| Before 20 weeks of pregnancy or the natural loss of fetal weight less than 500 g is called spontaneous abortion,which is a common adverse pregnancy outcome.When a woman has three or more spontaneous abortion histories with the same sex partner,it is called Recurrent Spontaneous Abortion(RSA).Missed abortion is a common clinical manifestation of RSA and a special type of abortion.It refers to that embryos or fetuses have died and can not discharge naturally in the uterine in time.Because the etiology of the disease is complicated,and the pathogenic mechanism is not yet clear,there is a lack of effective preventive measures and treatments.Therefore,research on the pathogenesis and treatment of RSA is one of the current hot issues in reproductive medicine.The success of pregnancy mainly depends on the activity of trophoblasts,the formation of normal embryos and the endometrium in a receiving state.Trophoblasts are the main fetal cells involved in the development of early embryonic villi which have proliferation and invasion characteristics like tumor cells.Insufficient infiltration of placental trophoblasts and poor vascular remodeling will lead to pregnancy complications,including RSA,preeclampsia,eclampsia and intrauterine growth retardation.Some researchers have discovered many risk factors that can cause trophoblast cell dysfunction,including trophoblastic cytotoxic cell immune response,trophoblast antigen and anti-granular cell antibodies,DNA damage,and abnormal angiogenesis that all be related to the occurrence of RSA.Moreover study suggested that the proliferation,migration and invasion capabilities of extravillous trophoblasts are closely related to the occurrence of RSA.Micro RNAs(miRNAs)are a class of non-coding small RNAs composed of19-24 nucleotides.They regulate gene expression by binding to specific sites in the3’UTR to inhibit the expression of messenger RNA(m RNA).miRNAs can regulate cell proliferation,differentiation and apoptosis,which may also regulate the proliferation,invasion and transfer of trophoblasts,and then participate in fertilized egg implantation and embryo development.At present,more and more evidencesshowed that a large number of miRNAs are expressed in human placental tissues.And the expression of miRNAs may be involved in the regulation of embryo implantation and development,then involved in the occurrence of RSA.This research will explore the function of some miRNA in RSA,and the main contents are as follows:1.Analysis of clinical case characteristics of RSAAnalyzing the clinical data of 190 patients of RSA,it was found that age was an independent risk factor for RSA and there is a positive relationship between the age and the probability of abnormal embryonic development.And the blood HCG levels of 14 days after ovulation(post-transplantation)could predict embryonic development.The proportion of abnormal embryonic development in people with infertility history higher than patients without infertility history.With the increase in the number of abortions(natural abortion or artificial abortion),the probability of embryo development stops increasing.The results of high-throughput sequencing(NGS)of aborted villi chromosomes showed that chromosome polyploidy is the most common abnormality.The method of NGS could find abnormal embryonic development caused by chromosome structure abnormalities and chromosome microdeletion/microduplication.2.Screening of different expressed miRNAs in spontaneous abortion villi tissueThree samples in the RSA patients group and the control group were selected for sequencing.The results found that there are a series of different expressed miRNAs in the spontaneous abortion villi tissues.There are a total of 33 different miRNAs related to early embryonic abortion,of which 17 miRNAs are up-regulated and 16 miRNAs are down-regulated in the villi tissues.Then 10 miRNAs with significant different expression were selected for verification by real time PCR(RT-PCR),and 9 of them were found to be consistent with the sequencing results.3.miRNA-515-5p targets the 3’UTR of MCL1 m RNAFluorescence quantitative results showed that miR-515-5p was highly expressed in the villi tissues of spontaneous abortions compared with the villi tissues of normal abortions,which was consistent with the sequencing results.According to the prediction of miRNA target gene prediction database,MCL1 may be the target gene of miR-515-5p.The dual luciferase reporter experiment confirmed that miR-515-5p directly targets the 3’UTR region of MCL1 m RNA.Through overexpression and silencing of miR-515-5p in the human trophoblast cell line HTR8-S/Vneo,miR-515-5p was found to inhibit the expression of MCL1.4.miRNA-515-5p regulates the proliferation and migration of human trophoblasts by targeting MCL1Fluorescence quantification,WB and immunohistochemistry were used to detect the expression of MCL1 in the tissues.The results showed that the expression level of MCL1 in the villi tissues of patients with spontaneous abortion was significantly reduced compare with the villi tissues of normal abortion.The over-expression and silencing of miRNA-515-5p were used to explore the effects of miRNA-515-5p on the proliferation,invasion and migration of HTR8-S/Vneo cells.Using CCK8 and flow cytometry to detect the proliferation of HTR8-S/Vneo cells,the results showed that miR-515-5p inhibited the proliferation of HTR8-S/Vneo cells.The Transwell invasion experiment was further used to study the effect of miR-515-5p on the invasion ability of HTR8-S/Vneo cells,the results showed that the invasion ability of HTR8-S/Vneo cells was enhanced after the expression of mi-515-5p was inhibited.The scratch healing experiment was used to detect the effect of miR-515-5p on the migration ability of HTR8-S/Vneo cells,the results showed that the migration ability of HTR8-S/Vneo cells is enhanced after inhibiting the expression of mi-515-5p.In order to further study whether miR-515-5p regulates the proliferation,invasion and migration of HTR8-S/Vneo cells by targeting MCL1,we adopted the method of over-expression of miR-515-5p and MCL1 at the same time to explore whether it is possible that over-expression MCL1 could rescue the effect of over-expression of miR-515-5p on the function of HTR8-S/Vneo cells.First,CCK8 and flow cytometry were used to detect the proliferation of HTR8-S/Vneo cells.The results showed that the over-expression of MCL1 rescued the inhibitory effect of miR-515-5p on the proliferation of HTR8-S/Vneo cells,indicating that miR-515-5p regulate the proliferation of HTR8-S/Vneo cells through MCL1.The Transwell invasion experiment was further used to study the effect of over-expression of MCL1 and miR-515-5p on the invasion ability of HTR8-S/Vneo cells,and the results showed that the over-expression of MCL1 rescued the invasion of HTR8-S/Vneo cells by miR-515-5p.Thses results indicated that miR-515-5p regulates HTR8-S/Vneo cell invasion through MCL1.The scratch healing experiment was used to detect the effect of over-expression of MCL1 and miR-515-5p on the migration ability ofHTR8-S/Vneo cells,and the results showed that the over-expression of MCL1 rescued the effect of miR-515-5p on the migration of HTR8-S/Vneo cells and indicated that miR-515-5p regulates HTR8-S/Vneo cell migration through MCL1.In summary,this study found that age is an independent risk factor for spontaneous abortion,and blood HCG levels of 14 days after ovulation(after transplantation)can predict embryonic development.With the increase in the number of abortions(natural abortion or artificial abortion),the probability of embryo development stops increasing.The results of high-throughput sequencing(NGS)of aborted villi chromosomes showed that chromosome polyploidy is the most common abnormality and NGS could find abnormal embryonic development caused by chromosome structure abnormalities and chromosome microdeletion/microduplication.miR-515-5p is highly expressed in villi tissue of spontaneous abortion,while MCL1 is low in in villi tissue of spontaneous abortion.It is further found that miR-515-5p inhibits the proliferation,migration and invasion of HTR8-S/Vneo trophoblast cells by targeting MCL1 gene.Therefore,miR-515-5p is a new regulator of MCL1,miR-515-5p may participate in the occurrence of spontaneous abortion by regulating the expression of MCL1,providing a possible theoretical basis for the pathogenesis of spontaneous abortion. |
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