measure the activity of matrix metalloproteinase (MMP) -2/9 in supernatant. ELISA was used to detect the levels of soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng). Results · SB203580 could promote the exogenous migration of human extravillous trophoblast cells in early pregnancy villus explants under oxidative stress. H/R could decrease the migration and invasion of HTR8/SVneo cells , and increase the phosphorylation level of p38 MAPK in HTR8/SVneo cells and the secretion of sFlt-1 and sEng. SB203580 could increase the activity of MMP2/9 in supernatant and cell migration and invasion, decrease the phosphorylation level of p38 MAPK in HTR8/SVneo cells under oxidative stress and the secretion of sFlt-1 and sEng. Conclusion · SB203580 can protect biological functions of human extravillous trophobalst cells under oxidative stress.
ABSTRACT
Human trophoblast cells were isolated and cultured in vitro in order to investigate possible pathogenesis of intrauterine infection caused by HCMV.Trophoblast cells were obtained by compound enzymes digestion and discontinuous percoll gradient.Cells and purity were identified by using immunocytochemistry assay with anti-CK7,Vim and β-hCG antibodies.HCMV AD169 strain replication in isolated trophoblast cells and cell apoptosis were detected at different time points post infection(p.i.).The results showed that highly purified trophoblast cells were obtained.Specific virus replication was increased dramatically at the 24th h p.i.,and then increased slowly during 48 h and 72 h.Apoptosis rate of trophoblast cells infected with HCMV was(34.68±3.14)% at 24th h p.i.,while that in control group was(15.32±2.34)%(P<0.05).It was suggested that highly purified trophoblast cells can be isolated by the simplified cell purification method.HCMV can infect human trophoblast cells,and be quickly replicated,resulting in the accelerated apoptosis of human trophoblast cells during early time.
ABSTRACT
m trophoblast cells stimulated by LPS.
ABSTRACT
AIM:To explore whether cyclosporine A (CsA) can regulate the expression of nometastatic gene23 H1 type (nm23-H1) in human choriocarcinoma Bewo cells,in order to seek new proof of treating trophoblast diseases.METHODS:The Bewo cells were divided into two groups. The vehicle control group,and the CsA group with different concentrations from 10-2 ?mol/L to 10 ?mol/L. The effect of CsA on the transcription of nm23-H1 was determined by reverse transcription-polymerase chain reaction (RT-PCR) after cultured for 48 h and protein level of nm23-H1 was determined by In-cell Western after cultured for 72 h.RESULTS:Compared to the vehicle group,CsA significantly downregulated the transcription and translation of nm23-H1 in a dose-dependent manner from 10-2 ?mol/L to 10 ?mol/L,and the inhibition reached its top when concentration of CsA was 1.0 ?mol/L (P