Identification Of MicroRAN In Miscarriage And Preganancy Decidua And MΙR-199 Functional Study

Natural state (non-human purpose cause) abortion is called spontaneous abortion. In all clinically recognized pregnancies, the incidence of spontaneous abortion is about 15%. Abortion occurred prior to pregnant 12 weeks is defined as early abortion, occurred from pregnancy 12 weeks to less than 28 weeks is called late abortion. MicroRNAs (miRNAs) are a class of endogenous small non-coding RNA which are completely or non-fully complementary bind to their target mRNA, regulate cell growth, differentiation, proliferation, apoptosis, and disease occurrence by targeting genes.Studies have showed that, in normal gestational placenta, villus and endometrium, the miRNA expressions have certain interactions with their associated target mRNAs, which imply that the miRNA irregular expressions probably involve in the occurrence of pathological pregnancy. However, due to the limited information mainly from microarray or real-time quantitative RT-PCR, the results are inconsistent in decidua. Sequencing analyses of miRNAs in human spontaneous aborted deciduas and early pregnancy deciduas have not been reported. In the present study, the differential expressions of miRNAs in spontaneous abortion deciduas and early pregnant deciduas were studied by sequencing analysis. The possible biological function of miRNA-199-5p and their candidate target mRNAs in the occurrence of spontaneous abortion were investigated.Objectives1. To analyze the full scale of human miRNAomes in normal pregnant and miscarriage deciduas by the use of an in-depth sequencing, and predict the target genes corresponding to the selected candidate miRNAs.2. To detect the the in vivo expression of the target genes.3. To evaluate the relationship between miRNA-199b-5p and SGK1 and their function in pregnancy maintenance in vivo and in vitro.4. To construct a MiR-199b-5p transgenic mouse model to evaluate the possible regulation of miRNA-199b-5p in the maintenance of pregnancy. Methods1. Deciduas derived from 6-8-week spontaneous miscarriages and normal pregnant women were compared by miRNA sequencing and the up-regulated and down-regulated miRNAs were classified.2. MicroRNA detection and identification. Ten miRNA were detected by real-time PCR in 29 cases of spontaneous abortion (abortion group) and 35 cases of early pregnancy decidua (pregnancy group).3. Sequencing data was analyzed by predictive bioimformatic tools. The candidate genes involved in pregnancy establishment and maintenance were identified from previous studies. The expressions of SGK1, IGF2R, MMP10 and LEPR were highlighted.4. Function studies of microRNA-199b-5p:miR-199b-5p were overexpressed or knock-down in HEK293T and Ishikawa cells. SGK1 expression levels were detected by RT-PCR and immunoblotting (Western blot). MiR-199b-5p trangenic mice model was established to determine their possible role in pregnancy maintenance in vivo.Results1. Total of 1921 known miRNAs and 123 novel miRNAs were analyzed in the study, in which 20 miRNAs were down-regulated and 17 miRNA were significantly up-regulated in the aborted group. The three most abundantly expressed miRNAs in the aborted group were hsa-let-7a-5p, hsa-let-7f-5p, and hsa-let-7g-5p, which accounted for 25.63%,21.99%, and 17.82% of the miRNAome, respectively.2. In the 10 miRNAs validated by qRT-PCR 7 of them expressed consistent with the sequencing results. Six miRNAs including let-7a-5p, let-7f-5p, let-7g-5p, let-7e-5p, let-7d-5p, and miR-98 were down-regulated (P<0.05), while miR-199b-5p was significantly up-regulated in the aborted group (P<0.05).3. Total of 108,787 predicted target genes were identified by Mireap analysis to the 37 differentially expressed miRNAs. Some candidate genes involved in decidualization and pregnancy maintenance were identified as potential targets for these differentially expressed microRNAs. We highlighted SGK1, IGF2R, MMP10 and LEPR as potential targets for microRNAs in the aborted deciduas, and proved that SGK1, MMP10 and LEPR decreased and IGF2R increased in aborted deciduas.4. When the HEK293T and Ishikawa cells were transfected with miR-199b-5p inhibitor the SGK1 mRNA and its protein expression increased, while transfected with miR-199b-5p mimic the SGK1 mRNA and its protein expression decreased. Furthermore, the SGK1 mRNA and its protein expression increased when the human endometrial stromal cells were infected with lentiviral miR-199b-5p inhibitor vectors.5. The SGK1 mRNA and protein expression were decreased in the miR-199b-5p transgenic mice.Conclusion1. The MiRNA expression profiles significantly altered between the miscarriage and normal pregnancy deciduas. The results open new insights into the pathogenic mechanisms of spontaneous miscarriages, which imply that miRNAs may be involved in the maintenance of early pregnancy.2. The candidate genes involved in decidualization and pregnancy maintenance were identified as potential targets for these differentially expressed microRNAs, and a collaborative network of microRNAs and mRNAs related to pregnancy maintenance was established. We highlighted the candidate genes as potential targets for microRNAs in the aborted deciduas.3. Significant inverse correlation was found between miR-199b-5p and SGK1 in vivo and in vitro.