| Preeclampsia is a pregnancy-specific disorder that can cause multiple organ to damage and threats to maternal and child’ health seriously, meanwhile, is responsible for significant maternal and fetal morbidity and mortality. It is characterized clinically by the onset of hypertension and proteinuria after 20 wk of gestation[1]. Currently, the etiology and pathogenesis of preeclampsia remains poorly understood, and also there is no clear early serology or clinical predictors. Treatment for this disorder is limited to symptomatic therapy or the only known cure is make the pregnancy terminated, The disease has become a thorny problem for global obstetrician[2]. But from the point of the clinical outcomes of preeclampsia, the related symptoms of disease will be rapid relief after the placenta is delivered, That suggests preeclampsia is the correlation placenta disease[9]. Research shows that in recent years, a variety of mi RNA may specially exist in the PE placenta tissue, which play an important role in the regulation of gene expression and then participate in the pathological process ofpreeclampsia[3-8]. The study found that mi RNA-18 b in the placenta tissue of patients with PE is a pathological lower expression[10]and the biological function of mi RNA-18 b has been tested in HTR-8/SVneo [25]. However, the target molecules of mi RNA-18 b have not been searched in a penetrating way. It is well known that the biological function of mi RNA-18 b is put to full use through gene silencing effect[37].And preliminary predict that one of mi RNA-18 b target genes may be Hypoxia-inducible factor 1α(HIF-1α).That HIF-1α is a target gene of mi RNA-18 b had been reported in a literature on c DNA cloning of HIF-1α in high altitude yak[11]. and there is evidence for the functional activity of HIF-1α over-expressed in PE placenta[12], which accurately reflect the regular relationships of target genes related to the specific mi RNA are consistent. Thus, we speculate that the expression of HIF-1αmay be regulated by mi RNA-18 b to participate in the development of preeclampsia.The purpose of this study is that the targeted relationship between mi RNA-18 b and HIF-1α can be verified so that a good new direction be found for prevention and treatment of preeclampsia.Objective1. Using bioinformatics method and related literature on PE-related mi R-18b-5p,HIF-1α has been preliminarily predicted as the target gene of mi R-18b-5p.The expression of HIF-1αwill be verified in the model of human trophoblastic cells(HTR-8/SVneo.2. Through the biology experiment method, the regulatory relations of mi R-18b-5p and its target gene HIF-1αcan be further verified so that the pathogenesis of PE-ralated mi R-18b-5p can be uncovered.Methods1. Using target gene prediction software, such as Target Scanã€mi RBase and so on, the target regulatory relationship between mi R-18b-5p with HIF-1α can be preliminarily proved.2. Using Reverse transcription-polymerase chain reaction technology(RT-PCR),the expression of HIF-1α will be detected in HTR-8/SVneo cell lines.3. Using the mi RNA over-expressing and inbibiting technology in HTR-8/SVneo cells, applying quantificational real-time polymerase chain reaction(real-time PCR)and western-blot technology, the target regulatory relationship between mi R-18b-5p with HIF-1α will be validated in m RNA level and protein level respectively.4. Using luciferase report gene method, to validate negative targeted regulation relationship between mi R-18b-5p with its target gene HIF-1α.Results1. According to prediction software technology of six kinds of target genes and taking the Overlapping portion of the four methods, the result shows that hif-1α could be one of mi R-18b-5p target genes.2. The results show that HIF-1αis expressed in HTR-8/SVneo cell lines by using RT-PCR technology.3. Using the mi RNA over-expressing and inhibiting technology in HTR-8/SVneo cells lines, the results of real-time PCR showed that the expression quantity of mi R-18b-5p in the transfected HTR-8 cells is increased and decreased regularly(P<0.05)and the m RNA expression level of HIF-1α in the mi R-18b-5p over-expressing group is increased significantly( P< 0.05) and yet the m RNA expression level of HIF-1α in the mi R-18b-5p inhibiting group is no significant difference(P>0.05).Western-blot results showed that the expression of HIF-1αin protein level is regularly similar to m RNA level expression. The above results indicate that the negative regulation relationship exist in mi R-18b-5p and HIF-1α.4. The results of luciferase report gene method show that compared with control group, the luciferase activity in a total of Wild type HIF-1αand mi R-18b-5p mimics transfection group dropped by about 25%, which is significantly different(P<0.05).But there is no obvious difference between a total of mutant HIF-1αand mi R-18b-5p mimics transfection group with control group(P>0.05).Conclusion1. The analysis results of target gene prediction software show that HIF-1α is one of mi R-18b-5p target genes and verified experimentally that there is basal expression of HIF-1α in HTR-8/SVneo cell lines, which provides the cell model of targeted relationship beween mi R-18b-5p and HIF-1α so as to reveal the pathological process of preeclampsia.2. The results of target genes validation show that mi R-18b-5p and HIF-1αin the m RNA and protein levels are negative targeted regulatory role. In VEGF signaling pathways, mi R-18b-5p may be involved in the pathogenesis of preeclampsia by regulating target genes HIF-1α, which contributes to elucidate the pathological mechanism of preeclampsia to lay a certain foundation of theory and experiment. |
