The Expression And Mechanism Research Of Mir-93 In Unexplained Recurrent Spontaneous Abortion

BackgroundRecurrent spontaneous abortion(RSA)refers to two or more consecutive spontaneous abortions in early pregnancy.The incidence of RSA in women of childbearing age is 2-5%,accounting for 15-20%of all spontaneous abortions.RSA is one of the most common abnormal pregnancies in clinical practice.The causes of recurrent abortion are very complex,including genetic factors(such as chromosomal abnormalities),endocrine factors(such as hypothyroidism),immune factors(autoimmune diseases),thrombotic diseases and infections.In addition,more than half of patients with unexplained recurrent spontaneous abortion(unexplained recurrent spontaneous abortion,URSA)are still unknown abortion。The etiology of URSA is complex and diverse,which brings great trouble to patients.At present,there is no clear method to predict URSA or effective methods to prevent the recurrence of URSA.Therefore,it is one of the hot issues in the field of Reproduction to find out the possible pathogenesis of URSA in the new field.There are many studies on the mechanism of URSA,among which microRNAs(miRNAs),as a kind of evolutionarily conserved non coding RNA(ncRNA),have been paid more and more attention because of their important role in gene expression regulation.miRNAs is a sequence of 18-25 nucleotides,which inhibits the expression of target genes by binding with their 3’UTR.Many studies have shown that the abnormal expression of miRNAs plays an important role in the occurrence and development of URSA.The successful implantation of an embryo is the key to the success of pregnancy.After entering the uterine cavity,the embryo contacts with the uterine epithelial cells and gradually embeds in it.The trophoblast cells reach the decidual tissue at 1/3 of the uterine base through physiological invasion,which promotes the recasting and expansion of spiral artery,and then satisfies the nutrition and blood supply between mother and fetus during normal physiological pregnancy.Trophoblast cells are the main functional cells of mammalian placenta.Their biological behavior is similar to tumor cells.The abnormal changes of proliferation,differentiation,invasion and apoptosis of trophoblast cells are considered to be the cytological basis of spontaneous abortion.A large number of miRNAs express in human placenta,which are involved in the proliferation,apoptosis,migration and invasion of trophoblast cells.The abnormal expression of miR-93 is related to the occurrence and development of many kinds of tumors,but the role of miR-93 in URSA has not been reported.In this study,we investigated the expression of miR-93 in human chorionic trophoblast cell line HTR-8/SVneo,and explored the mechanism of miR-93 affecting the function of HTR-8/SVneo cells.Part one Expression of miR-93 in villi of patients with unexplained recurrent abortionObjectiveTo investigate the expression of miR-93 in patients with unexplained recurrent abortion.Methods1.25 women with recurrent spontaneous abortion from 6 to 12 weeks of gestation were selected as RSA group,and 25 healthy pregnant women were selected as control group.2.The expression of miR-93 was detected by real-time PCR,and the location of miR-93 in villi was detected by fluorescence in situ hybridization.Results1.There was no significant difference in maternal age,gestational weeks and BMI between RSA group and normal control group(P>0.05).2.qRT-PCR showed that the expression of miR-93 in RSA group was significantly higher than that in normal pregnancy group(P<0.05).3.Fluorescence in situ hybridization showed that miR-93 was expressed in both RSA group and normal control group,mainly in cytoplasm.Compared with the normal control group,the expression of miR-93 in the villi of RSA group was significantly higher than that of the control group(P<0.05).ConclusionThe expression of miR-93 in the villi of RSA group was significantly higher than that in RSA group,indicating that the decrease of miR-93 may be related to the occurrence of RSA.Part two Effect of miR-93 on biological behavior of human chorionic trophoblast cells HTR-8/SVneoObjectiveTo investigate the effects of miR-93 on the proliferation,migration,invasion and apoptosis of human chorionic trophoblast cells HTR-8/SVneo.Methods1.HTR-8/SVneo cells were transfected with miR mimic negative control,miR-93 mimic,miR inhibitor negative control and miR-93 inhibitor.2.The expression of miR-93 was detected by qRT-PCR.3.MTT assay and EdU assay were used to detect the proliferation activity of the above four groups.4.Cell apoptosis was detected by flow cytometry.5.Transwell assay was used to detect the migration and invasion of the above four groups.Results1.qRT-PCR results showed that the expression of miR-93 in miR-93 mimic group was significantly higher than that in miR mimic negative control group(P<0.05),and the expression level of miR-93 in miR-93 inhibitor group was significantly lower than that in miR inhibitor negative control group(P<0.01).These results indicate that miR-93 mimic can significantly up-regulate the expression of miR-93,and miR-93 inhibitor can significantly inhibit the expression of miR-93,which can be used for subsequent functional experiments.2.Edu results showed that compared with the control group,miR-93 mimic group significantly inhibited the proliferation of HTR-8/SVneo cells(P<0.05),while miR-93 inhibitor group significantly promoted the proliferation of HTR-8/SVneo cells(P<0.05).These results indicate that miR-93 can inhibit the proliferation of HTR-8/SVneo cells.3.MTT results showed that there was no significant difference in cell viability between miR-93 mimic group and miR-93 inhibitor group(P>0.05).4.The results of flow cytometry showed that miR-93 mimic could significantly promote the early apoptosis level of HTR-8/SVneo cells(P<0.05),and could increase the late apoptosis level of HTR-8/SVneo cells,but the difference was not significant(P>0.05).Compared with Mir inhibitor negative control group,miR-93 inhibitor significantly inhibited the early and late apoptosis of HTR-8/SVneo cells(P<0.05).5.The results of cell migration and invasion showed that miR-93 mimic significantly inhibited cell migration compared with the control group(P<0.05),while miR-93 inhibitor could promote cell migration,but the difference was not significant(P>0.05).Compared with the control group,miR-93 mimic could significantly inhibit cell invasion(P<0.05),and miR-93 inhibitor could significantly promote cell invasion(P<0.05).In conclusion,miR-93 can inhibit the migration and invasion of HTR-8/SVneo cells.ConclusionmiR-93 inhibits the proliferation,migration and invasion of human chorionic trophoblast HTR-8/SVneo cells and promotes its apoptosis.Part three Effects of miR-93 on proliferation of HTR-8/SVneo cells by targeting BCL2L2ObjectiveTo investigate the targeting relationship between miR-93 and BCL2L2,and to investigate the effect of miR-93 on the proliferation of HTR-8/SVneo cells by inhibiting the expression of BCL2L2.Methods1.Bioinformatics analysis and prediction of miR-93 target gene.2.Construct the double luciferase reporter gene vector containing wild-type BCL2L2 3’UTR(WT BCL2L2-3’UTR),and construct the double luciferase reporter gene vector of mutant BCL2L2 3’UTR(MUT BCCL2L2-3’UTR).3.miR mimic negative control,miR-93 mimic,miR inhibitor negative control and miR-93 inhibitor were co transfected with WT BCL2L2-3’UTR or mut BCL2L2-3’UTR double luciferase reporter gene vectors,respectively,HTR-8/SVneo cells or HEK-293T cells were co-transfected with miR mimic negative control,miR-93 mimic,miR inhibitor negative control and miR-93 inhibitor.4.miR mimic negative control,miR-93 mimic,miR inhibitor negative control and miR-93 inhibitor were transfected into HTR-8/SVneo cells.The expression of BCL2L2 mRNA was detected by qRT-PCR and the expression of BCL2L2 protein was detected by Western blot.5.Synthesis of BCL2L2 siRNA,real-time PCR detection of BCL2L2 expression,to determine the most suitable BCL2L2 siRNA.BCL2L2 was ligated to pcDNA3.1 to construct the eukaryotic expression vector of BCL2L2.6.HTR-8/SVneo cells were divided into eight groups:miRNA negative control+pcDNA3.1,BCL2L2-pcDNA3.1,miR-93 mimic,BCL2L2-pcdna3.1+miR-93 mimic,miRNA inhibitor negative control+scrambled siRNA,BCL2L2 siRNA,miR-93 inhibitor,BCL2L2-sirna+miR-93 inhibitor.7.MTT assay and EdU assay were used to detect the proliferation of the cells.8.Immunohistochemistry was used to detect the expression of BCL2L2 in villi.Results1.In HEK-293T cells,compared with miR negative control group,miR-93 mimic luciferase activity had no significant difference(P>0.05),compared with miR inhibitor negative control,miR-93 inhibitor significantly increased luciferase activity(P<0.01);in HTR-8/SVneo cells,miR-93 mimic significantly inhibited luciferase activity(P<0.05),compared with Mir negative control group,miR-93 mimic significantly inhibited luciferase activity(P<0.05).Compared with inhibitor negative control,miR-93 inhibitor significantly increased luciferase activity(P<0.05).These results indicate that miR-93 can bind to 3’UTR of BCL2L2 and inhibit the expression of BCL2L2.2.In HTR-8/SVneo cells,compared with the control group co transfected with mut BCL2L2-3’UTR and miR-93 mimic,the luciferase activity of HTR-8/SVneo cells co transfected with miR-93 mimics and wt BCL2L2-3’TR was significantly decreased(P<0.05).Compared with the control group co transfected with mut BCL2L2-3’UTR and miR-93 inhibitor,miR-93 inhibitor and wt were co transfected into HTR-8/SVneo cells After BCL2L2-3’UTR,luciferase activity did not change significantly.Compared with the control group co transfected with mut BCL2L2-3’UTR and miR-93 mimic,the luciferase activity of hek-293t cells co transfected with miR-93 mimics and wt BCL2L2-3’UTR did not change significantly After BCL2L2-3’UTR,luciferase activity increased significantly(P<0.05).These results suggest that miR-93 inhibits its expression by binding to specific regions of BCL2L2,which is the target gene of miR-93.3.Compared with miR mimic negative control,the expression of BCL2L2 protein in miR-93 mimic transfection group was significantly lower than that in miR inhibitor negative control group(P<0.01).Compared with miR inhibitor negative control,BCL2L2 protein expression in miR-93 inhibitor transfected group was significantly increased(P<0.01).The results showed that miR-93 could inhibit the expression of endogenous BCL2L2 protein.Compared with miR mimic negative control,the expression of BCL2L2 mRNA in miR-93 mimic group was not significantly changed.Compared with miR inhibitor negative control,the expression of BCL2L2 mRNA in miR-93 inhibitor group was significantly increased(P<0.01).These results indicate that miR-93 can significantly inhibit the expression of BCL2L2.4.Compared with the control group,the proliferation of transfected cells was significantly inhibited(P<0.05).Compared with miR-93 mimic group,the proliferation of cells transfected with BCL2L2-pcDNA3.1 and miR-93 mimic was significantly increased.Compared with the control group,BCL2L2 siRNA significantly promoted the proliferation of HTR-8/SVneo cells(P<0.05).Compared with miR-93 inhibitor group,the proliferation level of cells transfected with BCL2L2 siRNA and miR-93 inhibitor was significantly decreased.These results suggest that miR-93 inhibits the proliferation of HTR-8/SVneo cells by inhibiting the expression of BCL2L2:5.Compared with the control group,BCL2L2 siRNA significantly promoted the proliferation of HTR-8/SVneo cells(P<0.05).Compared with miR-93 inhibitor group,miR-93 mimic transfection significantly inhibited cell proliferation(P<0.05),while BCL2L2 siRNA and miR-93 inhibitor group significantly decreased cell proliferation.These results suggest that miR-93 inhibits the proliferation of HTR-8/SVneo cells by inhibiting the expression of BCL2L2.ConclusionBCL2L2 is the target gene of miR-93.miR-93 inhibits the proliferation of HTR-8/SVneo cells by inhibiting the expression of BCL2L2.