Fibronectin 1(FN1)Inhibits The Apoptosis Of Human Trophoblast Cells By Activating The PI3K/Akt Signaling Pathway

Trophoblasts are a special type of placental cells,that play important roles in embryo implantation and the formation of the maternal-fetal interface.After the blastocyst implants into the endometrium,trophoblast cells differentiate into extravillous and chorionic trophoblast cells.Extravillous trophoblast cells play a key role in invading the decidual stroma and blood vessels of the uterus.There are two types of chorionic trophoblast cells:syncytiotrophoblast cells in the outer layer and cytotrophoblast cells in the inner layer.Syncytiotrophoblast cells are located on the surface of the chorion and directly participate in material exchange between the mother and fetus.Cytotrophoblast cells,which are located in the inner layer,have greater proliferation ability and under certain conditions,can differentiate into extravillous trophoblast cells and syncytiotrophoblast cells.The dynamic balance between trophoblast cell proliferation and apoptosis is critical for maintaining of pregnancy,and disruption of this balance can lead to complications,such as preeclampsia or abortion.Studies in China and abroad have shown that the trophoblast apoptosis index is significantly higher in cases of spontaneous abortion than in normal pregnancies.Fibronectin1 is a high molecular weight glycoproteinthat has numerous biological functions.FN1 is present in the extracellular matrix and plays key roles in cell adhesion,growth,migration and differentiation and is involved in maintaining cell morphology.FN1 is significantly up-regulated in uterine leiomyoma and is associated with trophoblast invasion,uterine proliferation and adhesion function.However,the effect of FN1 on trophoblast cells and the underlying molecular mechanism are unknown.Part Ⅰ Bioinformatics analysis shows that FN1 regulates the proliferation and apoptosis of trophoblast cellsObjective:Identifying the key genes and pathways involved in trophoblast differentiation by analyzing GSE127170 data set in GEO.Methods:The GSE127170 data set included 12 samples,including untreated JEG-3 cells and forkolin treated JEG-3 cells,untreated BeWo cells and forkolin treated BeWo cells,untreated JEG-3 and BeWo cells and forkolin treated JEG-3 and BeWo cells.Firstly,DEGs were analyzed,then PPI network was constructed and hub gene was identified.Finally,Gene Ontology(GO)and Kyoto Encyclopedia of genes and genomes(KEGG)pathways were obtained by gene set enrichment analysis(GSEA).Results:1.In gene set 1 of GSE127170 data set,the results showed 903 differentially expressed genes(DEGs)(410 P<0.05 and |logfc |>1)were obtained,including 493 up-regulated and 410 down regulated genes.A total of 17 overlapping Hub genes were identified:Lyn proto oncogene,SCR family tyrosine kinase(Lyn),fibronectin 1(FN1),etc.2.In gene set 2 of GSE127170 data set,the results showed that there were 440 differential genes(DEGs),including 260 up-regulated genes and 180 down regulated genes.A total of 16 overlapping Hub genes were identified:Yap1,itga1 and FN1.3.In gene set 2 of GSE127170 data set,there were 388 differentially expressed genes(DEGs),including 238 up-regulated genes and 150 down regulated genes.A total of 15 overlapping hub genes were identified:placental growth factor(PGF),colony stimulating factor 1 receptor(CSF1R),and FN1.4.Go and KEGG enrichment analysis showed that TGFB1,FN1 and KLF4 were overlapping Hub genes from gene set 1,2 and 3.FN1 was mainly enriched in PI3K/Akt signaling pathway.TGFB1,FN1,and KLF4 was mainly enriched biological process(BP)term including regulation ERK1 and ERK2 cascade,response to wounding,ameboidal-type cell migration,negative regulation of cytokine production,blood vessel development,regulation of cell adhesion,embryonic morphogenesis,ERK1 and ERE2 cascade,blood vessel morphogenesis,and regulation of cytokine production using Metascape website.Conclusion:TGFB1,FN1 and KLF4 are the key genes involved in the regulation of trophoblast proliferation and apoptosis.Go and KEGG analysis showed that FN1 mainly affected PI3K/Akt signaling pathway and participated in cell proliferation and apoptosis.Part Ⅱ FN1 was down-regulated in chorionic villus tissues from the placentas of patients with SA and regulated cell viability and apoptosis in JEG-3 and BeWo cellsObjective:To detect the expression and significance of FN1 in chorionic villus tissues and trophoblast cells.Methods:1.65 cases of spontaneous abortion(SA group)and 65 cases of induced abortion(IA group)were collected.The chorionic villus tissues were taken and the histopathological changes were observed by HE staining.2.The expression of FN1 in chorionic villus tissues of SA group and IA group was detected by immunohistochemistry(IHC).3.The expression of FN1 mRNA in serum of patients was detected by real-time fluorescent quantitative PCR(qRT-PCR).4.The expression of FN1 was up-regulated or down regulated by transfection of FN1 or siFN1 in human trophoblast cells JEG-3 and BeWo.The transfection efficiency was detected by Western blot and qRT-PCR 5.Cell viability was detected by CCK-8 assay.6.Apoptosis was detected by flow cytometry.7.Western blot was used to detect the expression of cleaved Caspase-3,Bax,Bcl-2,PCNA and Ki67.8.Pearson’s x 2 was used to analyze the relationship between the expression of FN1 and the clinical features of SA patients.Results:1.There was no change in the morphology of the chorionic villus tissues of patients in the IA group.In contrast,the morphology of chorionic villus tissues in the SA group were significantly changed.Microscopic examination showed different degrees of degenerative changes,proliferation,and degeneration in the chorionic villus tissues.2.Compared with IA group,the chorionic villus tissues and surrounding tissues of SA group only showed weak FN1 staining.3.The expression level of FN1 mRNA in serum of SA group was significantly higher than that of IA group.4.FN1 expression was not related to age(P=0.314)or gestational cycle(P=0.718).5.In JEG-3 and BeWo cells,up-regulation of FN1 expression enhanced cell viability,while down-regulation of FN1 expression inhibited cell viability.6.In JEG-3 and BeWo cells,up-regulation of FN1 expression inhibited apoptosis,while down-regulation of FN1 expression increased apoptosis.7.In JEG-3 and BeWo cells,down-regulation of FN1 expression promoted the expression of Cleaved caspase-3 and Bax,and inhibited the expression of Bcl-2,PCNA and Ki67.In contrast,up-regulation of FN1 expression inhibited the expression of Cleaved caspase-3 and Bax,and increased the expression of Bcl-2,PCNA and Ki67.Conclusion:The expression of FN1 is down regulated in chorionic villus tissues of SA patients.FN1 regulated the cell viability and apoptosis of trophoblast cells by regulating the expression of proliferation and apoptosis related proteins.Part Ⅲ FN1 regulated cell viability and apoptosis in JEG-3 and BeWo cells by altering the activation of the PI3K/Akt signaling pathwayObjective:To investigate the molecular mechanism of FN1 on the cell viability and apoptosis of JEG-3 and BeWo cells.Methods:1.Western blot was used to detect the expression of p-PI3K/PI3K and p-Akt/Akt in JEG-3 and BeWo cells.2.Transfected JEG-3(NC/FN1)and BeWo(siNC/siFN1)cells were treated with PI3K/Akt signaling pathway inhibitor LY294002 and activator IGF-1,respectively.3.Cell viability was detected by CCK-8 assay.4.Cell apoptosis was detected by flow cytometry.5.Western blot was used to detect the expression of cleaved Caspase-3,Bax,Bcl-2,PCNA and Ki67.Results:1.The up-regulation of FN1 in JEG-3 and BeWo cells promoted the phosphorylation of PI3K and Akt.2.In JEG-3 and BeWo cells,LY294002 and IGF-1 could successfully restore the effect of FN1 on trophoblast cell viability.3.In JEG-3 and BeWo cells,LY294002 and IGF-1 could successfully restore the effect of FN1 expression on trophoblast apoptosis.4.In JEG-3 and BeWo cells,blocking PI3K/Akt signaling pathway restored the effect of FN1 on the expression of cleaved Caspase-3,Bax,Bcl-2,PCNA and Ki67.Conclusion:FN1 regulated cell viability and apoptosis in JEG-3 and BeWo cells by altering the activation of the PI3K/Akt signaling pathway.Part Ⅳ FN1 knockout increased apoptosis of trophoblast cells in chorionic villus tissues from SA miceObjective:To investigate the effect of FN1 on trophoblast apoptosis in vivo.Methods:1 According to different treatments,the pregnant mice were randomly divided into normal pregnancy group(NP),spontaneous abortion group(SA),normal pregnancy FN1-/-mice group and FN1-/-SA mice group,with 6 mice in each group.After induced abortion,the mice were killed,and the embryo absorption rate was calculated by dividing the number of absorbed embryos by the number of implanted embryos Step experiment.2.The histopathologic changes in the chorionic villus tissues of mice in the NP,FN1-/-,SA,and FN1-/-_SA groups were observed by H&E staining.3.The expression of FN1 in chorionic villus tissues of NP,FN1-/-,SA,and FN1-/-_SA groups mice was detected using qRT-PCR.4.The percentage of cleaved caspase-3 positive cells in chorionic villus tissues of NP,FN1-/-,SA,and FN1-/-_SA groups mice was detected using IHC.Results:1.The organization the chorionic villus tissues of mice in the NP group were normal,and the cell arrangement was tight.However,there were more histopathological changes in the chorionic villus tissues of the FN1-/-group than in those of the NP group but less than in those of the SA and FN1-/--SA groups.The pathological changes in the chorionic villus tissues of the FN1-/--SA group were greater than the changes in those of the SA group.2.Comparison of the FN1 expression levels in the chorionic villus tissues from mice of these groups showed a gradual decrease across the NP,SA,FN1-/-,and FN1-/-_SA groups.3.SA or FN1 knockout alone increased the levels of cleaved caspase-3 in chorionic villus tissues,and FN1 knockout further increased cleaved caspase-3 levels in the chorionic villus tissues of SA mice.Conclusion:FN1 affected the pathological changes and apoptosis of chorionic villus tissues in vivo.