The Role Of Long Non-coding RNA In Maternal-fetal Immune Tolerance And Recurrent Spontaneous Abortion

BackgroundAs the only exception of immunology,maternal-fetal immune tolerance has been the focus and difficulty of immunological research.During normal physiological pregnancy,embryos contain half of the paternal genetic material but do not cause maternal immune system attack.The immune tolerance balance established by decidual immune cells at the maternal-fetal interface plays an important role in this process.Therefore,it is of great clinical value to explore the causes and mechanisms underlying the formation of immune tolerance at the maternal-fetal interface.The maternal-fetal interface is mainly composed of three types of cells:embryo-derived trophoblast cells,maternal-derived decidual immune cells（DIC）and decidual stromal cells（DSC）.The maintenance of trophoblast cells function also plays an important role in successful pregnancy.Trophoblast cells can be divided into syncytiotrophoblastls and cytotrophoblasts through the process of proliferation and differentiation.Syncytiotrophoblast cells form extravillous trophoblast cells,which form cell columns through proliferation or migrate and implant into the maternal endometrium,so as to establish a close connection between the fetus and the mother.Therefore,the ability of proliferation,invasion and migration of trophoblast cells is the key to ensure normal pregnancy.Once the immune tolerant microenvironment of the maternal-fetal interface is broken or the function of trophoblast cells is impaired,recurrent abortion may occur.Long non-coding RNA（lncRNA）has gradually entered people’s field of vision in recent years.It is considered as an important regulatory factor especially in the occurrence and development of tumor.It can regulate the expression of proteins and genes at transcriptional and post-transcriptional levels.However,so far,studies on the establishment and maintenance of lncRNA in the balance of immune tolerance at the maternal-fetal interface and recurrent spontaneous abortion are scarce.During our study,we found that the decidual macrophages in normal pregnancy were mainly M2,while the proportion of M2 in decidual macrophages in patients with recurrent spontaneous abortion was significantly down-regulated.This suggested that M2 decidual macrophages played an important role in maintaining normal pregnancy.In this study,we further explored the specific causes and mechanisms of M2 polarization of decidual macrophages.We found that exosomes secreted by trophoblast cells could induce M2 polarization of macrophages in vitro.Through exosome microarray analysis,we found that lncRNA ZEB2-AS1 was enriched in trophoblast cells derived exosomes of normal pregnancy,and further proved that ZEB2-AS1 played an important role in inducing M2 polarization of decidual macrophages and maintaining the immune tolerance microenvironment at the maternal-fetal interface.On the other hand,by analyzing the exosome microarray,we found that the expression of lncRNA LINC01088 in trophoblast cells derived exosomes of patients with recurrent spontaneous abortion was up-regulated.Therefore,we further detected the expression of LINC01088 in villus tissues of patients with recurrent spontaneous abortion,which was consistent with the microarray results,the expression of LINC01088 was up-regulated in villus tissue of patients with recurrent spontaneous abortion.The effects of LINC01088 on the proliferation,invasion and migration of trophoblast cells were further investigated,and the mechanism of the effects was discussed deeply.In this study,the effect of lncRNA on the establishment of immune tolerance balance at the maternal-fetal interface and function of trophoblast cells were analyzed.From maintaining normal pregnancy to causing recurrent spontaneous abortion,the role of lncRNA in the maternal-fetal interface was discussed in various aspects,providing a new direction and strategy for the treatment and diagnosis of recurrent spontaneous abortion.Objective:Normal pregnancy is inseparable from the establishment of immune tolerance at the maternal-fetal interface.During gestation,a variety of maternal and fetal cells work together and coordinate with each other,forming the maternal-fetal interface microenvironment with the advantage of immune tolerance.Once the advantage of immune tolerance in the maternal-fetal interface microenvironment is broken,recurrent spontaneous abortion may occur.Decidual macrophages account for about 25%of the decidual immune cells,and immunosuppressive macrophages（M2）are predominant.In our study,we found that the proportion of M2 in decidual immune cells of patients with recurrent spontaneous abortion was significantly decreased compared with normal pregnancy.These results indicated that M2 decidual macrophages played an important role in maintaining normal pregnancy,while the reason for the M2 polarization of decidual macrophages remained unclear.Exosomes can carry abundant information from original cells and play an important role in intercellular information communication.LncRNAs are important component of exosomes.Whether trophoblast cells derived exosomes promote M2 polarization of decidual macrophages?What role does lncRNA in exosomes play in this process?The purpose of this study was to investigate the regulation of trophoblast cells derived exosomal lncRNA on decidual macrophage polarization and its role in maintaining maternal-fetal interface immune tolerance.Methods:1.CD14+monocytes were extracted from the decidua tissues of normal pregnancy and recurrent spontaneous abortion patients,and the expression of M2 macrophages in the decidua tissues was detected by fluorescence quantitative PCR.2.The morphology,size,particle size distribution and expression of markers of trophoblast cells derived exosomes were detected by transmission electron microscopy,nano-particle size analysis and western blotting.3.After the exosomes were labeled with DiI,the uptake of exosomes from trophoblast cells by macrophages was observed by confocal microscopy.4.The effects of trophoblast cells derived exosomes on macrophage polarization were detected by fluorescence quantitative PCR and western blotting.5.Trophoblast cells derived exosomes from normal pregnancy and recurrent spontaneous abortion patients were detected by microarray,and screened lncRNA ZEB2-AS1.The overexpression plasmid of ZEB2-AS1 was designed and transfected into macrophages.The effect of ZEB2-AS1 on the polarization of macrophages was detected by fluorescence quantitative PCR.6.In the macrophages incubated with trophoblast cells derived exosomes and transfected with ZEB2-AS1 overexpressed plasmid,western blotting was used to detect the changes of signaling pathway molecules in the macrophages.7.CCK-8 and EdU assays were performed to detect the effect of macrophages after M2 polarization induced by trophoblast cells derived exosomes on the proliferation of trophoblast cells.Results:1.The amount of M2 was reduced in decidual macrophages in patients with recurrent spontaneous abortion.2.Trophoblast cells derived exosomes had a double-layer membrane structure with a diameter of about 100nm,and could express exosomal biomarkers HSP90,TSG101 and CD9.3.Macrophages could uptake trophoblast cells derived exosomes.4.Macrophages incubated with trophoblast cells derived exosomes presented a similar appearance to M2,and the fluorescence quantitative PCR results showed that the expression of M2-related markers increased after exosomes incubation,while M1-related markers had no significant effect.Results of western blotting showed that the expression of arginase-1 in macrophages was up-regulated after exosome incubation.5.In exosomes microarray detection,144 lncRNAs were up-regulated and 230 lncRNAs were down-regulated in the recurrent spontaneous abortion group compared with the normal pregnancy group.6.LncRNA ZEB2-AS1 was detected in M2 macrophages induced by trophoblast cells derived exosomes,which was highly expressed and consistent with the microarray results.Moreover,after transfection of the overexpressed plasmid in macrophages,the fluorescence quantitative PCR results showed that the expression of M2-related markers increased.7.In the macrophages incubated with trophoblast cells derived exosomes and transfected with ZEB2-AS1 overexpressed plasmid,results of western blotting showed that JNK and p38 signaling pathways were activated.8.After the incubation of trophoblast cells,macrophages could also work on trophoblast cells and promote the proliferation of trophoblast cells to maintain the normal pregnancy.Conclusions:Trophoblast cells derived exosomes could carry lncRNA ZEB2-AS1 into macrophages,and promoted the M2 polarization of macrophages by activating JNK and p38 signaling pathways,so as to maintain a normal maternal-fetal immune tolerance microenvironment.Objective:Long noncoding RNA（lncRNA）is a non-coding RNA with a length of more than 200 nucleotides with no protein coding function.At present,many studies have proved that lncRNA plays an important role in the pathological process of many diseases.In our study,we found that the expression of lncRNA LINC01088 was up-regulated in villus tissues of patients with recurrent spontaneous abortion.What effects would the abnormally high expression of LINC01088 have on the biological function of trophoblast cells?Whether it can lead to recurrent spontaneous abortion?It’s unclear.In order to answer these questions,the effects of LINC01088 on the proliferation,invasion,migration and the ability to secrete biological active factors of trophoblast cells were examined.The functions of LINC01088 in trophoblast cells were discussed in various aspects.Methods:1.The cell models of LINC01088 overexpressed were successfully constructed by transfection of LINC01088 overexpressed plasmid.Meanwhile,LINC01088 knockout trophoblast cell lines were constructed by CRISPR/Cas9.2.In the cells with LINC01088 overexpressed or knocked out,CCK-8,EdU,clone formation assay and other methods were used to detect the effect of LINC01088 on the proliferation ability of trophoblast cells.3.In trophoblast cells overexpressed by LINC01088,the effects of LINC01088 on the cell cycle and apoptosis of trophoblast cells were detected by flow cytometry,and the expressions of related molecules in cell cycle and apoptosis were detected by fluorescence quantitative PCR and western blotting.4.In the cells or villus tissues overexpressed or knocked out by LINC01088,the effects of LINC01088 on the invasion and migration ability of trophoblast cells and villus tissues were detected by transwell and villus tissue in vitro culture experiments.5.In the trophoblast cells overexpressed by LINC01088,the effect of LINC01088 on the ability of trophoblast cells to secrete biological active factors was preliminarily detected by fluorescence quantitative PCR assays,and whether the secretion pattern of trophoblast cells changed after LINC01088 overexpressed was observed.Results:1.The expression of LINC01088 in villus tissue of patients with recurrent spontaneous abortion was significantly higher than normal pregnancy.2.Cell models with LINC01088 overexpressed or knocked out were successfully constructed.3.The results of CCK-8,EdU and clone formation assay showed that the proliferation ability of HTR8/SVneo and JAR was significantly weakened in trophoblast cells overexpressed by LINC01088,while the proliferation ability of HTR8/SVneo and JAR was significantly enhanced after LINC01088 was knocked out.4.LINC01088 could inhibit the progression of trophoblast cells’ cell cycle,especially the progression from G1 to S,meanwhile the expression of cyclin E1,CDK2,CDK4 and CDK2 were repressed.5.LINC01088 could significantly promote the apoptosis of trophoblast cells,especially the late apoptosis of trophoblast cells.At the same time,LINC01088 could inhibit the anti-apoptotic molecule Bcl2,and promote the expression of pro-apoptotic molecule Bax and the activation of caspase3.6.LINC01088 suppressed the invasion and migration of trophoblast cells.7.LINC01088 had little effect on the mRNA levels of CCL5,TGF-β,IL-10 and IL-35 in trophoblast cells,but significantly reduced the expression of CXCL12.Conclusions:The expression of LINC01088 was increased in villus tissue of patients with recurrent spontaneous abortion.The highly expressed LINC01088 could significantly inhibit the proliferation,invasion and migration of trophoblast cells,restrain the cell cycle,promote the apoptosis of trophoblast cells and lead to recurrent spontaneous abortion.Meanwhile,LINC01088 could inhibit the expression of CXCL12 in trophoblast cells and change the secretion pattern of trophoblast cells.Objective:In the second part,we had confirmed that LINC01088 could significantly inhibit the proliferation,invasion and migration of trophoblast cells,restrain the cell cycle,promote the apoptosis of trophoblast cells and cause recurrent spontaneous abortion.But the specific mechanism is still unclear.LncRNA has abundant transcripts and a variety of regulatory mechanisms.It can participate in gene regulation through transcription,post-transcriptional regulation,epigenetic regulation and other aspects,thus playing an important role in the pathological process of many diseases.In this study,the localization of LINC01088 in cells were firstly disscussed,and RNA pull-down combined with protein spectrum analysis and western blotting were used to find out the proteins that bind to LINC01088 and might work.The mechanism of LINC01088 was explored gradually.Methods:1.Cytoplasmic and nuclear RNA of trophoblast cells were isolated,and fluorescence quantitative PCR was performed to determine the location of LINC01088 in trophoblast cells.2.RNA pull-down and protein spectrum were used to detect the proteins bind to LINC01088.Western blotting was used to verify the results,and the role of arginase-1 which binds to LINC01088 was identified.3.The effects of LINC01088 on the stability of arginase-1 were detected by fluorescence quantitative PCR and western blotting.4.The effect of LINC01088 on nitric oxide/nitric oxide synthase system was detected by western blotting and nitric oxide detection kit.5.The signaling pathways and molecules that played a role in LINC01088 inhibiting the proliferation,invasion and migration of trophoblast cells and inducing recurrent spontaneous abortion in trophoblast cells were detected by western blotting.Results:1.In HTR8/SVneo and JAR cells,LINC01088 was mainly located in the nucleus.2.LINC01088 could bind to arginase-1 and enhance the stability of arginase-1,but there was no significant effect on the expression level of mRNA.3.LINC01088 could inhibit the expression of endothelial nitric oxide synthase protein level,resulting in the simultaneous decrease of nitric oxide level.4.LINC01088 could significantly activate JNK/p38 MAPK signaling pathway,however it had little effect on β-catenin,NF-κB,p65,IκB and Erk MAPK pathway molecules and their phosphorylation state.Conclusions:In trophoblast cells,LINC01088 was mainly expressed in the nucleus and could bind with arginase-1 to enhance the stability of arginase-1,thus reducing the expression of nitric oxide synthase and resulting in decreased expression of nitric oxide.Reduced nitric oxide could activate the JNK/P38 MAPK signaling pathway,leading to impaired functions such as proliferation,invasion and migration of trophoblast cells and further inducing recurrent spontaneous abortion.