The Expression And Mechanism Research Of MicroRNA-575 In Missed Abortion

Objective:This study examined the expression of miR-575 in patients with missed abortion(MA)and analyzed its clinical significance,and analyzed the effect of miR-575 on human chorionic cellsapoptosis and its mechanism,which could provide a reliable basis for clinical prevention and treatment of missed abortion.Method:In this study,30 patients(MA group)and 30fertile women(control group)were selected from the group of seven gestational weeks in our hospital obstetrics and gynecology clinic from March 2015 to October 2015.(1)The expression of miR-575 was detected by fluorescence quantitative PCR.The apoptosis rate of miR-575 was detected by TUNEL method,and the correlation between miR-575 and tissue apoptosis was analyzed.(2)Human choriocarcinoma cells JEG-3were transfected with miR-575 scramble,inhibitor and mimic.JEG-3 cells were divided into control group,miR-575 scramble group,miR-575 inhibitor group and miR-575 mimic group.The expression of miR-575 mRNA and protein was detected by fluorescence quantitative PCRand western blot in four groups cells.The MTT assay was used to detect the proliferative activity of the cells.Flow cytometry was used to detect the apoptotic rate.The expression of Bcl-2,Bax,p53,VEGF,Ang-2 mRNA and protein were detected byfluorescence quantitative PCR and western blot.(3)Bioinformatics software predicts the target gene of miR-575.The SOD23'UTR WT and SOD2 3'UTR Mut plasmids containing the luciferase reporter gene wereco-transfected JEG-3cellswith miR-575 respectivelyto theluciferase expression level.The expression of SOD2 mRNA and protein in control group,miR-575 scramble group,miR-575 inhibitor group and miR-575 mimic group were detected by fluorescence quantitative PCR and western blot respectively.JEG-3 cells were transfected intopc-SOD2 and the expression of SOD2 mRNA and protein in control group and pc-SOD2 group was detected.JEG-3 cells were divided into control group,inhibitor group and miR-575 inhibitor + pc-SOD2 group.The expression of Ang-2,VEGF mRNA and protein were detected and the apoptosis rate was detected by flow cytometry.Results:(1)The expression of miR-575 in the villi tissue of MA group was significantly higher than that in the normal control group(P = 0.033).Compared with the normal control group,the apoptotic rate of villi cells in the MA group was significantly higher(P = 0.008).Spearman rank correlation analysis showed that the apoptotic rate of villous cells in patients with MA was positively correlated with the expression of miR-575(r = 0.419,P = 0.001).(2)Compared with miR-575 scramble group,the expression of miR-575 in inhibitor group was significantly decreased(t =3.217,P = 0.044),and the apoptosis rate of miR-575 inhibitor groupwas significantly lower(t = 3.566,P = 0.042).The expression of miR-575 in mimic group was significantly increased(t = 4.773,P = 0.007),and the apoptotic rate was significantly higher(t = 3.859,P = 0.023).Compared with miR-575 scramble group,the proliferation rate of miR-575 in inhibitor group cell was significantly increased at 48,72 and 96 h(P<0.05),while that of miR-575 in mimic group cells was significantly decreased(P<0.05).Compared with miR-575 scramble group,the expression of Bcl-2,Ang-2 and VEGF mRNA and protein in inhibitor group was significantly increased,and the expression of Bax and p53 mRNA and protein was significantly decreased,and the expression of Bcl-2,Ang-2 and VEGF mRNA and protein in mimicgroup was significantly decreased,and the expression of Bax and p53 mRNA and protein was significantly increased(P<0.05).(3)SOD2 is predicted as a target gene for miR-575.Compared with the normal control group,the expression of SOD2 mRNA(t = 3.122,P = 0.048)and protein(t = 3.245,P = 0.046)were significantly decreased in the villi tissue of MA group.The expression of luciferase in miR-575 inhibitor + SOD2 3'UTR WT group was significantly lower than that in miR-control + SOD2 3'UTR WT group(t = 4.618,P = 0,005).There was no significant difference between the expression level of luciferase in miR-575 inhibitor+ SOD2 3'UTR Mut group and miR-control + SOD2 3'UTR Mut group(t = 0.245,T=0.911).Compared with the control group,the expression of SOD2 rmRNA(t =0.349,P = 0.044)and protein(t = 0.388,P = 0.040)in pc-SOD2 group was significantly higher.Compared with Scramble group,the expression of SOD2 mRNA and protein were significantly decreased in miR-575 mimic group(P<0.01),on the contrary increased in miR-575 inhibitor group(P<0.01).Theexpression of Ang-2,VEGF mRNA and protein in the inhibitor group was significantly higher than that in the control group(P<0.05),and the apoptotic rate was significantly decreased(P<0.01).The expression of Ang-2,VEGF mRNA and protein in theinhibitor +pc-SOD2 group was significantly decreased than that in the control group(P<0.05),and the apoptotic rate was not statistically different(P>0.05).Conclusion:MiR-575 may inhibit the expression of SOD2 by targeting,and then reduce the expression of Ang-2 to promote the apoptosis of villous trophoblast cells,and restrain the angiogenesis by controling the expression of VEGF,and finally lead to the occurrence of missed abortion.