The Expression Of MiRNAs And Its Target Genes In Villi And HTR-8 Cells In Missed Abortion

Objectives 1.To analyze the relevant clinical data and examination indexes related to missed abortion and provide reference advice for preventing the occurrence of missed abortion;2.To explore the correlation between miRNAs and the occurrence of missed abortion;3.To explore the regulation effects of miR-3651 on related target genes.Methods 1.121 patients with missed abortion were recruited as a case group in the family planning clinic of the First Affiliated Hospital of Guangxi Medical UniversityJingxi City People’s Hospital,Debao County Maternal and Child Health Hospital Gynecology Clinic from December 2016 to December2017.98 pregnant women who terminated pregnancy were recruited as the control group.Chi-square test and t test were used to compare the differences of clinical data and examination indexes between the case group and the control group.SPSS 16.0 was used for statistical analysis.A two-tailed p-value of p<0.05 was used as the statistical significance level.2.The villi of 30 case group and 30 control group were collected.Real-time PCR was used to detect the expression of differential miRNA in the selected GEO chip in the two group.The differences of miRNA expression between the two groups were compared by t test For the differential miRNAs,their related target gene were screened bybioinformatics methods.3.Select human trophoblast cell line HTR-8 as the cell model.Using the transient transfection technology,the miR-3651 over expression in the cell was miR-3651 mimics group,miR-3651 inhibits expression was miR-3651 inhibitor group.The negative control group(NC)and blank control group were set to control the experimental conditions.Total RNA was extracted in the cell of all groups after transfection extraction.The expression level of miR-3651 and forecast target gene in all group RNA by RT-PCR.The expression level of each group were compared by t test,which would show miR-3651 regulation function of related target genes.Result1.By comparing the clinical data of the case group and the control group,it was found that the age of ≥ 35 years old,BMI,abortion history,stillbirth history,and infection during pregnancy were statistically significant(p<0.05).Comparing clinical examination indexes in the case group and the control group,it showed that the differences among human chorionic gonadotropin(β-hCG),platelet volume(PCT),platelet volume distribution(PDW),fibrinogen(FIB),prothrombin activity(PTA)were statistically significant(p<0.05).2.Real-time fluorescent quantitative PCR technique was used to verify the expression of differential miRNAs in the villi of case group and control group.The result showed that miR-3651 and miR-4417 were expressed in the chorionic villi of the casen group and the control group.The relative expression level of miR-3651 in the villi was 1.19±0.52 in the case group.The relative expression level in the control group was 0.86±0.46.The relative expression level of miR-4417 in chorionic villi was 1.04±0.60 in the case group and 0.50±0.35 in the control group.The express level of miR-3651 and miR-4417 in the case group was significantly higher than the control group’s,the difference wasstatistically significant by t test(P<0.05).16 miR-3651 target genes and 8miR-4417 target genes were predicted by bioinformatics methods.3.Transient transfection technique was used to over-express and inhibit miR-3651 expression in human trophoblast cell line HTR-8.RT-PCR was used to detect the expression level of miR-3651 and SEC63,ATP6AP2 in transfected cells.The expression level of mRNA showed that the expression level of miR-3651 in the overexpression group was higher than that in the negative control group.The expression levels of SEC63 and ATP6AP2 mRNA were decreased,and the difference was statistically significant(p<0.05).The expression of miR-3651 decreased,while the expression of SEC63 and ATP6AP2 mRNA increased.The difference was statistically significant(p<0.05).conclusion1.The incidence of missed abortion is affected by many factors.Older pregnancy,multiple abortions,history of stillbirth,infection during pregnancy,abnormal hormone levels,abnormal coagulation may be related to the occurrence of missed abortion.2.The expression of miRNA-3651 and miRNA-4417 in villi of the case group was higher than that of the control group,suggesting that miRNA-3651 and miRNA-4417 may be related to the occurrence of missed abortion;3.miR-3651 has a regulatory effect on target genes SEC63 and ATP6AP2.At mRNA level,miR-3651 down-regulates the expression of SEC63 and ATP6AP2.