Mechanism Of JAZF1 Deficiency-Mediated Decidualization Homeostasis Imbalance Of Endometrial Stromal Cells In Recurrent Spontaneous Abortion

BackgroundDecidualization of human endometrial stromal cells is crucial for the establishment and maintenance of pregnancy,which is accompanied by cell proliferation,differentiation and apoptosis.The balance between these processesis conducive to the maintenance of proper decidualization,while abnormal decidualization homeostasis may lead to abortion.JAZF1 has been reported to be associated with recurrent spontaneous abortion,and previous studies have shown that downregulation of JAZF1 expression can induce apoptosis in various cell types,such as pancreatic β cells and human spermatogonia stem cells.However,the role and mechanism of JAZF1 in the regulation of decidualization homeostasis in endometrial stromal cells and recurrent spontaneous abortion have not been elucidated.ObjectiveTo analyze the role and mechanism of JAZF1 in decidualization of endometrial stromal cells and recurrent spontaneous abortion,and to explore how JAZF1 regulates the function of stromal cells to maintain decidua homeostasis and pregnancy.Methods1.The expression and intracellular localization of JAZF1 in endometrium during proliferative and secretory phases and decidua of early pregnancy and decidualization in vitro were detected by using 10× single-cell transcriptional sequencing data,q RTPCR,western blot and immunohistochemistry.The correlation between JAZF1 expression and recurrent spontaneous abortion was also analyzed.2.JAZF1 was knocked down in human endometrial stromal cell lines using si RNA and CRISPR/Cas 9 techniques or overexpressed by lentivirus.The apoptosis and decidualization levels of cells were detected by q RT-PCR,western blot and flow cytometry.3.Combined with RNA-seq,CUT & Tag-seq,immunoprecipitation,mass spectrometry and dual luciferase reporter assays,the molecular mechanism of JAZF1 regulation on endometrial stromal cells survival and decidualization function was revealed.Results1.Aberrantly decreased level of JAZF1 in decidual stromal cells of RSA patients1)The 10× single-cell transcriptional sequencing data of decidua tissues in early pregnancy showed that JAZF1 expression was downregulated in all subtypes of decidua stromal cells in recurrent spontaneous abortion,with statistical differences.q RT-PCR,western blot,immunohistochemistry,and immunofluorescence also indicated that JAZF1 expression was downregulated in decidual stromal cells of recurrent spontaneous abortion,with statistical differences.2)q RT-PCR,western blot and immunofluorescence showed that JAZF1 expression increased with the upregulation of IGFBP1,PRL and FOXO1 after decidualization in human endometrial stromal cell lines and primary endometrial stromal cell lines in vitro,with statistical differences.3)Immunohistochemistry indicated that JAZF1 expression was upregulated in endometrial stromal cells at the early secretory phase compared with that in proliferative phase.The results were consistent with public endometrium microarray data and endometrial single-cell sequencing results,with statistical differences.4)Immunohistochemistry indicated that JAZF1 expression was significantly downregulated in endometrial stromal cells at proliferative and early secretory phases in patients with recurrent spontaneous abortion compared with normal women,with statistical differences.2.JAZF1 deficiency induced cell death through the activation of mitochondrial apoptosis pathway,resulting in decidualization defects1)JAZF1 was knocked down,and decidualization was induced in human endometrial stromal cell lines and primary endometrial stromal cell lines.q RT-PCR and western blot indicated increased expressions of pro-apoptotic molecules BAX,cleavedcaspase 3 and cyt c,while the expression of anti-apoptotic molecule BCL2 decreased,with statistical differences.Flow cytometry showed that the proportion of apoptotic cells increased,and CCK8 analysis showed that the proliferation ability of cells decreased after JAZF1 knockdown,with statistical difference.2)JAZF1 was knocked down,and decidualization was induced in human endometrial stromal cell lines and primary endometrial stromal cell lines.q RT-PCR,western blot and ELISA indicated that the expressions of decidualization markers IGFBP1,PRL and FOXO1 were upregulated,with statistical differences.3)JAZF1 was overexpressed,and decidualization was induced in human endometrial stromal cell lines.q RT-PCR and western blot indicated that the expression of proapoptotic molecule cyt c decreased,while the expression of anti-apoptotic molecule BCL2 increased,with statistical differences.Flow cytometry showed that the proportion of apoptotic cells decreased,and CCK8 analysis showed that the proliferation capacity of cells increased after JAZF1 overexpression,with statistical differences.4)JAZF1 was overexpressed,and decidualization was induced in human endometrial stromal cell lines.q RT-PCR,western blot and ELISA indicated that the expressions of decidualization markers IGFBP1,PRL and FOXO1 were downregulated,with statistical differences.5)RNA-seq analysis indicated that the expressions of pro-apoptotic genes BAX,TP53 and CDKN1A were upregulated,while the anti-apoptotic gene BCL2 was downregulated after JAZF1 knockdown,with statistical differences.GO analysis suggested that the upregulated differentially expressed genes after JAZF1 knockdown were enriched in the apoptotic signal pathway,and the same differentially expressed genes after JAZF1 knockdown and downregulated after JAZF1 overexpression were still enriched in the apoptotic activation signal pathway.96)After JAZF1 was knocked down in human endometrial stromal cell lines,a pancaspase inhibitor was added and decidualization was induced.q RT-PCR and western blot indicated decreased expression of pro-apoptotic molecule cleaved caspase 3 and decidualization markers IGFBP1 and PRL,with statistical differences.7)The supernatant of human endometrial stromal cell lines and primary endometrial stromal cell lines with JAZF1-knockdown after decidualization was co-cultured with HTR-8/SVneo.The results indicated that trophoblast invasion ability was downregulated,while the stromal cells with JAZF1-overexpression upregulated it,with statistical differences.3.Apoptosis levels in decidual stromal cells of recurrent spontaneous abortion were upregulated1)The 10× single-cell transcriptional sequencing data of decidua tissues in early pregnancy showed that the expression of pro-apoptotic gene BAX in decidua stromal cells of recurrent spontaneous abortion was upregulated,with statistical difference.GO analysis showed that the differentially expressed genes of the three subtypes of decidual stromal cells were enriched in the apoptotic activation signal pathway,with statistical differences.2)q RT-PCR,western blot and immunohistochemistry indicated that the expression levels of pro-apoptotic molecules BAX,cleaved-caspase 3 and cyt c were upregulated,while the anti-apoptotic molecule BCL2 was downregulated in decidual stromal cells of recurrent spontaneous abortion,with statistical differences.3)TUNEL indicated that the apoptotic level in decidua of recurrent spontaneous abortion was upregulated,with statistical difference.4.JAZF1 bound to the G0S2 promoter and inhibits its transcription in decidualized stromal cells1)Combined analysis of CUT & Tag-seq and RNA-seq showed that the same differentially expressed genes that upregulated after JAZF1 knockdown and increased JAZF1 binding after decidualization of stromal cells were enriched in the cell apoptosis activation signal pathway,including BCL2L11,TRIB3,IL19 and G0S2.Among them,G0S2 has the largest difference ratio after JAZF1 knockdown,and JAZF1 had potential binding sites in the G0S2 promoter sequence,showing statistical differences.2)JAZF1 was knocked down,and decidualization was induced in human endometrial stromal cell lines and primary endometrial stromal cell lines.q RT-PCR and western blot indicated that G0S2 expression was upregulated,with statistical differences.3)The 10× single-cell transcriptional sequencing data of decidua tissues in early pregnancy showed that G0S2 expression was up-regulated in decidua stromal cells of recurrent spontaneous abortion,with statistical difference.q RT-PCR,western blot and immunohistochemistry indicated that G0S2 expression level in decidua stromal cells of recurrent spontaneous abortion was upregulated,with statistical differences.5.JAZF1 deficiency induces apoptosis through activation of G0S2,leading to decidualization defects1)G0S2 was overexpressed and decidualization was induced in human endometrial stromal cell lines.q RT-PCR and western blot indicated upregulated expression of proapoptotic molecules BAX,cleaved caspase 3 and cyt c,and downregulated expression of anti-apoptotic molecule BCL2,with statistical differences.After overexpression of G0S2,flow cytometry showed an increased proportion of apoptotic cells,while CCK8 analysis showed a decreased proliferation ability,with statistical differences.2)G0S2 was overexpressed and decidualization was induced in human endometrial stromal cell lines.q RT-PCR,western blot and ELISA indicated that the expressions of decidualization markers IGFBP1,PRL and FOXO1 were upregulated,with statistical differences.3)JAZF1 was knockdown in human endometrial stromal cell lines and primary endometrial stromal cell lines followed by treatment with G0S2 inhibitors,or coknockdown of JAZF1 and G0S2,and induced decidualization.q RT-PCR and western blot indicated that the expressions of pro-apoptotic molecules BAX,cleaved-caspase3 and cyt c were downregulated,while the anti-apoptotic molecule BCL2 was upregulated,with statistical differences.After JAZF1 knockdown and G0S2 inhibition or co-knockdown JAZF1 and G0S2,flow cytometry showed that the proportion of apoptotic cells decreased,and CCK8 showed that cell proliferation was upregulated,with statistical differences.4)JAZF1 was knockdown in human endometrial stromal cell lines and primary endometrial stromal cell lines followed by treatment with G0S2 inhibitors,or coknockdown of JAZF1 and G0S2,and induced decidualization.q RT-PCR and western blot indicated downregulated expression of decidualization marker IGFBP1,with statistical differences.There was no statistical difference in PRL expression.6.JAZF1 binds to Purβ to inhibit the transcription of G0S2 and maintain stromal cells’ function1)Human protein microarray,co-immunoprecipitation and mass spectrometry showed that JAZF1 bound to the transcription factor Purβ in decidualized stromal cells with statistical difference.2)JAZF1 and Purβ were co-knockdown in human endometrial stromal cell lines and primary endometrial stromal cell lines during decidualization.q RT-PCR and western blot indicated that the expressions of pro-apoptotic molecules BAX,cleaved-caspase3 and cyt c were downregulated,while the anti-apoptotic molecule BCL2 was upregulated,with statistical difference.After co-knockdown of JAZF1 and Purβ,flow cytometry showed that the proportion of apoptotic cells decreased,and CCK8 analysis showed that the proliferation ability of cells was upregulated,with statistical differences.3)JAZF1 and Purβ were co-knockdown in human endometrial stromal cell lines and primary endometrial stromal cell lines during decidualization.q RT-PCR and western blot indicated that decidualization markers IGFBP1 and PRL were down-regulated,with statistical differences.4)Immunohistochemistry indicated that Purβ widely expressed in all cell types of decidual tissue,including cytoplasm and nucleus.5)Dual luciferase reporter genes suggested that transcription factor Purβ bound to the G0S2 promoter region,with statistical difference.After JAZF1 knockdown,transcription factor Purβ upregulated the fluorescence activity of G0S2 promoter region,with statistical difference.Conclusion Low JAZF1,high G0S2,excessive apoptosis and decidualization defects were observed in decidual stromal cells of recurrent spontaneous abortion.In vitro studies have confirmed that JAZF1 inhibits G0S2 transcription by restricting the activity of transcription factor Purβ,which is involved in the survival of human endometrial stromal cells and the regulation of decidualization.These findings have important clinical implications for the understanding of pathology of recurrent spontaneous abortion and the development of new treatment strategies.