BBOX1-AS1 Regulates The Molecular Mechanism Of GADD45A By Binding To HnRNPK And Its Role In Recurrent Pregnancy Loss

BackgroundRecurrent pregnancy loss(RPL)is defined as two or more consecutive pregnancy failures occurring before 24 weeks of gestation.It is a common pathological problem during pregnancy and its clinical etiology is complex and unclear.Currently known causes include chromosomal abnormalities of parents or embryos,malformations of reproductive anatomy organs,infectious factors,immune factors and maternal endocrine abnormalities.In addition,the advanced age of the pregnant woman is also a high-risk factor for RPL.The etiology is still unknown in 50%of patients,which seriously affects the physical and mental health of women of childbearing age,and the incidence has been on the rise in recent years.Therefore,there is an urgent need for us to investigate its pathogenesis in depth and provide new ideas for clinical treatment.As a medium of communication between fetus and mother,the normal physiological state of the maternal-fetal interface is closely related to the establishment and maintenance of a successful pregnancy.It is composed of fetal-derived trophoblast cells,maternal decidual stromal cells,decidual immune active cells,epithelial cells and stromal cells,which can secrete various cytokines,chemokines,hormones,etc.Together,they constitute the special immune microenvironment at the maternal-fetal interface and maintaining normal fetal development.Trophoblast cells are the only cells of fetal origin.Moderate proliferation,migration and invasion of trophoblast cells during early gestation are essential for placental formation and fetal development.The maintenance of normal biological function of trophoblast cells is regulated by a variety of factors,and trophoblast dysfunction leads to a series of pregnancy-related disorders,including fetal growth restriction(FGR),preeclampsia(PE),recurrent pregnancy loss,etc.Therefore,the study of trophoblast function may provide theoretical basis for refining the pathogenesis of RPL.With the widespread application of high-throughput sequencing and microarray technologies in human disease research,studies have found that only about 2%of the human genome encodes for proteins,while at least 90%of the genome is transcribed into non-coding RNAs.LncRNAs are greater than 200 nucleotides in length but do not encode proteins.LncRNAs interact with DNA,RNA and protein,participate in the regulation of protein-coding genes at various levels,including epigenetic,transcriptional and post-transcriptional.It is involved in participate in cell proliferation,chromatin remodeling,genomic imprinting and other biological processes.Studies have shown that abnormal regulation of lncRNAs is related to the occurrence of various diseases,such as tumors and cardiovascular diseases.In recent years,the role of lncRNAs in reproductive diseases has received attention.In this study,lncRNA expression profile microarrays were used to screen differentially expressed lncRNAs in the villous tissues of RPL and normal pregnancy control group,it was found that the expression of BBOX1-AS1 in the villous tissues and serum of RPL patients was significantly increased.The effect of BBOX1-AS1 on the biological function of trophoblast cells was detected through cell function assays and villus explants culture,the specific mechanism was further explored by RNA-Seq,RNA pull-down assay,mass spectrometry and double luciferase reporter assay.In conclusion,this study reveals that the BBOX1-AS1/hnRNPK/GADD45A regulatory axis plays an important role in regulating trophoblast dysfunction,and providing new insights for improving the pathogenesis of RPL.Section Ⅰ:BBOX1-AS1 affects the biological function of trophoblast cells and is associated with recurrent pregnancy lossObjective:The normal physiological function of trophoblast cells is the basis of embryo implantation and normal development.In recent years,lncRNAs have been found to be closely related to the occurrence and regulation of pregnancy-related diseases.In order to explore the role of lncRNA in RPL,lncRNA expression profile microarray was used to screen lncRNA with differential expression in villous tissues of normal pregnancy control group and RPL,and it was found that lncRNA BBOX1-AS1 was abnormally highly expressed in villous tissues of RPL.At present,the role of BBOX1-AS1 in RPL has not been studied,and the effect of abnormal expression of BBOX1-AS1 on the biological function of trophoblast cells remains unclear.In this part of the study,we explored the expression of BBOX1-AS1 in villus tissue and serum of RPL,the effects of BBOX1-AS1 on biological functions such as cell proliferation,migration,invasion and apoptosis were investigated by trophoblast cell model.The regulation of BBOX1-AS1 on the biological function of trophoblast cells was investigated.Methods:1.LncRNA expression profile microarray was used to detect the expression of lncRNA in villus tissues of RPL and normal pregnancy control group,and screen out the differentially expressed lncRNAs in the two groups;the results were verified by quantitative real-time PCR(qRT-PCR).2.qRT-PCR was used to detect the expression of BBOX1-AS1 in villus tissue and serum of RPL,in addition,the expression of BBOX1-AS1 in four trophoblast cell lines was examined.3.BBOX1-AS1 overexpression and knockdown trophoblast cell models were constructed,the effects of BBOX1-AS1 on trophoblast cell proliferation and apoptosis were detected by CCK-8 assay,clone formation assay,EdU assay and flow cytometry.4.Transwell assay and tubular formation assay were used to determine the effects of BBOX1-AS1 on trophoblast migration,invasion and tubular formation ability.5.The effect of BBOX1-AS1 on trophoblast migration of villous explants was detected by villous explants culture.6.The effect of BBOX1-AS1 overexpression on the expression of trophoblast-derived cytokines and chemokines was examined by qRT-PCR.Results:1.Compared with normal pregnancy controls,LncRNA BBOX1-AS1,DANT1 and OXSM were upregulated in villus tissues of RPL,and their expression trend was consistent with the microarray results,especially BBOX1-AS1 expression was most significantly elevated.2.Compared with normal pregnancy controls,the expression of BBOX1-AS 1 was increased in the serum of RPL,and ROC curve analysis showed that serum BBOX1-AS1 had higher diagnostic efficacy for RPL(AUC=0.814)3.The CCK-8 assay,clone formation assay and EdU assay showed that overexpression of BBOX1-AS1 inhibited the proliferation ability of HTR8/SVneo and JAR cells;while knockdown of BBOX1-AS1 significantly enhanced the proliferation ability of HTR8/SVneo and JAR cells.4.BBOX1-AS1 overexpression promoted the expression of the pro-apoptotic molecule BAX and the activation of caspase-3,and down-regulated the expression of the anti-apoptotic molecule BCL2,which in turn promoted the apoptosis of trophoblast cells.5.BBOX1-AS1 inhibits the migration,invasion and tube formation ability of trophoblast cells.6.BBOX1-AS1 promotes the expression of trophoblast-derived IL-1β.Conclusions:A high throughput lncRNA expression profiling microarray was used to screen differentially expressed lncRNAs in the villous tissue of RPL and normal early pregnant women,and found that BBOX1-AS1 expression was significantly elevated in both villous tissue and serum of RPL.The results of cell function assays and chorionic villus explant culture showed that BBOX1-AS1 could regulate the proliferation,migration and invasion of trophoblast cells.The increased expression of BBOX1-AS1 in the serum of RPL suggests that BBOX1-AS1 may be a potential target for the diagnosis of RPL.Section Ⅱ:BBOX1-AS1 regulates the expression of the downstream target gene GADD45AObjective:In the first part of the study,we found that BBOX1-AS1 regulates the biological function of trophoblast cells,but the specific regulatory mechanism is not clear.Studies have shown that antisense lncRNA exerts biological effects through cis-regulation or trans-regulation of gene expression.To investigate the specific mechanism by which BBOX1-AS1 exerts its biological effects,in this part of the study,RNA-seq was performed on trophoblast cells overexpressing BBOX1-AS1 to analyze and screen downstream target genes,and explored the effects of BBOX1-AS1 on downstream signaling pathways to further elucidate the mechanism of BBOX1-AS1 affecting the biological functions of trophoblast cells.Methods:1.qRT-PCR was used to detect the effect of BBOX1-AS1 overexpression on BBOX1 mRNA level in trophoblast cells.2.RNA-seq and bioinformatics analysis of BBOX1-AS1 regulated gene expression profile and related signaling pathways.3.qRT-PCR and western blot assay were used to detect the expression of downstream target genes in villus tissues of RPL.4.The expression of GADD45A in the placenta of abortion prone pregnant mice was detected by immunohistochemical staining.5.Western blot assay was used to detect the effect of GADD45A on the expression of MAPK signaling pathway related proteins.6.The effects of GADD45A on apoptosis,migration and invasion of trophoblast cells were determined by flow cytometry and transwell assay.7.Knockdown GADD45A in BBOX1-AS1 overexpressing trophoblast cells and its effect on the biological function of trophoblast cells was evaluated by clone formation assay,transwell assay,tubular formation assay and villus explants culture.Results:1.BBOX1-AS1 overexpression had no significant effect on BBOX1 mRNA levels in trophoblast cells.2.RNA-seq results showed that 320 genes were up-regulated and 376 genes were down-regulated(fold change>2,p<0.05).KEGG signaling pathway analysis showed that differentially expressed genes were highly correlated with MAPK signaling pathway.3.BBOX1-AS1 regulates p38 and JNK MAPK signaling pathways,and promotes the expression of target gene GADD45A.4.The mRNA and protein levels of GADD45A in villus tissue of RPL were higher than normal pregnancy controls.5.Immunohistochemical staining showed that the expression of GADD45A was elevated in placental tissues of abortion prone pregnant mice.6.Western blot showed that BBOX1-AS1 regulates p38 and JNK MAPK signaling pathways through GADD45A.7.Overexpression of GADD45A promoted trophoblast apoptosis and inhibited trophoblast migration and invasion ability.8.Rescue assay results showed that BBOX1-AS1 regulates biological functions such as proliferation,migration,invasion and tube formation of trophoblast cells through GADD45A.Conclusions:RNA-seq was performed on HTR-8/SVneo cells overexpressing BBOX1-AS1 showed that BBOX1-AS1 was involved in regulating p38 and JNK MAPK signaling pathways,and promoting the expression of target gene GADD45A.It was further found that GADD45A inhibit the proliferation,migration and invasion of trophoblast cells.In addition,GADD45A knockdown reversed the inhibitory effect of BBOX1-AS1 on the proliferation,migration,invasion and tubular formation of trophoblast cells.These results suggest that BBOX1-AS1 regulates the biological functions of trophoblast cells through G ADD45 A.Section III:BBOX1-AS1 regulates the stability of GADD45A mRNA by binding hnRNPKObjective:The above study found that BBOX1-AS1 regulates the expression of the downstream target gene GADD45A,which in turn regulates the biological function of trophoblast cells,but the specific regulatory mechanism remains unclear.Studies have shown that lncRNA regulates gene expression at multiple levels,including epigenetic,transcriptional and post-transcriptional levels.In this part of the study,we will further clarify the localization of BBOX1-AS 1 in trophoblast cells,identify the binding proteins to BBOX1-AS 1 by RNA pull-down assay,protein mass spectrometry and RIP assay,and further explore the possible mechanism.Methods:1.Fluorescence in situ hybridization(FISH)and subcellular fractionation assay were used to detect the subcellular localization of BBOX1-AS1 in trophoblast cells.2.Dual luciferase reporter assay was used to detect the effect of BBOX1-AS1 on GADD45A gene promoter activity.3.Trophoblast cells overexpressing BBOX1-AS1 were treated with actinomycin D,and GADD45A mRNA levels were detected by qRT-PCR;trophoblast cells overexpressing BBOX1-AS1 were treated with cycloheximide,and GADD45A protein levels were detected by western blot assay.4.RNA pull-down assays,protein mass spectrometry and RIP assays were performed to identify proteins binding to BBOX1-AS1.5.Trophoblast cells with hnRNPK knockdown were treated with actinomycin D,and GADD45A mRNA levels were detected by qRT-PCR.Results:1.RNA FISH and subcellular fractionation assay showed that BBOX1-AS1 existed in both the cytoplasm and nucleus of trophoblast cells.2.Dual luciferase reporter assay showed that BBOX1-AS1 had no significant effect on the promoter activity of GADD45A gene.3.BBOX1-AS1 overexpression promoted the stability of GADD45A mRNA and had no significant effect on GADD45 A protein stability.4.BBOX1-AS1 binding to hnRNPK protein promotes the stability of GADD45A mRNA,Conclusions:BBOX1-AS1 is localized in the cytoplasm and nucleus of trophoblast cells.Further studies revealed that BBOX1-AS1 binding to hnRNPK protein and promoting the stability of GADD45A mRNA,which in turn mediates the abnormal biological function of trophoblast cells.These results suggest that the BBOXl-AS1/hnRNPK/GADD45A regulatory axis plays an important role in trophoblast-induced RPL.