| Objective:To investigate the effects of CoCl2-induced hypoxia in the proliferation ability of choriocarcinoma cell line JEG-3in vitro. Then establish the hypoxic model of JEG-3cells in vitro.Explore the possible causes that cobalt chloride-induced hypoxia affect invasion of the choriocarcinoma JEG-3cell line.Methods:(1) Choriocarcinoma JEG-3cell line was incubated in cell culture mediums with different concentrations of CoCl2(the final concentrations of CoCl2was0,50,100,150,200μmol/l,respectively) for different time (24,48,72,96hours,respectively).Then CCK-8assay was used to examine the proliferation of JEG-3cell line.(2) Choriocarcinoma JEG-3cell line was incubated with150μmol/l CoCl2for24hours,48hours and72hours respectively to establish different degrees of hypoxia models of choriocarcinoma JEG-3cell line. The expressions of HIF-la and VEGF at the levels of transcription and translation were analyzed by real-time PCR and western blotting. Transwell method was performed to evalutate the invasiveness of the hypoxia model of choriocarcinoma JEG-3cell line.Results:(1) CCK-8assay showed that CoCl2could promote the growth and proliferation of choriocarcinoma JEG-3cell line with a concentration and time-dependent manner in some certain concentrations(<150μmol/l) and within72hours.With the concentration increasing (up to200μmol/l) or the time extending(96h),the proliferation of JEG-3cells was inhibited.(2) Real-time PCR results showed that CoCl2(150μmol/l)did not stimulate mRNA expression of HIF-la in72hours. Compared with the mRNA expression of the normoxic group,hypoxia24hours group, hypoxia24hours group, hypoxia48hours group, hypoxia72hours group were1.17times,0.98times,1.04times, respectively.However the expression of VEGF mRNA was increased, compared with normoxic group. Compared with the VEGF mRNA expression of normoxic group, hypoxia24hours group, hypoxia24hours group, hypoxia48hours group, hypoxia72hours group were2.62times,4.79times,5.66times, respectively.(3) Western blot results showed that the protein expression of both HIF-la and VEGF were increased under the hypoxia condition. The IOD of HIF-1alpha in hypoxia24hours group, hypoxia48hours group, hypoxia72hours group were (0.24±0.01),(0.64±0.03),(0.65±0.02) respectively. And the protein level of HIF-1a peaked when treated during48hours. It was not statistically significant difference (p>0.05)between the hypoxia48h group and hypoxia72h group. However compared with the hypoxia groups and normoxic group(0.26±0.01),the difference was statistically significant (p<0.05).The expression of VEGF protein was increasing with the hypoxic hours going.The IOD of VEGF in hypoxia24hours group, hypoxia48hours group, hypoxia72hours group were (0.46±0.02),(0.94±0.03),(1.26±0.02) respectively.The difference was statistically significant between the hypoxia groups and the normoxic group(0.28±0.02)(p<0.05).(4) The transwell invasive assay results showed that with the prolonged hypoxia time the JEG-3trans-membrane behavior enhanced.[Normoxia group (31±5),hypoxia24h group (33±4),hypoxia48h group (40±3),hypoxia72h group (50±5)at400×].However the difference between normoxia group and hypoxia24h group was no statistically significant(p>0.05); the difference between hypoxia48h group and hypoxia72h group was significant(p<0.05);the variations of both hypoxia48h group and72h group compared with the24h group were significant(p<0.05).Conclusion:1. The hypoxia model of choriocarcinoma JEG-3cells in vitro could be established by CoCl2.The proliferation activity of choriocarcinoma JEG-3cell was stimulated within some certain CoCl2concentrations and time.2. The expression of HIF-1α mRNA was not significantly increased, however the expression of protein increased significantly in hypoxia.It means that the expression of HIF-1α was regulated at translation level not at transcription level in hypoxia. The expression of VEGF significantly increased at both transcription level and translation level in hypoxia.3. Hypoxia could promote the invasiveness of choriocarcinoma JEG-3cells, which may be related with the enhanced HIF-1α and VEGF protein expressions. |
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