| Studies have shown that the normal development of the placenta is closely related to the change of oxygen pressure.The placenta is exposed to anoxic environment at 8 to 10 weeks of gestation,and the oxygen pressure increases after 10 weeks.The change of oxygen pressure can affect the proliferation and invasion ability of trophoblast cells,and futher affect the development of the placenta.Preeclampsia(PE)is a pregnancy-specific complication with global incidence ranging from 5%?10%,which is characterized by the newly occurring hypertension,edema and proteinuria after 20 weeks of gestation.Although it leads to morbidity and mortality of mothers and perinatal infants,the pathogenesis of PE has not been fully elucidated.Currently,it is widely believed that preeclampsia is associated with insufficient trophoblastic invasion,helical artery recasting disorder and shallow placental implantation resulting in continuous hypoxia and developmental disorders of placenta.hypoxia inducible factor-1 alpha(HIF-1?)is a critical transcriptional factor for cells to adapt to a low oxygen environment,which can activate the transcription of downstream target genes and regulate cell proliferation,invasion,apoptosis and other abilities.Studies have shown that the expression of HIF-la is elevated in placental tissues and maternal serum of PE and is closely related to the development of PE.Forkhead box protein O3a(FOXO3a),a member of the Forkhead box protein O family,can regulate hypoxia,oxidative stress,heat shock and other stress responses.FOXO3a is down-regulated in a variety of cancers such as prostate cancer and breast cancer,which can inhibit the proliferation and migration of cancer cells,promote the apoptosis of cancer cells,and thus inhibit the occurrence and development of cancer.It is considered as a new target for cancer diagnosis and treatment.Some studies have found that FOXO3a is an important target for HIF-1?-mediated cellular stress response.For example,HIF-la could regulate the expression of FOXO3a to promote apoptosis of umbilical vein endothelial cells(HUVECs)and cardiac microvascular endothelial cells(CMECs)under hypoxia.However,the effect of HIF-1? regulating FOXO3a on the trophoblast cell biological function under hypoxia conditions has not been reported.ObjectivesTo investigate the effect of HIF-l? on the expression of FOXO3a in trophoblast cells and the invasion and apoptosis of cells under hypoxia conditions.And further explore its role in the development of preeclampsia.Materials and methods1 MaterialsIn this study,placental tissue and HTR8-SVneo cells were selected as research subjects.The study was approved by the Ethics Committee of the Third Affiliated Hospital of Zhengzhou University and obtained informed consent from the patient.30 women with PE as the PE group and 30 women with normal pregnancy constituted the control group from May 2017 to December 2018 in the Third Affiliated Hospital of Zhengzhou University,were recruited in the study.The diagnose of preeclampsia was based on the eighth edition of gynecology and obstetrics.HTR8-SVneo cells was from human early pregnant extravillous trophoblast and was purchased form ATCC,USA.2 Methods2.1 The mRNA and protein expression of FOXO3a in placental tissues.The qRT-PCR and Western blot were used to detect the mRNA and protein expression of FOXO3a in placental tissues.Immunohistochemistry was used to detect the expression and localization of FOXO3a in two groups of placental tissues.2.2 The hypoxia model was establishedHTR8-SVneo cells were treated with 0 ?mol/L,125 ?mol/L,250 ?mol/L and 500?mol/L cobalt chloride(CoCl2)for 24h,48h and 72h.The CCK8 were used to detect the cell viability.The drug treatment concentration and time of cell viability inhibition rate at 50%(IC50)were selected to establish an extracellular hypoxia model.As a result,the 250 ?mol/L for 48h was selected for subsequent experiments.2.3 The protein expression of HIF-1? and FOXO3a in HTR8-SVneocells treated cobalt chloride.The Western blot was used to detect the protein expression of HIF-la and FOXO3a in HTR8-SVneo cells treated with 0,125,250 and 500 ?mol/L cobalt chloride for 48h.The Western blot was used to detect the protein expression of HIF-1? and FOXO3a in HTR8-SVneo cells treated with 250?mol/L cobalt chloride for Oh,24h,48h and 72h.2.4 The Change of invasion and apoptosis in HTR8-SVneo cells after transfection with FOXO3a siRNA under hypoxiaThe experiment was divided into four groups,named normoxic group,hypoxic group,hypoxic NC group and hypoxic siFOXO3a group.HTR8-SVneo cells were transfected with FOXO3a siRNA under hypoxia conditions.The mRNA and protein expression of FOXO3a was separately detected by Real-time quantitative PCR and Western blot;the invasion ability was detected by Transwell;the apoptosis rate of cells was detected by flow cytometry.2.5 The Change of invasion and apoptosis in HTR8-SVneo cells after transfection with HIF-1? siRNA under hypoxiaThe experiment was divided into four groups,named normoxic group,hypoxic group,hypoxic NC group and hypoxic siHIF-la group.HTR8-SVneo cells were transfected with HIF-1? siRNA under hypoxia conditions.The protein expression of HIF-1? and FOXO3a was detected by Western blot;the invasion ability was detected by Transwell;the apoptosis rate of cells was detected by flow cytometry.3 Statistics analysisThe data was analyzed by Graphpad prism 7.0 software.All the result were expressed as mean±standard deviation.Comparision between the two groups used the independent samples t-test.One-way ANOVA was used for data from multi-groups and a Bonferroni's correction post hoc test.With ?=0.05 for the test standards.Results1 Comparison of general clinical data and the expression of FOXO3a in the control group and PE group1.1 Comparison of general clinical data among the two groupsThe average age of control group and PE group were respectively(32.45 ± 4.66)years and(32.90 ± 5.41)years,the gestational age were respectively(39.00 ± 0.50)weeks and(32.43 ± 1.59)weeks,the systolic blood pressure were respectively(114.72± 7.26)mmHg and(162.29± 13.79)mmHg,the diastolic blood pressure were respectively(72.52±8.22)mmHg and(102.81 ± 9.53)mmHg,the proteinuria were(0.08 ± 0.04)g/24h and(4.99 ±2.96)g/24h,the fetal weights were(3518.28 ± 350.87)g and(1457.74±376.13)g.The average age among the two groups had no statistically significant(P>0.05).The systolic blood pressure,diastolic blood pressure and proteinuria in PE group were higher than control group and the differences were statistically significant(P<0.05).The gestational age and fetal weights in PE group were lower than control group,and the differences were statistically significant(P<0.05).1.2 The mRNA and protien expression of FOXO3a in the two groups placental tissuesThe mRNA and protien expression of FOXo3a in PE group(2.33 ± 0.99,1.29 ±0.43)was higher than the control group(0.89 ± 0.28,0.25±0.18),and the differences were statistically significant(P<0.05).1.3 The localization and expression of FOXO3a in the two groups placental tissuesThe protein expression of FOXO3a was located in the cytoplasm and nucleus of syncytiotrophoblast cytotrophoblast and extravillous trophoblast.And the staining intensity of FOXO3a in the PE group(4.62±2.29)was higher than the control group(1.45 ± 1.30),and the differences were statistically significant(P<0.05).2 The cell activity and the protien expression of HIF-1? and FOXO3a in HTR8-SVneo cells under hypoxia2.1 The cell viability in HTR8-SVneo cells under hypoxiaCobalt chloride treated for 24h:the cell viability of 125?mol/L group(1.08±0.08)was higher than 0?mol/L group(0.86±0.12),and the differences were statistically significant(P<0.05);the cell viability of 500?mol/L group(0.60±0.09)was lower than 0?mol/L group,and the differences were statistically significant(P<0.05).Cobalt chloride treated for 48h:the cell viability in 250?mol/L group(0.52 ±0.10),500?mol/L group(0.33±0.08)were lower than 0?mol/L group(1.07±0.18),and the differences were statistically significant(P<0.05).Cobalt chloride treated for 72h:the cell viability in 125?mol/L group(0.81±0.09),250?mol/L group(0.38±0.08),500?mol/L group(0.22±0.07)were lower than 0?mol/L group(1.12±0.12),and the differences were statistically significant(P<0.05).2.2 The protien expression of HIF-1? and FOXO3a in HTR8-SVneo cells treated cobalt chloride of different concentration for 48hThe protein expression of HIF-1? and FOXO3a in 125?mol/L group(0.99±0.15,0.67±0.07),250?mol/L group(1·15±0.11,0.94±0.16)and 500?mol/L group(0.63±0.08,1,18±0.17)were higher than 0?mol/L group(0.41±0.05,0.37±0.06),and the differences were statistically significant(P<0.05).2.3 The protien expression of HIF-1? and FOXO3a in HTR8-SVneo cells treated 250?mol/L cobalt chloride for different timesThe protein expression of HIF-1? and FOXO3a in 24h group(1.11±0.15,0.85±0.08),48h group(1.21±0.14,1.24±0.16)and 72h group(1.51±0.14,1.48±0.13)were higher than 0h group(0.43±0.10,0.42 ± 0.10),and the differences were statistically significant(P<0.05).3 The mRNA and protien expression of FOXO3a in HTR8-SVneo cells after transfection with FOXO3a siRNA under hypoxiaThe mRNA and protien expression of FOXO3a in normoxia group(0.99 ± 0.11,0.33 ± 0.04),hypoxia group(1.84 ± 0.57,1.08 ± 0.15)and hypoxia NC group(2.10 ±0.35,1.02 ± 1.14)were higher than the hypoxia siFOXO3a group(0.21 ± 0.66,0),and the differences were statistically significant(P<0.05).The mRNA and protien expression of FOXO3a in hypoxia group and hypoxia NC group were higher than the normoxia group,and the differences were statistically significant(P<0.05).4 Change of the invasion and apoptosis in HTR8-SVneo cells after transfection with FOXO3a siRNA under hypoxiaThe numbers of invasive cell in hypoxia group(35.83 ± 4.71),hypoxia NC group(38.33± 4.55)and hypoxia siFOXO3a group(58.33± 6.28)were lower than the normoxia group(72.83± 9.87),and the differences were statistically significant(P<0.05);the numbers of invasive cell in the hypoxia siFOXO3a group were higher than the hypoxia group and hypoxia NC group,and the diferences were statistically significant(P<0.05).The percentage of apoptosis in hypoxia group(27.20 ± 1.43),hypoxia NC group(28.29 ± 1.33)and hypoxia siFOXO3a group(12.17±1.40)were higher than the normoxia group(8.06± 1.21),and the differences were statistically significant(P<0.05);the percentage of apoptosis in the hypoxia siFOXO3a group were lower than the hypoxia group and hypoxia NC group,and the differences were statistically significant(P<0.05).5 The mRNA and protien expression of HIF-la and FOXO3a in HTR8-SVneo cells after transfection with HIF-1? siRNA under hypoxia5.1 The mRNA and protien expression of HIF-la in HTR8-SVneo cells after transfection with HIF-1? siRNA under hypoxiaThe mRNA and protien expression of HIF-1? in normoxia group(1.04 ± 0.08,0.40± 0.05),hypoxia group(1.10 ± 0.17,1.09 ± 0.14)and hypoxia NC group(1.18±0.16,1.04 ± 0.15)were higher than the hypoxia siHIF-la group(0.29± 0.06,0.12±0.06),and the differences were statistically significant(P<0.05).The protien expression of HIF-1 a in hypoxia group and hypoxia NC group were higher than the normoxia group,and the differences were statistically significant(P<0.05).5.2 The protien expression of FOXO3a in HTR8-SVneo cells after transfection with HIF-1? siRNA under hypoxiaThe protien expression of FOXO3a in normoxia group(0.27 ± 0.05),hypoxia group(1.02±0.07)and hypoxia NC group(1.07±1.11)were higher than the hypoxia siHIF-1? group(0.13 ± 0.03),and the differences were statistically significant(P<0.05).The protien expression of FOXO3a in hypoxia group and hypoxia NC group were higher than the normoxia group,and the differences were statistically significant(P<0.05).6 Change of the invasion and apoptosis in HTR8-SVneo cells after transfection with HIF-1? siRNA under hypoxiaThe numbers of invasive cell in hypoxia group(32.33 ± 4.50),hypoxia NC group(36.17 ± 5.31)and hypoxia siHIF-la group(51.50±6.78)were lower than the normoxia group(68.17± 10.09),and the differences were statistically significant(P<0.05);the numbers of invasive cell in the hypoxia siHIF-1? group were higher than the hypoxia group and hypoxia NC group,and the differences were statistically significant(P<0.05).The percentage of apoptosis in hypoxia group(27.20±1.43),hypoxia NC group(27.88±1.22),and hypoxia siHIF-1? group(14.36± 1.07)were higher than the normoxia group(8.06 ±1.21),and the differences were statistically significant(P<0.05);the percentage of apoptosis in the hypoxia siHIF-1? group were lower than the hypoxia group and hypoxia NC group,and the differences were statistically significant(P<0.05).ConclusionHIF-1? could inhibit trophoblastic invasion and promote trophoblastic apoptosis by regulating the expression of FOXO3a under hypoxia conditions,which could be associated with the development of PE. |
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