| Preeclampsia (PE) is a pregnancy-specific disease which has high incidence as well as severe clinical manifestations and it is one of the main reason leads to the death of maternal and perinatal[1]. The pathogenic factors leads to PE are divergent, but we currently considered that the reason leads to production of PE perhaps is placental dysfunction and insufficient vascular remodeling, which is the main reason which caused trophoblast dysfunction. MicroRNA are a class of small molecule non-coding RNA, which plays an important role in the regulation of gene expression[2]. The recently researches showed that a variety of miRNA may specific expressed in human placenta, its unique features of gene regulation may be closely related with the incidence of PE, which is including miR-18a pathological expressed in the PE placenta of pregnant women[3]. Additionally, other found that miR-18a inhibited the expression of estrogen receptorα(estrogen receptor alpha, ERα/ER1) to promote the proliferation of hepatoma cells in liver cancer patients and the targeted regulation relationship between them as well as the specific targets of regulation of them[4]. ER may be related to incidence of PE[5]. At present, in the world-wide there are also no reports in series about research of PE-related miRNA and its target genes related to disease-miRNA functional research in disease. Therefore, it is necessary to deeply study the mechanisms of the role of PE-related miRNA played in the development of the disease, so as to provide new evidence and clues for research the pathogenesis of PE.Objective:1. To investigate the differential expression of ERαwhich is the targeted gene of PE-related miR-18a in normal and PE placenta, and the differences of estradiol (E2) levels in maternal plasma which effects through ERαbetween normal and preeclampsia pregnancy women, furthermore, to validate the correlation of miR-18a and its target gene ERαwith the incidence of PE.2. To investigate the regulatory role of miR-18a affects on human trophoblast cell, and to clarify the role of the miR-18a in the development process of PE.Methods:1. To prove target regulatory relationship between miR-18a and ERαagain by six miRNA target gene prediction software (TargetScan and so on).2. To detect plasma levels of estradiol of group of normal term pregnant women and the group of pregnant women with preeclampsia by using automatic electrochemiluminescence immunoassay system. To detect expression comparison of ERαmRNA and histological localization of ERαin placenta of 21 normal pregnant women(control group) and 23 pregnant women(PE group) with preeclampsia by reverse transcription - polymerase chain reaction and immunohistochemistry. To detect expression comparison of ERαbetween the control group and PE group on the protein level by Western-blot.3. To over-express and inhibit the expression of miR-18a in human trophoblastic tumor cell line (JEG-3), to detect the expression levels of miR-18a and ERαin JEG-3 cell, to validate negative regulation relationships between miR-18a and ERαon mRNA and protein levels.4. To detect JEG-3 cell proliferation, apoptosis and invasive changes by using flow cytometry and Tran-swell chamber after over-expressing and inhibiting the expression of miR-18a in JEG-3 cell.Results:1. Using six target gene prediction software and taking the intersection, four of which confirmed that the targeting regulatory relations between miR-18a and ERαand it is consistent with the reported results [6].2. The detection of plasma levels of estradiol showed that plasma estrogen levels of normal pregnant women were significantly higher than the group of pregnant women with preeclampsia(P<0.05). The detection of immunohistochemistry showed that there are expressions of ERαboth in the two groups, additionally, the expressions of ERαin the cytoplasm or nucleus of cytotrophoblast of the group of pregnant women with preeclampsia were higher than the group of normal pregnant women(P<0.05).The results of RT-PCR and Western-blot showed that both of the two groups expressed ERαin the mRNA and protein levels, but the expressions of ERαof the group of pregnant women with preeclampsia were higher than the group of normal pregnant women(P<0.05).3. After over-expressing and inhibiting the expression of miR-18a in JEG-3 cell, the results of RT-PCR showed that in the transfected JEG-3 cells, the expression of miR-18a is regulated, then increased and decreased significantly; the expression on ERαmRNA levels in transfected cells were significantly different (P <0.05), by negative regulation of miR-18a is Significant; Western-blot results showed that the expression of ERαin the protein level is also negatively regulated by miR-18a. 4. Flow cytometry results showed that after over expression and inhibition of miR-18a, there is no significant difference in proliferation of each experimental group and control group cells (P> 0.05); down regulated the expression of miR-18a significantly increased the cells wither Death rate (P <0.05); cell infiltration showed that down-regulation of the expression of miR-18a can significantly reduce the JEG-3 cell invasion (P <0.05).Conclusion:1. The expression of mRNA and protein of ERαin the group of pregnant women with preeclampsia were higher than the group of normal pregnant women, but the plasma levels of estradiol in the group of pregnant women with preeclampsia were lower than the group of normal pregnant women, which showed that pathological higher expression of mRNA of ERαin the group of pregnant women with preeclampsia may be related negatively with the lower plasma levels of estradiol in the group of normal pregnant women, what's more, in the pathological state of preeclampsia, the lower plasma levels of estradiol in the body may lead to compensatory increase of ERα. The ERαin placental villous cytotrophoblast, villous capillary wall cells, and villous interstitial fibrosis fibroblasts located by immunohistochemistry as the mediated central for ER to play its function may be involved in the occurrence and development of hypertensive diseases of pregnancy through mediating role between each other.2. Experimental results showed that as the target role of miR-18a, ERαis a clear negative regulatory role of miR-18a in the mRNA and protein levels.3. Down regulation of expression of miR-18a in JEG-3 cells could increase the apoptosis rate and reduce the invasion; therefore, miR-18a which is low pathological expression in the PE placenta possibly participated in the pathogenesis of PE through its function of trophoblast cells, additionally, what's we found could be the theoretical and experimental basis for the gene therapy used in diseases of preeclampsia. |
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