The Study Of MiR-365 Mediated SGK1 Expression In Trophoblast Apoptosis

Part I Expression analysis of mi R-365 in spontaneous abortion decidua and its effect on the trophoblast apoptosisObjective: To explore the potential role of dysregulated mi RNAs in decidua of spontaneous abortion as an approach to elucidate the role of mi R-365 in trophoblast apoptosis behind spontaneous abortion.Methods: mi RNA microarry was used to screen the dysregulated mi RNAs in spontaneous abortion decidua,the results of microrry were verified by quantitative real-time PCR(q RT-PCR);p53-and MDM2-promoter luciferase reporter system were used to screen p53-and MDM2-related mi RNAs;mi R-365-lentivirus vector was constructed,and after transfected HTR-8/SVneo cell,the m RNA and protein expression of p53 and MDM2 were detected by q RT-PCR and Western Blot(WB),respectively,and apoptosis was checked by flow cytometry at different time points,transmission electron microscope observed the ultrastructural changes.Results:7 up-regulated mi RNAs and 10 down-regulated mi RNAs were screened from the spontaneous abortion decidua,in which mi R-365 could reduce the activity of MDM2 significantly(P < 0.01),and significantly increase the activity of P53(P <0.01);MDM2 m RNA and protein levels were significant low expression(P < 0.01),while p53 m RNA and protein levels were significant high expression in the HTR-8/SVneo cells overexpressing mi R-365,and all of them were time dependent;The results of flow cytometry and transmission electron microscopy showed that overexpressed mi R-365 induced trophoblast apoptosis in a time dependent manner.Conclusions: These differentially expressed mi RNAs in spontaneous abortion decidua,suggesting that mi RNA may be related to the spontaneous abortion;The up-regulated mi R-365 promotes the trophoblas apoptosis,suggesting that the high expression of mi R-365 in decidua may be involved in the pathogenesis of spontaneous abortion by increasing the trophoblast apoptosis.Part II Preliminary study on the mechanism of mi R-365 regulating SGK1 signaling pathway on trophoblast apoptosisObjective: To explore the potential target genes of mi R-365 inducing trophoblastic apoptosis,and to elucidate the molecular mechanism of mi R-365 mediated trophoblas tapoptosis by Serum-and glucocorticoid-inducible kinase 1(SGK1).Methods: Bioinformatics analysis was performed to predict the potential target genes of mi R-365;mi R-365 overexpression cell model was constructed and the expression of candidate target genes was detected by q RT-PCR,and the conservatism and homology of mi R-365 and its target genes were predicted by bioinformatics analysisd;SGK1 and SGK3 3’UTR luciferase report system was constructed to screen the mi R-365 target gene;q RT-PCR and WB detected the expression of SGK1 m RNA and protein in spontaneous abortion decidua;flow cytometry to detect the cell apoptosis and transmission electron microscope to observe the ultrastructural changes of trophoblast cells at different time points after transfection of Pgenesil-1-SGK1-sh RNA;HTR-8/SVneo cells were co-transfected with mi R-365 and SGK1 lentivirus,p53/MDM2 expression were detected by q RT-PCR and WB,apoptosis were detected by flow cytometry,and ultrastructural changes of trophoblast cells were observed by transmission electron microscope.Results: Among the 5 candidate target genes,overexpressed mi R-365 could significantly inhibit the expression of SGK1 and SGK3(P<0.01),but had little effect on MAP3K13,MAPK2 and MAPK11P1 L,and the complementary binding sequence of mi R-365 and SGK1,SGK3 was highly conserved and homologous up to 100%;Overexpression of mi R-365 significantly inhibited the luciferase activity of SGK1(P< 0.01),but had no significant effect on SGK3 activity;The expression of SGK1 m RNA and protein in the decidua of spontaneous abortion was significantly down-regulated(P < 0.01),and was negatively correlated with the expression of mi R-365;Flow cytometry and transmission electron microscopy showed that knockout SGK1 would induce apoptosis in a time dependent manner;SGK1overexpression can reverse the effect of mi R-365 overexpression on p53 and MDM2 m RNA and protein expression,as well as on the apoptosis of trophoblast cells.Conclusion: mi R-365 target regulating SGK1 by specifically binding its 3’UTR sequence;SGK1 gene knockout is similar to the effect of mi R-365 overexpression in regulating trophoblast apoptosis,suggesting that SGK1 can be used as a functional target for mi R-365;Overexpressed SGK1 can reverse the effect of mi R-365 overexpression on MDM2 and P53 induced apoptosis,suggesting that the role of mi R-365 in spontaneous abortion may be achieve by SGK1 inducing trophoblastic apoptosis.