Studies On The Expression And Function Of MicroRNA In Placentas With Preeclampsia

Preeclampsia (PE) is a pregnancy specific disorder which has a high incidence, approximately 9.7% in China[1]. It is the second cause of maternal mortality and is a major threat to maternal and fetus health. The aetiology and pathogenesis of this disease has remained unknown. Evidence showed that the disorder of trophoblast function is associated with the pathogenesis of PE. MicroRNA (miRNA) are noncoding RNAs of 21 to 24 nucleotides, which function as negative regulators of gene expression by antisense complimentarily to specific messenger RNAs and subsequent induction of translation inhibition, which can also be associated with transcript destabilization. It has been reported previously that miR-210 and miR-182 are expressed differentially in the human placentas of patients with PE compared with controls using real time RT-PCR analysis[2], suggesting that PE is associated with alterations in placental microRNA expression. However, neither a comprehensive list of the human miRNA nor the function study of placental microRNA has been screened in PE placentas. Thus, it is necessary to unravel the expression and function of the miRNA in PE placentas. Our study may provide a useful tool in studing the aetiology of PE.Objective1. To investigate the expression of miRNA 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 miRNA in the regulation of target gene expression and functions of trophoblast function.Methods1. MicroRNA microarray (Exiqon, release 8.1) analysis was performed on 8 of the matched tissue pairs from the control and sPE groups. Two softwares for bioinformatics anaylsis, targetscan and PITA, were used for miRNA target prediction.2. Expression of ten miRNA in fifteen placentas from severe PE (sPE group), eight placentas from mild PE (mPE group) and eleven from gestational normal pregnancies (control group) were confirmed using real time RT-PCR.3. The differentially expressed miRNA in PE placentas, miR-152, was selected for target verification. We transfected pre-miR-152 into JEG-3 cells, and then determined the levels of HLA-G mRNA and protein in JEG-3 cells using RT-PCR and Western blot analysis, respectively. The verification of miRNA-152 target site was performed using luciferase reporter vector.4. After transfection of pre-miR-152 into JEG-3 cells, invasion assay and NK cell cytotoxicity assay were performed to investigate the effect of miR-152 on JEG-3 cell invasion and whether overexpression of miR-152 in JEG-3 cells could be associated with increased susceptibility to NK lysis.Results1. Thirty-four microRNA were differentially expressed in preeclamptic placentas compared with that of normal placentas. Of these, 11 were overexpressed and 23 were underexpressed in preeclamptic pregnancies. The potential targets of miR-30a-3p, miR-152 and miR-182, including IGF1, HLA-G and ESR1, respectively, were predicted using bioinformatics anaylsis. 2. Among the ten miRNA which selected for real-time RT-PCR verification, 8 of 10 real-time RT-PCR data were concordant with the microarray data: sPE-overexpressed miR-210, miR-152, miR-518b and sPE-underexpressed miR-377, miR-411, miR-542-3p, miR-18a, miR-363 (P<0.05); mPE-overexpressed miR-152 and mPE-underexpressed miR-210, miR-377, miR-411 (P<0.05).3. We transfected pre-miR-152 into JEG-3 cells. RT-PCR analysis showed that the transfection was effective, while no significant changes were observed in the mRNA level of HLA-G (P>0.05). However, the decreased HLA-G protein level was observed using western blot analysis (P<0.05). We performed a pMIR-REPORT luciferase reporter assay and observed a significant decrease (≈47%) of luciferase activity in the presence of pre-miR-152 in JEG-3 cells. These results experimentally confirm that HLA-G is a direct target for miR-152 in JEG-3 cells.4. The results of invasion assay showed that there was no difference in invasion among the test groups (P>0.05). However, after transfection of pre-miR-152 into JEG-3 cells, the lytic activity of NK-92 cells toward JEG-3 cells was dramatically enhanced (P<0.05) versus that in the control groups.Conclusion1. The results show that different microRNA are deregulated in preeclamptic pregnancies, suggesting the involvement of these microRNA in the pathogenesis of preeclampsia.2. These results experimentally confirm that HLA-G is a direct target for miR-152, suggesting the potential role of miR-152 in the etiology of PE through regulating HLA-G.3. Our results showed that overexpression of miR-152 led to increased NK cell mediated cytolysis in JEG-3 cells through regulating HLA-G. This study may provide a useful tool in the gene treatment of PE and choriocarcinoma.