| Ovarian cancer is a common gynecologic malignant tumor,with a high mortality rate,and the current treatment of the disease is mainly surgery and chemotherapy.Chemotherapy resistance makes the treatment effect is not optimistic.Although the research of ovarian cancer has been greatly developed,the specific pathogenesis of ovarian cancer is still unclear,which brings great difficulties to the prevention and treatment of ovarian cancer.As a kind of solid tumor,there is a certain degree of hypoxia in ovarian cancer.Hypoxia microenvironment plays an important role in the occurrence and development of tumor,which has been studied in a series of biological behaviors such as apoptosis,invasion and migration of tumor cells.Therefore,it is important to study the related biological behaviors of ovarian cancer cells in hypoxic ovarian cancer cells.SMG-1 is an important regulatory factor in the nonsense-mediated mRNA decay（NMD）.The nonsense-mediated mRNA decay is an effcetive mRNA surveillance mechanism that detects and degrades mRNAs with premature termination codons（PTC）and protects cells from the potentially deleterious effects of truncated proteins.SMG-1 is also a member of the phosphatidylinositol 3-kinase-related kinase family.Like other PIKK kinases,SMG-1 plays a role in the stress response to DNA damage and maintains genomic stability.In addition,SMG-1 is involved in many biological processes,such as oxidative stress,hypoxia,cell proliferation and apoptosis.A number of studies about this gene have been introduced into the field of cancer.STAT3 is an important nuclear transcription factor,which can be continuously activated in many human tumors.Activated JAK kinases phosphorylate STAT3 and form a homo-or hetero-dimerization of p-STAT3.Then the p-STAT3 can translocate into the nucleus and bind to specific promoter sequences,playing the transcriptional regulatory function.STAT3 is the convergence point of many oncogenic signaling pathways,which can control the intracellular signal transduction pathways of several proinflammatory cytokines and growth factors.Abnormal activation of STAT3 can be found in human ovarian cancer cells,the abnormal activation of STAT3 can affect the proliferation and apoptosis of ovarian cancer cells.ObjectiveIn this study,qRT-PCR and Western blot were used to investigate the expression of SMG-1 in ovarian cancer cells under hypoxic condition and detect the expression of STAT3 after the inhibition of SMG-1 gene.Flow cytometry and CCK-8 assay were used to detect the effect of SMG-1 gene on the proliferation and apoptosis of hypoxic ovarian cancer cells and to investigate the effect of silencing SMG-1 gene on the expression of STAT3 and apoptosis in hypoxic ovarian cancer cells.Materials and Methods 1 MaterialsThe human ovarian cancer cell line SKOV3 purchased from the American Type Culture Collection were maintained with RPMI1640 complete medium containing 10%FBS at 37℃ under the saturated humidity of the air with 5% CO2.2 MethodsIn this study,RPMI1640 medium（containing 10% fetal bovine serum）was used to culture SKOV3 cells,and the cells were used for intervene experiments that in the logarithmic phase.2.1 Groups and processing method of hypoxiaIn the hypoxia part of this study,the logarithmic growth phase cells were digested by 0.25% trypsin and then seed the cells in 6-well plates at a concentration of 1.0×105cells/ml.When the cells were filled with 50%-60%,we can start with hypoxia.Grouped as follows:normal group and hypoxia group.Adding cobalt chloride in hypoxia group（final concentration of 150 μmol/L）,normoxia group without treatment.Total mRNA and total protein were extracted after hypoxia treatment with 48 h.Firstly,the effect of hypoxia was verified.If the expression of HIF-1α protein was significantly higher than that of non hypoxic cells after hypoxia（P<0.05）,we determined the success of hypoxia.Then the expression of SMG-1 mRNA and protein was detected in two groups of cells.2.2 Groups and processing method of transfectionIn the transfection part of this study,the logarithmic growth phase cells were digested by 0.25% trypsin and then seed the cells in 6-well plates at a concentration of 1.0×105cells/ml.The cells were cultured by the RPMI1640 medium（containing 10% fetal bovine serum）.After the cells were attached,the cobalt chloride（150 μmol/L）was used to establish the hypoxia model.After hypoxia treatment for 24 h,transfection was carried out,and the transfection method was referred to the Lipofectamine2000 specification.Grouped as follows:group A,without siRNA and Lipofectamine2000;group B,with negative control siRNA and Lipofectamine2000;group C,with transfection reagent;group D,with transfection reagent Lipofectamine2000 and SMG-1 siRNA.After transfection with 6h,the culture medium was replaced by the fresh complete culture medium（containing 10% fetal bovine serum and 150 μmol/L of cobalt chloride）and the cells were placed In the incubator for 48 hours.qRT-PCR and Western blot were used to verify the silence effect of SMG-1 siRNA in protein and mRNA levels.The expression of SMG-1 in D group was significantly different from that in A,B and C group（P<0.05）and the expression inhibition rate of SMG-1 in D group was 50% higher than that in group A,we can say the SMG-1 gene was silenced effectively.Flow cytometry and CCK-8 assay were used to investigate the apoptosis rate and the inhibitory rate of cell proliferation.qRT-PCR and Western blot were used to investigate the expression of STAT3 mRNA and STAT3 protein.3 Statistical analysisIn this study,SPSS21.0 software was used to analyze the experimental data.All data were expressed as the （?）±s.The comparison between the two groups was analyzed by two independent samples t test.One-way ANOVA was used to compare the data between multiple groups.Significance was considered when P<0.05.Results 1 Expression of SMG-1 in SKOV3 cells after hypoxia treatment 1.1 The relative expression level of SMG-1mRNA in SKOV3 cells after hypoxia treatmentThe relative expression level of SMG-1 mRNA in hypoxia group was 4.19±0.94,and the normoxia group was 1.12±0.17.The level of the hypoxia group was significantly higher than the normoxia group（P<0.05）.1.2 The relative expression level of SMG-1 protein in SKOV3 cells after hypoxia treatmentThe relative expression level of SMG-1 protein in hypoxia group was 0.58±0.11,and the normoxia group was 0.26 ± 0.06.The level of the hypoxia group was significantly higher than the normoxia group（P<0.05）.2 Effect of silencing SMG-1on cell apoptosis rate of SKOV3 cells under hypoxiaFlow cytometry showed that the apoptosis rate of group A was（5.7±1.22）%,group B was（6.6 ± 0.87）%,group C was（6.9 ± 1.35）%,and group D was（10.2±0.70）%.The apoptosis rate of group D was significantly higher than that of group A,group B and group C（P<0.05）.3 Effect of silencing SMG-1on the inhibitory rate of cell proliferation of SKOV3 cells under hypoxiaCCK-8 test showed that the inhibition rate of cell proliferation in group B was（22.54±5.74）%,group C was（22.56±1.11）%,and group D was（42.17±5.03）%.The inhibition rate of cell proliferation in group D was significantly higher than that in group B and group C（P<0.05）.As a reference,the cell proliferation inhibition rate of group A was 0%.4 Effect of silencing SMG-1on the relative expression level of SMG-1 mRNA and STAT3 mRNA under hypoxia 4.1 Effect of silencing SMG-1on the relative expression level of SMG-1 mRNA under hypoxiaReal-time PCR analysis indicated that the expression level of SMG-1 mRNA in group A was 1.00±0.07,the group B was 1.02±0.13,the group C was 0.88±0.07,and the group D was 0.46±0.04.The expression level of SMG-1 mRNA in group D was significantly lower than the other three groups（P<0.05）.4.2 Effect of silencing SMG-1on the relative expression level of STAT3 mRNA under hypoxiaReal-time PCR analysis indicated that the expression level of STAT3 mRNA in group A was 1.00±0.05,the group B was 0.98±0.10,the group C was 0.86±0.04,and the group D was 0.68 ± 0.08.The expression level of STAT3 mRNA in group D was significantly lower than the other three groups（P<0.05）.5 Effect of silencing SMG-1on the protein expression level of SMG-1 、STAT3 and p-STAT3 under hypoxia 5.1 Effect of silencing SMG-1on the protein expression level of SMG-1 under hypoxiaThe result of western blot displayed that the expression level of SMG-1 protein in group A was 0.38±0.06,the group B was 0.37±0.06,the group C was 0.38±0.03,and the group D was 0.16±0.04.The expression level of SMG-1 protein in group D was significantly lower than the other three groups（P<0.05）.5.2 Effect of silencing SMG-1on the protein expression level of STAT3 under hypoxiaThe result of western blot displayed that the expression level of STAT3 protein in group A was 0.59±0.05,the group B was 0.63±0.05,the group C was 0.71±0.05,and the group D was 0.46±0.03.The expression level of STAT3 protein in the group D was significantly lower than the other three groups（P<0.05）.5.3 Effect of silencing SMG-1on the protein expression level of p-STAT3 under hypoxiaThe result of western blot displayed that the expression level of p-STAT3 protein in group A was 0.56±0.03,the group B was 0.62±0.04,the group C was 0.61±0.04,and the group D was 0.34±0.02.The expression level of p-STAT3 protein in the group D was significantly lower than the other three groups（P<0.05）.ConclusionsThe expression level of SMG-1 gene is affected by hypoxia.Inhibition of SMG-1 can affect the expression and activation level of STAT3 under hypoxia,and thus participate in the apoptosis of SKOV3 cells together. |
