| Preeclampsia(PE)is generally defined as a new hypertension(diastolic blood pressure of ?90 mm Hg)and substantial proteinuria(?300 mg in 24 h)at or after 20 weeks' gestation complicating 2–8% of pregnancies and is a major contributor to maternal mortality worldwidealong with the other hypertensive disorders of pregnancy.Altho?gh the mechanism of PE has not yet been fully elucidated,due to the symptoms of PE reduced or disappeared with the delivery of the placenta,so the placenta is thought to play a key role in the pathogenesis of PE.The main cells consisting of placenta is throphoblasts which include villous trophoblastsand extrovillous trophoblast(EVT).During pregnancy,EVT invades into the uterine tissue and migrates to the maternal uterine spiral to displace and replace the endothelial cell lining of the spiral arteries.This remodeling of the spiral arteries plays a essential role in the formation and development of the placenta.Many studies demonstrate that adequate invasion of EVT causes shallow implantation and insufficient remodeling of vascular,which leads to several diseases of pregnancy,including PE.Autophagy is a process of self-degradation of cellular components to maintain cellular homeostasis,which provides energy for cell survival under adverse condition such as hypoxia,immune injury and starvation.However,both adequate autophagy and excessive autophagy affect the cell function and involve in the pathogenesis of many diseases.Several tudies have shown that the expression of LC3-II protein was increased in the placentas from PE compared with the placentas from normal pregnancies,this reveals that increased autophagic activity in placenta is associated with the pathogenesis of PE.The expression of micro RNA-34a(miR-34a)decreased in the placentas from PE,and research in the human retinoblastoma cells has identified that miR-34 a suppresses autophagy by targeted regulation of HMGB1.Nevertheless,the relationship among miR-34 a and HMGB1 and autophagy in PE has not been reported.Objective To investigate the effect of miR-34 a on regulating HMGB1 expression and autophagy in human trophoblast cells,then to explore the possible mechanism of miR-34 a involved in the pathogenesis of preeclampsia.Materials and methods 1 Materials JAR and JEG-3 cells purchased from the Beijing Ding Guo Chang Sheng Biotechnology were cultured with RPMI1640 medium at 37?under the saturated humidity of the air with 5% CO2.2 Methods JAR and JEG-3 cells were cultured with RPMI1640 medium at 37?under the saturated humidity of the air with 5% CO2,then cells were digested and seeded to 6-well cell culture plate,when the cells fused to 70%,they were divided into two part for experiments.2.1 Effect of miR-34 a on expression of HMGB1 in JAR and JEG-3 cells JAR and JEG-3 cells were divided into five groups,respectively:miR-34 a mimics group(transfected with miR-34 a mimics),NC group(transfected with miR-34 a mimics negative control),miR-34 a inhibitors group(transfected withmiR-34 a inhibitors),INC group(transfected withmiR-34 a inhibitors negative control)and blank control group(transfected with nothing).Real time PCR and Western blot were used to detect the expression level of HMGB1 m RNA and HMGB1 protein.2.2 Effect of hypoxia on autophagy in JAR and JEG-3 cells JAR and JEG-3 cells were divided into two groups:normal group(without Co Cl2),hypoxia group(treated with 300?mol/l Co Cl2).Western blot were used to detect the expression level of HMGB1 and LC3 protein,transmission electron microscopy was used to observe the number of autophagic vesicles.2.3 Effect of miR-34 a on autophagy in JAR and JEG-3 cells JAR and JEG-3 cells were divided into four groups,respectively:miR-34 a mimics group,NC group,miR-34 a inhibitors group and INC group,48 hours later,Co Cl2 was used to treated cells for 2 hours at the concentration of 300?mol/L,then to detect the expression level of HMGB1 and LC3 protein by Western blot,transmission electron microscopy was used to analyze the change of the number of autophagic vesicles.3 Statistics analysis Statistical analysis was performed using the SPSS version 21.0 software package.All the result were presented as mean ± standard deviation(SD).One-way ANOVA was used for data from multi-group,when the data from the two groups,statistical differences were calculated using calculated by comparing the means using Student's t test.?=0.05 was considered statistically significant.Result1 Expression of HMGB1 m RNA and protein in JAR and JEG-3 cells after transfected with miR-34 a mimics and inhibitors The expression level of HMGB1 m RNA and protein in JAR cells:compared with blank control group(1.01±0.15,1.01±0.10)and NC group(1.11±0.34,0.91±0.08),which were decreased in miR-34 a mimics group(0.37±0.13,0.59±0.12)and the difference had statistical significance(P<0.05).Compared with blank control group and INC group(0.99±0.16,1.03±0.12),which were increased in miR-34 a inhibitors group(1.62±0.25,1.47±0.13)and the difference had statistical significance(P<0.05).The expression level of HMGB1 m RNA and protein in JEG-3 cells: compared with blank control group(1.00±0.13,0.97±0.16)and NC group(1.00±0.15,0.92±0.13),which were decreased in miR-34 a mimics group(0.56±0.15,0.64±0.06).Compared with blank control group and INC group(1.01±0.14,1.04±0.07),which were increased in miR-34 a inhibitors group(2.12±0.59,1.37±0.05)and the difference had statistical significance(P<0.05).2 Expression of HMGB1 and LC3 protein and the number of autophagic vesicles in JAR and JEG-3 cells under hypoxia In JAR cells,the expression of HMGB1 protein and LC3?/ LC3? protein in hypoxia group(1.20±0.10,1.06±0.08)was increased than that in normal group(0.62±0.07,0.45±0.13)and the difference had statistical significance(P<0.05).The number of autophagic vesicles in hypoxia group(0.90±0.87)was increased than that in normal group(0.20±0.42)and the difference had statistical significance(P<0.05).In JEG-3 cells,the expression of HMGB1 protein and LC3?/ LC3? protein in hypoxia group(1.32±0.21,1.23±0.97)was increased than that in normal group(0.70±0.15,0.65±0.19),the number of autophagic vesicles in hypoxia group(1.20±1.03)was increased than that in normal group(0.10±0.32)and the difference had statistical significance(P<0.05).3 Expression of HMGB1 and LC3 protein in JAR and JEG-3 cells after transfected with miR-34 a mimics and inhibitors The expression of HMGB1 protein and LC3?/ LC3?in JAR cells: Compared with NC group(1.04±0.04,1.01±0.03),which was suppressed in miR-34 a mimics group(0.56±0.12,0.48±0.02)and the difference had statistical significance(P<0.05).Compared with INC group(0.82±0.17,0.99±0.06),which was increased in miR-34 a inhibitors group(1.70±0.24,1.48±0.09)and the difference had statistical significance(P<0.05).The expression of HMGB1 protein and LC3?/ LC3?in JEG-3 cells: Compared with NC group(1.24±0.28,1.12±0.12),which was suppressed in miR-34 a mimics group(0.48±0.18,0.32±0.87).Compared with INC group(1.46±0.21,1.07±0.11),which was increased in miR-34 a inhibitors group(2.13±0.27,1.52±0.15)and the difference had statistical significance(P<0.05).4 Autophagic vesicles in each group of JAR and JEG-3 cells after transfected with miR-34 a mimics and inhibitors The number of autophagic vesicles in JAR and JEG-3 cells:Compared with NC group(0.70±0.82,1.00±0.82),which were decreased in miR-34 a mimics group(0.10±0.32,0.10±0.31),but compared with INC group(0.40±0.52,0.50±0.53)which were increased in miR-34 a inhibitors group(1.40±0.84,1.50±1.08)and the difference had statistical significance(P<0.05).Conclusion miR-34 a can regulate the expression of HMGB1 and affect the cell autophagy in JAR and JEG-3 cells. |
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