Expression Of MiR-4417 In Missed Abortion And Its Effect On Cell Proliferation,Apoptosis And Migration

Objective 1.The expression level of miR-4417 and its related the target genes in embryo villus tissues of the missed abortion and the normal with early pregnancy were analysed,to explore the correlation between miR-4417 and missed abortion and miR-4417 of its regulatory role in its target genes;2.The overexpression level of miR-4417 in JEG-3 cell system was established,and to explore the effect of miR-4417 on proliferation,apoptosis and migration of cell biology functions.Methods:1.Case control study was adopted and 50 fetal villi samples from missed abortion patients and 50 fetal villi samples from healthy pregnant women with early pregnancy and accepting artificial termination of pregnancy voluntarily were collected in the family planning outpatient department of the first affiliated hospital of guangxi medical university,and general information,previous history and clinical data of the subjects were also collected.Real-time fluorescence quantitative PCR(RT-PCR)was used to detect the expression level of miR-4417 which was screened by the GEO database and its related target genes which were predicted by bioinformatics method such as RAB6A?RAB6C and CHST2 in the fetal villi tissue of the missed abortion patients and the healthy pregnant women with early pregnancy,and the expression difference of miR-4417 and its related target genes between the two groups was analyzed by independent sample t test,and the Pearson correlation statistical methods was used to analysed the correlation of the expression of miR-4417and its related target genes such as RAB6A,RAB6C,CHST2 in the fetal villi tissue;2.JEG-3 cells of human villous carcinoma was selected as the research model to study the effect of miR-4417 on the biological behavior of cells.Transient transfection technology was used to establish the overexpression system of miR-4417 in JEG-3 cells,and negative control group�miR-4417 mimics NC group and blank control group�the control group were set up.The expression level of miR-4417 in the cell system was detected by RT-PCR for verification.Cell proliferation experiment,cell apoptosis experiment and cell migration experiment were carried out in this cell system,and the cells proliferation activity rate,cells apoptosis rate and migration area of cells were calculated respectively,and to explore the influence of miR-4417 on the biological behavior of JEG-3 cells.Results 1.The results of comparison of general conditions between the case group and the normal control group showed that there were no statistically significant difference between the two groups in the gestational weeks,the age of menarche,the regularity of menstruation,dysmenorrhea,the pregnancy and abortion frequency(P>0.05),and there was a statistical difference in the age between the two groups(t=-3.414,P<0.05).2.PCR technology was used to detect the expression level of miR-4417 and its predicted target genes in the fetal villi tissue of the case group and the normal control group.The results showed that the relative expression level of miR-4417 in the case group was significantly higher than that in the normal control group(t=-2.35,P=0.022).The relative expression level of RAB6A in the case group was significantly lower than that in the normal control group(t=2.37,P=0.020).The relative expression level of RAB6C in the case group was significantly lower than that in the normal control group(t=2.697,P=0.009),however the expression level of CHST2 was not statistically different between the two groups(P>0.05).3.The results of the correlation analysis of the relative expression level of miR-4417 and its predicted target genes in fetal villi tissue of the missed abortion patients and the normal control group showed that the relative expression level of miR-4417 was negatively correlated with the relative expression level of RAB6A in the fetal villi tissue of the missed abortion patients and the normal control group(r=-0.286,P=0.007),and the relative expression level of miR-441 7 was also negatively correlated with the relative expression level of RAB6C(r=-0.223,P=0.026).4.The results of the age stratification analysis of the expression level of miR-4417 in the fetal villi tissue of the case group and the normal control group showed that the relative expression level of miR-4417 in the fetal villi tissue of the case group and the normal control group had no statistical significance between the ages of 19?24 years and 25?30 years(P>0.05).Between the ages of 31 and 35 years,the relative expression level of miR-4417 in the fetal villi tissue of the case group was significantly higher than that in the normal control group(t=-3.21,P=0.004).5.The expression level of miR-4417 in JEG-3 cells which was transfected with miR-4417 mimics was detected by the RT-PCR.The results showed that there was no statistical difference in the relative expression levels of miR-4417 in the control group and the miR-4417 mimics NC group,and the relative expression level of miR-4417 mimics in JEG-3 cells was increased significantly compared with miR-4417 mimics NC group and the control group(P<0.001)respectively,and the overexpression system of miR-4417 is successfully constructed in JEG-3 cells.6.The results of cell proliferation experiment show that:after 72 hours of cell culture,the difference of cell proliferation ability in miR-4417 mimics group,the control group and miR-4417 mimics NC group was statistically significant(F=68.27,P<0.001),after analysing the results of pairwise comparison,the difference of each group of proliferation ability of cells between three groups was statistically significant(P<0.001),and the JEG-3 cell proliferation ability of miR-4417 mimics group was lower than the control group(P<0.001);After 96 hours of cell culture,the results of comparing the difference of the cell proliferation ability of miR-4417 mimics group,the control group and the miR-4417 mimics NC group showed that it is statistically significant(F=50.17,P<0.001),and after analysing the results of pairwise comparison,the difference of each group of proliferation ability of cells between three groups was statistically significant(P<0.001),and the proliferation ability of the JEG-3 cell in miR-4417 mimics group was lower than the control group(P<0.001).7.The results of cell apoptosis experiment showed that the difference of the early apoptosis rate of cells in miR-4417 mimics group,miR-4417 mimics NC group and the control group was statistically significant(F=239.26,P<0.001),after analysing the results of pairwise comparison,the difference of each group of the early apoptosis rate of cells between three groups had statistical significance(P<0.05),and the early apoptosis rate of cells in miR-4417 mimics group was lower than that in the control group(P=0.018).There was a statistically significant difference in the late apoptosis rate of cells in the three groups(F=113.81,P<0.001),after analysing the results of pairwise comparison,there was a statistical difference between the late apoptosis rate of cells of the miR-4417 mimics group and the miR-4417 mimics NC group(P<0.001),and there was a statistical difference between the late apoptosis rate of cells of the control group and the miR-4417 mimics NC group(P<0.001).There was no statistical difference in the late apoptosis rate of cells of the miR-4417 mimics group and the control group(P=0.996).The difference of total apoptosis rate of cells in the three groups was statistically significant(F=114.81,P<0.001),after analysing the results of pairwise comparison,the difference of each group of the total apoptosis rate of cells between three groups had statistical significance(P<0.05),and the total apoptosis rate of cells in the miR-4417mimics group was lower than that in the control group(P=0.027).8.The results of cell migration experiment showed that there was no statistical significance in the area of cell migration of the miR-4417 mimics group,the miR-4417 mimics NC group and the control group after 24 hours(F=1.24,P=0.354),after analysing the results of pairwise comparison,there were no statistically significant differences between the cell migration area of the three groups(P>0.05);There was no statistically significant difference in the cell migration area between the three groups after 48 hours(F=3.41,P=0.102),and after analysing the results of pairwise comparison,there were no statistically significant differences between the cell migration area of the three groups(P>0.05).Conclusion 1.The high expression level of miR-4417 in fetal villi tissue may be related to the occurrence of missed abortion.And the relative expression level of miR-4417 in the fetal villi tissue of the case group was significantly higher than that its in the normal control group in the layer between the age of 31 and 35 years old(t=-3.21,P=0.004).2.RAB6A and RAB6C may be the targets of miR-4417,and miR-4417 may negatively regulate the mRNA expression levels of RAB6A and RAB6C,and them may be the cause of missed abortion.3.In JEG-3 cells,upregulation of miR-4417 expression could inhibit the proliferation and apoptosis of JEG-3 cells,but it may not affect the migration of JEG-3 cells.