Studies On The Expression And Function Of RSA-related MiRNA-27a And Leptin

Objective:1. To investigate the different expression ofiniRNA-27a in the chorionic villi of Recurrent spontaneous abortion.and normal pregnancy, and verify the results of gene chip.2. Over-expressing and inhibiting the expression of miRNA-27a in HTR-8cells.To investigate the influence of miRNA-27a on the Leptin, to explore the relationship between miRNA-27a and leptin regulation3.To.study the.impact.of miRNA-27a on apoptosis.ability of.HTR-8cell, to investigate the role of miRNA-27a in recurrent spontaneous abortion, and provide a theoretical basis for the study of recurrent spontaneous abortion. Methods:1.To detect the expression of miRNA-27a in villus of15cases of normal pregnant women(control group) and15cases of patients with recurrent spontaneous abortion(test group)by real time fluorescent quantitative PCR(RT-PCR).2.To over-express and inhibit the expression of miR-27a in HTR-8cells, to detect the expression levels of miR-27a and Leptin mRNA in HTR-8cells by RT-PCR, and to detect the expression levels of leptin protein by Western-blot, in order to validate negative regulation relationships between miR-27a and leptin.3.To..detect.HTR-8cell.apoptosis.changes.by.using.flow.cytometry.after..over-expressing.and.inhibiting.the..expression.of..miR-27a.in HTR-8cell.Results:1. The result of RT-PCR showed that both of the two groups expressed miR-27a in the mRNA level, but the expression of miR-27a of the.group.of patients.with recurrent.spontaneous.abortion.was higher than the group of normal.pregnant.women(P<0.001),the.difference.has.statisti cal significance 2. After over-expressing the expression of miRNA-27a in human trophoblast cell line (HTR-8), RT-PCR showed the expression of miRNA-27a increased (P<0.05), and the expression of Leptin mRNA decreased (P<0.05), the difference was statistically significant; after inhibiting the expression of miRNA-27a, RT-PCR showed that the expression of miRNA-27a decreased (P＜0.05), and the expression of Leptin mRNA increased (P＜0.05), the difference has statistical significance; Western-blot test results showed that over-expressing the expression of miRNA-27a, the expression of Leptin protein decreased (P<0.05), after inhibiting the expression of miRNA-27a, the expression of Leptin protein increased (P<0.05), the difference was statistically significant. That miRNA-27a and Leptin had negative regulation on mRNA and protein level.3.The results of flow cytometry showed that after over-expressing the expression of miRNA-27a, the apoptosis rate of cells increased (P<0.05); and after inhibiting the expression of miRNA-27a, the apoptosis rate of cells decreased (P<0.05), the difference was statistically significant.Conclusion:1. The expression of miR-27a in the RSA group was higher than the control group, which showed that the higher expression of miRNA-27a may be associated with RSA.2. As the target role of miR-27a, leptin was a clear negative regulatory role of miR-27a in the mRNA and protein levels.3. Up regulated the expression of miR-27a in HTR-8cells could increase the apoptosis rate of HTR-8cells, miRNA-27a which is pathological higher expression in the patients with recurrent spontaneous abortion possibly participated in the pathogenesis of RSA through its function for trophoblast cells.