| The tropoblast cell is the main component of the placenta, which plays a pivotal role in material exchange on the maternal-fetal interface. Early placental development occurs in a predominantly low oxygen environment. At 8-13 weeks of pregnancy, however, the invasion of tropoblast enhanced with pregnant time, the uterine vascular modifications are remodeling, and the increased of oxygen in order to maintain villous trophoblast implantation. Persistent hypoxia promotes a range of malignant responses in pregnancy such as proliferation, survival and apoptosis. A variety of diseases during pregnancy, such as preeclampsia(PE) and fetal growth restriction(FGR), have found shallow trophoblast invasion, thus lead to the placental blood perfusion is limited, shallow placental implantation and excessive apoptosis of pregnancy, eventually lead to adverse pregnancy outcome.SMG-1 belongs to a family of phosphoinositide 3-Kinase-related kinases(PIKK), which is a serine/threonine kinases. Initially, it is described as the main kinase involved in nonsense-mediated m RNA decay(NMD). In addition, more and more researches have found that SMG-1 is involved in cell proliferation, apoptosis and of many other physiological functions, and may be regulated in the hypoxic, DNA damage, ultraviolet radiation and other external stimuli environment. Recent studies have shown that SMG-1 plays a certain potential role in tumor suppression.Signal transducer and activator of transcription 3(Stat3) is an important intracellular transcription factor, The binding of Stat3 at receptor phosphotyrosine sites leads to the phosphorylation of tyrosine 705 in the C-terminal domain of Stat3, and this phosphorylation activates Stat3, tyrosine kinases, such as JAK and Src, leading to Stat3 phosphorylation via the tyrosine phosphorylation cascade. Phosphorylation of a single serine(Ser727) in the C-terminal transactivation domain of Stat3 by numerous serine kinases including m TOR, protein kinase C(PKC), and CDK5 allows for the maximal activation of transcription of responsive genes. The Stat3 family of transcription factors also controls numerous physiological processes including proliferation, inflammation, and is aberrantly expressed in pathological conditions such as cancer and preeclampsia. In recent years, it also found that Stat3 is involved in a variety of hypoxic cells and hypoxic animal models.People regard the tropoblast cell lines as the model of research for the tropoblast. In this report, we select JAR cells as the material, which belongs to human choriocarcinoma transformed cells, and derived from trophoblast cell lines. People choose full term or early abortion placental as the model of primary tropoblast cells. However, the tropoblast cell number of this term is diminished, and it’s hard to proliferate in vitro, adherent in ordinary culture medium. It is available to influence by environment make it limited in researches. More studies have selected human choriocarcinoma cell line as the research objects.Objective The aims of this study using Flow Cytometer, Real time PCR and Western Blot methods to investigate the impact of SMG-1 on the level of apoptosis on normal oxygen and/or hypoxia conditions, and the expression of Stat3 and p-Stat3 in choriocarcinoma JAR cell lines. In order to explore the relationship between SMG-1 and JAR cell apoptosis on the normal and/or hypoxia conditions.Materials and Methods 1 Materials JAR cell lines purchased from the Beijing Ding Guo Changsheng Biotechnology(ATCC, HTB-144) were maintained with high sugar complete medium(DMEM-H 10% FBS, 100U/ml penicillin, 100U/ml streptomycin) at 37°C under the moist atmosphere of air with 5%CO2. 2 Methods Human choriocarcinoma JAR cell lines were seeded into 6-well cell culture plate, When the cells was fused to 70%, JAR cells were cultured on the normal conditions(37℃、5% CO2,95% atmosphere) and/or hypoxia conditions which is published by Co Cl2. Grouped as follows, normal conditions: Transfected SMG-1 si RNA group, transfected scramble group(negative control) and blank control group; The hypoxia conditions: normal oxygen group and hypoxia group. Flow cytometry was used to detect the level of apoptosis. Using Real time-PCR and Western Blot methods to detect the m RNA expression of SMG-1 and Stat3, and the protein expression of SMG-1, Stat3 and p-Stat3. 3 Statistical analysis Statistical analysis was performed using the SPSS version 17.0 software package. All data were expressed as the `x±s. Real time-PCR were analysis by 2-ΔΔCT. when the data from the multi-group, statistical differences were calculated using one-way ANOVA. when the data from the two groups, statistical differences were calculated using calculated by comparing the means using Student’s t test. A α=0.05 was considered statistically significant.Results 1 Effect of SMG-1 on cell apoptosis rate of JAR cells under normal oxygen Silencing the gene expression of SMG-1 use transfected with SMG-1 si RNA. The JAR cell apoptosis rate were detected with flow Cytometry, and the analysis demonstrated that the apoptosis rate in the transfection group was(14.69±2.641)%, the blank control group was(5.43±1.043)%, and the negative control group was(7.26±1.214)%. The transfection group was significantly up-regulated compared to the blank control group and negative control group(P < 0.05). However, no significant difference was observed in the blank control group and negative control group(P>0.05). Thus the cell apoptosis rate was up-regulated when the SMG-1 gene was inhibited in JAR cell lines. 2 Effect of SMG-1 on Stat3 m RNA expression of JAR cells under normal oxygen Silencing the gene expression of SMG-1 use transfected with SMG-1 si RNA. The level of m RNA were detected by Real time PCR, the m RNA expression of SMG-1 in the si RNA group was 0.430±0.040, and the negative control group was 0.949±0.047. the transfection group was significantly down-regulated compared to the blank control group and negative control group(P < 0.05). And the gene expression of Stat3 was 0.667±0.069, the negative control group was 1.083±0.147, the m RNA expression of Stat3 in the si RNA group was also decreased compared with blank control group and negative control group(P<0.05). Thus the relative Stat3 m RNA expression was decreased when the SMG-1 gene was inhibited in JAR cell lines. 3 Effect of SMG-1 on Stat3 and p-Stat3 protein expression of JAR cells under normal oxygen Silencing the gene expression of SMG-1 use transfected with SMG-1 si RNA.Western blot analysis demonstrated that the protein expression of Stat3 in the transfection with transfection group was 1.0564±0.022, the blank control group was 1.789±0.046 and negative control group was 1.872±0.036. Comparing to the blank control group, the protein expression of Stat3 in the transfection group was significantly down-regulated(P<0.05); And the protein expression of p-Stat3 in the transfection group was 0.157±0.026, and the blank control group was 0.430±0.010, the negative control group was 0.326±0.043, Comparing to the blank control group, the protein expression of p-Stat3 in the si RNA group was also significantly down-regulated(P<0.05); However, no significant difference was observed between the blank control group and negative control group(P>0.05). Thus the relative Stat3 and p-Stat3 protein expression were decreased when the SMG-1 gene was inhibited in JAR cell lines. 4 Effect of cell apoptosis rate of JAR cells under hypoxia JAR cells were cultured in low oxygen, Flow Cytometry analysis demonstrated that the apoptosis rate in the low oxygen group was(17.83±2.041)%, the control group was(4.02±1.242)%. Comparing to the control group, the hypoxia group was significantly increased, the data was statistically significant(P<0. 05). Thus the cell apoptosis rate was up-regulated when the JAR cell lines were cultured in hypoxia condition. 5 Effect of SMG-1 and Stat3 m RNA expression of JAR cells under hypoxia JAR cells were cultured in low oxygen, the level of m RNA were detected by Real time PCR. Compared with control group, the m RNA expression of SMG-1 in the hypoxia group(3.569±0.264) was increased significantly,(P<0.05); And the m RNA expression of Stat3 in the hypoxia group(3.569±0.264) was also increased significantly compared with control group(P<0.05). Thus both of the relative SMG-1 and Stat3 m RNA expression were increased when the JAR cell lines were cultured in hypoxia condition.6 Effect of SMG-1, Stat3 and p-Stat3 protein expression of JAR cells under hypoxia JAR cells were cultured in low oxygen, Western blot analysis demonstrated that the protein expression of SMG-1 in the hypoxia group was 0.435±0.020, the control group was 0.260±0.003. Comparing to the control group, the low oxygen group was significantly increased, the data was statistically significant(P<0.05); while the protein expression of Stat3 was 1.274±0.0240, and the protein expression of p-Stat3 was 0.822±0.065 in the low oxygen group. Comparing to the control group, the protein expression of Stat3 and p-Stat3 in the low oxygen group was significantly decreased(P<0.05). Thus the relative SMG-1 protein expression was increased, while the relative Stat3 and p-Stat3 protein expression were decreased when the JAR cell lines were cultured in hypoxia condition.Conclusions Regulatory mechanism of hypoxia on gestational trophoblastic apoptosis is extremely complex. In our study, we have faound that both of SMG-1 and Stat3 were involved in the mechanism of hypoxia choriocarcinoma JAR cell lines. Inhibition of the SMG-1 could reduced the Stat3 and p-Stat3 expression, thus improving the level of apoptosis in JAR cell lines. |
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