Research On The Regulation Of MiR-29b On The Function Of Trophoblast Cells Contributing To Pathogenesis Of Pre-eclampsia

Background:Pre-eclampsia (PE) is a disease of pregnancy characterizedby hypertension and proteinuria, developing after20weeks of gestation. It has been estimated that5%-7%of pregnancies world wide are complicated by this disorder, resμlting in a very large disease burden. Its pathogenesis is incompletely understood, identification of biomarkers for PE diagnosis is of particμlar interest. Recent studies on microRNA (miRNA) offer the possibility for developing a new class of molecμlar markers for diagnosis of PE. MiRNAs are short (19-25nucleotides), single-stranded, and non-protein-coding RNAs that regμlate gene expression by binding to the3’ untranslated region of the target mRNAs. They function in diverse biological processes, including development, differentiation, apoptosis, and oncogenesis. Recent research shows that many miRNAs are expressed abundantly in the human placenta, however, published data on miRNAs in human PE is surprisingly sparse. This lack of data prompted us to determine and compare the expression profile of miRNA in placenta of Chinese patients with PE compared with normal placenta tissue. Moreover, until now, the effects of miRNAs in mediating trophoblast cells were addressed sparsely. Hence, it was necessary to further disclose their potential roles in the pathogenesis of PE. Objective:1. The etiology of PE is uncertain. Many people focused on the roles of miRNAs in clinical manifestation. MiRNA microarray analysis was performed to study the miRNA’s expression profiles in placentas from women with severe PE and normal pregnancy and discuss the roles of miRNA in the pathogenesis of PE.2. To investigate the roles of miR-29b in functions of trophoblast function and disclose the underlying mechanism.3. To examine the expression of miR-29b and its target genes in PE placenta and control and identify the correlation of miR-29b with these genes in patients explore the effects of miR-29b on the onset of severe PE.Methods:1. Placental tissue was obtained from women who were hospitalized in the Department of Gynecology and Obstetrics of the Affiliated Drum Tower Hospital of Nanjing University Medical School between March2005and August2008at the time of cesarean section. We selected24pregnancies complicated by severe late-onset PE and26pregnant women with normal term pregnancy were recruited as the control group. Eight samples, four normal placentae (4normal placentae were pooled to form a control group) and four placentae from women with severe PE were assayed using a miRNA microarray chip. The differently expressed miRNA were further analyzed by significance analysis of microarrays and cluster analysis.2. Expression of seven most up-regμlated miRNAs in twenty four placentas from severe PE(sPE group) and twenty from gestational normal pregnancies(control group) were confirmed using real time RT-PCR. Prediction of target genes of these miRNAs was done using computer based programs including Targetscan, Pictar and miRBase. Function and possible signal pathway of these target genes were further analyzed by gene ontology (GO). 3. The differentially expressed miRNA in PE placentas, miR-29b, was selected for target verification and pathway analysis. Human trophoblast cell lines (HTR-8/SVneo, BeWo and JAR cells) were selected for in vitro experiments. The use of gene transfection technology enabled the high or low expression of miR-29b in trophoblast cells. On this basis, we detected the effects of miR-29b on biological behavior such as proliferation, apoptosis, cycle, invasion and angiogenesis. Bioinformatics predicts the target genes of miR-29b. The expression of target genes were identified by RT-PCR and Western blot and ELISA analysis, respectively. The verification of miRNA-29b target site was performed using luciferase reporter vector. The effects of miR-29b on ERK and FAK signal pathway were assayed by Western blot.4. The expression of miR-29b and its target genes in PE placenta and control were detected by real time RT-PCR and the correlation of miR-29b with these genes in patients were explored by Pearson correlation and linear regression analysis.Results:1. Twenty-seven microRNA were differentially expressed in preeclamptic placentas compared with that of normal placentas. Of these, twenty were overexpressed and seven were underexpressed in preeclamptic pregnancies. Among the seven miRNA (miR-16, miR-29b, miR-195, miR-26b, miR-181a, miR-335and miR-222) which selected for real-time RT-PCR verification, real-time RT-PCR data were concordant with the microarray data. The expression of these miRNAs were significantly different in severe PE compared with normal placenta (P<0.05). The fold change are2.51,2.51,3.32,2.25,4.67,2.22,2.58respectively. The potential targets of miRNAs were predicted using Targetscan, Pictar and miRBase. In addition, we used CapitalBio Molecμle Annotation System V4.0to perform gene ontology (GO) analysis on the gene target lists of these miRNAs and found that specific biological process categories were enriched such as cell death, cellular physiological process, signal transduction and cell proliferation.2. We found a significant up-regulation of miR-29b in Chinese severe PE patients placenta. In addition, we investigated the biological functions of these target genes using GO and observed enrichment for genes implicated in important cellμlar functions, such as proliferation, cycle progression, apoptosis and migration. The use of gene transfection technology enabled the high expression of miR-29b can enhance the apoptosis of trophoblast cells. Whereas, miR-29b inhibitors have opposite resμlts. SiRNA-Mcl-1also induced the apoptosis of trophoblast cells. MiR-29b inhibits invasion of trophoblast cell lines and decreases capillary tube and network formation of HTR-8/SVneo. However, no significant change was observed on cell proliferation and cell cycle after up-regμlation of miR-29b. MiR-29b targets MMP2, Mcl-1, VEGFA and integrin β1in mRNA and protein levels. Luciferase experiments show that MMP2, Mcl-1, VEGFA and integrin β1are direct targets of miR-29b. Western analysis revealed that transfection of HTR-8/SVneo and BeWo cells with pre-miR-29b reduced the expression of pFAK in both two cell lines, while pERK1/2expression was unaffected (both P<0.01).3. The expression of these target genes Mcl-1, MMP2, VEGFA and integrin β1in24severe PE patients were markedly lower compared to26healthy controls (both P<0.01). The Pearson correlation analysis indicate an inverse correlation between miR-29b and these genes (miR-29b and Mcl-1mRNA:r=-0.6688, P<0.001; miR-29b and MMP2mRNA:r=-0.8080, P<0.001; miR-29b and VEGFA mRNA:r=-0.7190, P<0.001; miR-29b and integrin β1mRNA:r=-0.7586, P<0.01).Conclusions:1. These data show that different miRNAs are deregμlated in severe preeclamptic pregnancies, it provides the preliminary screen for the roles of miRNAs in trophoblast cells.2. MiR-29b may contribute to onset of PE through repression of invasion and angiogensis of trophoblast cells and enhancement of apoptosis of trophoblast cells by regulating these taget genes (MMP2, Mcl-1, VEGF and integrin β1)which are involved in these processes.3. According to the roles of miR-29b in pathogenesis of PE, it maybe become the key diagnosis and treatment target of PE.