| Preeclampsia(PE)is one of the specific diseases during pregnancy(systolic blood pressure>140mm Hg and/or diastolic blood pressure>90mm Hg with proteinuria> 0.3 g / 24 h or randomized proteinuria + after 20 weeks gestation)and is the leading cause of morbidity and mortality in mother and child.At present,the pathogenesis of PE is not yet fully clear,but once the placenta is delivered,the symptoms of preeclampsia will be reduced or even disappeared,suggesting that placenta plays a crucial role in the pathogenesis of preeclampsia.The placenta is mainly composed of trophoblasts,studies have shown that insufficient invasion of trophoblast can cause placental implantation too shallow,and then affect the placental development,eventually leading to preeclampsia and other pregnancy diseases.MicroRNAs(miRNAs)are a class of endogenous,non-coding,single-stranded small RNAs of about 22 nucleotides in length that play an important role in biological activity by regulating target m RNAs.In recent years,more and more studies have shown that the expression of miRNAs in human placenta can affect the function of trophoblast invasion,proliferation and protein synthesis.In the PE occurs,some miRNAs appear abnormal expression,miR-34 a is one of them.Sun M’s research shows that miR-34 a is significantly elevated in placental tissue of PE,and miR-34 a is closely related to the invasion and migration function of trophoblast.In breast cancer,miR-34 a can target Notch1 to affect cell invasion and proliferation.However,whether miR-34 a is associated with Notch1 in PE and how to regulate the biological functions of trophoblasts has not been reported yet.Objective To investigate the effect of Micro RNA-34 a regulate Notch1 combined with the research on placental tissues of women with preeclampsia and the ability of invasion and proliferation of human chorionic JEG-3 trophoblast cells.Materials and methods 1 Materials We selected placental tissues and JEG-3 cells in this study.Our hospital ethics committee was approved this study,all of specimen providers are informed consent.All the placenta tissues were choosen from September 2016 to March 2017 at the Third Affiliated Hospital of Zhengzhou University.There was a number of 30 cases of a normal pregnancy was chosen as normal group,those 30 patients with preeclampsia was chosen as PE group,The diagnose of preeclampsia was based on the eighth edition of gynecology and obstetrics.JEG-3 cells was purchased from Dingguo company in Beijing,its biological traits close to the original isolated trophoblast cells.2 Methods Placenta tissues biopsies were obtained from women with a normal pregnancy and those with PE,Real-time PCR was used to evaluate the expression level of miR-34 a miRNA and Notch1 m RNA,Notch1 protein expression and orientation were detected by immunohistochemistry.Chemically synthesized miR-34 a mimic,miR-34 a inhibitor and Notch1 si RNA were transfected into JEG-3 cells.The expression of miR-34 a miRNA and Notch1 m RNA were detected by Real-time PCR.The expression of Notch1 protein were detected by Western blot.The effect on the ability of invasion and migration were analyzed by Transwell migration assay and Wound healing assay.The effect on the ability of proliferation were detected by CCK8.3 Statistics analysis Statistical analysis was analyzed by SPSS 21.0 software.All the result were expressed as mean ± standard deviation.Comparision between the two groups using the independent samples t-test and Chi-square(χ2).One-way ANOVA was used for data from multi-group.With α=0.05 for the test standards.Result 1 General clinical data and detection index comparison among the two groups of pregnant women 1.1 General clinical data comparison among the two groups The average age of control group and PE group were(29.25±2.45)years and(30.63±3.51)years respectively,the gestational age were(37.06±1.62)weeks and(34.38±2.32)weeks,the systolic blood pressure were(112.48±3.76)mm Hg and(170.14±4.69)mm Hg,the diastolic blood pressure were(69.32±3.67)mm Hg and(115.31±4.58)mm Hg,the proteinuria were(0)g/24 h and(5.67±3.25)g/24 h,the fetal weights were(3504.05±436.31)g and(3025.62±629.05)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(P<0.05).The gestational age and fetal weights in PE group were lower than control group(P<0.05).1.2 The expression of miR-34 a miRNA and Notch1 m RNA in placenta tissues The results indicated that compared with the control group(1.02±0.22),the expression of miR-34 a miRNA(1.84±0.26)in PE group increased(P<0.05).Compared with the control group(1.01±0.16),the expression of Notch1 m RNA(0.63±0.13)in PE group decreased(P<0.05).1.3 The localization and expression of Notch1 protein in placenta tissues Notch1 can be detected in the two groups.It was mainly localized in the cell cytoplasm and cell membrane of trophoblasts cells and vascular endothelial cell of the placenta tissues.The expression of Notch1 protein in PE group was lower than the control group,the difference was statistically significant(P<0.05).2 The expression of Notch1 m RNA and protein of JEG-3 cells after the transfection of miR-34 a The results of Real-time PCR and Western blot showed that the expression of Notch1 m RNA and protein of JEG-3 cells in miR-34 a mimics group(0.46±0.08,0.52± 0.08)was decreased than that in blank control group(1.00,1.37 ±0.09),lipo2000(0.94 ± 0.14,1.31 ± 0.09)group and NC group(0.89 ± 0.13,1.34 ±0.10),the difference was statistically significant(P<0.05).Compared with blank control group(1.00,1.04 ± 0.08),lipo2000(0.94 ± 0.14,1.00 ± 0.08)group and INC group(0.90±0.13,1.04±0.07),which were increased in miR-34 a inhibitors group(1.42±0.12,1.86±0.11),the difference was statistically significant(P<0.05).3 Effect on the ability of invasion,migration and proliferation of JEG-3 cells after the transfection of miR-34 a The results of transwell invasion assay and wound healing assay showed that the number of transmembrane cells and relative migration distance in miR-34 a mimics group(78.38 ± 6.26,22.82 ± 2.57)was decreased than that in blank control group(120.88 ± 8.20,49.62 ± 3.81),lipo2000(113.51 ± 8.62,47.63 ± 4.02)group and NC group(114.13±5.64,52.09±5.79)(P<0.05),and the results of CCK8 at three time points showed that the proliferation was inhibited,the difference was statistically significant(P<0.05).Compared to the other groups,the number of transmembrane cells and relative migration distancewhich were increaded in miR-34 a inhibitors group (162.88±7.61,62.46±3.76)(P<0.05),and the results of CCK8 at three time points showed that the proliferation was promoted(P<0.05).4 The expression of Notch1 m RNA and protein of JEG-3 cells after the transfection of Notch1 si RNA The results of Real-time PCR and Western blot showed that the expression of Notch1 m RNA and protein of JEG-3 cells in Notch1 si RNA group(0.40±0.32,0.46±0.08)was decreased than that in blank control group(1.00,1.39±0.05),lipo2000(0.94±0.14,1.35±0.11)group and si-NC group(0.91±0.10,1.32±0.15),the difference was statistically significant(P<0.05).5 Effect on the ability of invasion,migration and proliferation of JEG-3 cells after the transfection of Notch1 si RNA The results of transwell invasion assay and wound healing assay showed that the number of transmembrane cells and relative migration distance in Notch1 si RNA group(82.00 ± 7.11,23.21 ± 2.59)was decreased than that in blank control group(120.88 ± 8.20,49.62 ± 3.81),lipo2000(113.51 ± 8.62,47.63 ± 4.02)group and NC group(112.63±8.58,46.08±6.18)(P<0.05),and the results of CCK8 at three time points showed that the proliferation was inhibited,the difference was statistically significant(P<0.05).Conclusion miR-34 a may inhibits the invasion and proliferation of trophoblast cells by regulating Notch1 contributing to the development of preeclampsia. |
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