| Objective: Ovarian cancer has complex pathogeneses in which numerous physiological processes,including cell proliferation,migration,division,apoptosis and chemoresistance,are dysregulated.Unfortunately,these processes remain unclear,which contributes to the unsatisfactory therapeutic effect of ovarian cancer.This study focused on the mechanisms of cell proliferation,migration and invasion disorders in the development of ovarian cancer.BMP9,as one of TGF-β superfamily and BMPs family members,plays a diverse and significant array of biological roles,including cell differentiation,proliferation,apoptosis,tumorigenesis,and metabolism.However,the role and mechanism of BMP9 in ovarian cancer progression remains unclear.Notch1 signaling pathway plays a critical role in the development,including chemotherapy resistance,angiogenesis and other processes of ovarian cancer.The interaction of BMP9 and Notch1 signaling in ovarian cancer progression has not been elucidated.Therefore,this study aims to explore the interaction and mechanism of BMP9-Notch1 in the progression of ovarian cancer using transcriptome and metabolome sequencing.Methods:(1)In this study,the expression levels of BMP9 in ovarian cancer tissues and cells were detected by Tq-PCR,WB,immunohistochemistry assay.The correlation between BMP9 and clinical prognosis was detected by prognostic survival analysis.Overexpressing and silencing BMP9 recombinant adenovirus and stable cell lines were constructed and the effect of BMP9 on ovarian cancer cell proliferation,migration and invasion was detected by WST-1 assay,crystal violet staining,cell clone forming,wound healing assay and transwell assay.(2)Immunohistochemistry and prognostic survival analysis of clinical tissues in HPA database were used to detect the expression of Notch1 in ovarian cancer tissues and its correlation with clinical prognosis.The expression of Notch receptors and ligands in ovarian cancer cell lines was mearsured by Tq-PCR assay.Tq-PCR and WB assay were used to verify the interaction between overexpressing BMP9 and Notch receptors and ligands.Notch signaling pathway inhibitor DBZ was chosed to test the biological effect of DBZ on BMP9 in ovarian cancer cells by crystal violet staining,WST-1 assay and transwell assay.Dominant negative Notch1(dn Notch1)recombinant adenovirus was constructed,and the effect of BMP9-Notch1 signaling pathway on cell proliferation,migration,invasion,tumor growth and metastasis of ovarian cancer was detected by WST-1 assay,wound healing assay,cell cycle assay,cell clone forming,transwell assay,subcutaneous xenografts and intraperitoneal metastasis model in nude mice.(3)Transcriptome sequencing was used to detect downstream target genes of BMP9 in ovarian cancer cells.Tq-PCR assay was used to verify the expression of ID2 and other related target genes.CBio Portal database was used to analyze the co-expression relationship between target genes of overexpressing BMP9 and Notch1.Tq-PCR assay was used to detect the expression of ID2,c-Myc,Cyclin A2,MMP9 and p27 of BMP9-Notch1 signaling on ovarian cancer cells.KEGG pathway enrichment and protein interaction analysis were performed to evaluate the crosstalk between BMP9,Notch1 and PI3K/AKT signaling pathway.Western blot assay verified the expression of PI3 K,p-PI3 K,AKT and p-AKT proteins,through which BMP9-Notch1 signaling acts on ovarian cancer cells.The IC50 value of PI3 K inhibitor LY294002 was detected by IC50 assay.Recombinant adenoviruses overexpressing BMP9 and Notch1 intracellular domain(NICD1)were constructed and the effect of LY294002 on the proliferation,migration and invasion of BMP9 and NICD1 in ovarian cancer cells was detected by WST-1 and transwell assay.Western blot assay was used to verify the expression of PI3 K,p-PI3 K,AKT,p-AKT,c-Myc,Cyclin A2,MMP9 of BMP9/NICD1 and LY294002 in ovarian cancer cells.(4)The differential metabolites in SKOV3 cells overexpressing BMP9 were detected by metabolomics sequencing.Correlation analysis between differential transcriptomic genes and metabolites was used to explore the molecular mechanism of BMP9-Notch1 signaling in ovarian cancer cells.Results:(1)This study found that the expression of BMP9 was increased in human ovarian cancer tissues and cells.High expression of BMP9 was significantly associated with high pathological grade and shortened progression-free survival.Silence or overexpression of BMP9 could significantly inhibite or promote cell proliferation,migration and invasion of ovarian cancer in vitro.(2)Notch1 expression was significantly increased in human ovarian cancer tissues,and its expression level was negatively correlated with progression-free survival of ovarian cancer patients.Different Notch receptors and/or ligands were expressed in human ovarian cancer cells,and the expression of Notch1 in SKOV3 and OVCAR3 cells was significantly increased.Overexpression of BMP9 significantly up-regulated m RNA expression of Notch1 and Jag2 and induced the accumulation of NICD1 protein.DBZ or dn Notch1 significantly inhibited cell proliferation and migration of ovarian cancer.The effect of BMP9 overexpressing on the proliferation and migration of ovarian cancer cells was inhibited by DBZ.BMP9 promoted cell proliferation,clone formation,cell cycle S/G2 phase progression,wound healing,migration and invasion of ovarian cancer cells,which could be inhibited by dn Notch1.In vivo experiments confirmed that overexpression of BMP9 promoted tumor growth and intraperitoneal implant metastasis of ovarian cancer,which was inhibited by dn Notch1.(3)Transcriptomic sequencing detected 25 up-regulated and 27 down-regulated downstream target genes of BMP9 overexpressing in SKOV3 cells.Among the up-regulated genes,the expression of ID2 target gene was significantly correlated with the BMP9-Notch1 signaling axis.BMP9-Notch1 signaling played a role in ovarian cancer cells by up-regulating ID2,c-Myc,Cyclin A2,MMP9 and decreasing p27 expression.KEGG pathway enrichment and protein interaction analysis showed BMP9 and Notch1 were closely related to PI3K/AKT signaling pathway.PI3 K inhibitor LY294002 inhibited the effect of BMP9 and NICD1 overexpressing on the proliferation,migration and invasion of ovarian cancer cells.Overexpression of BMP9 and NICD1 induced phosphorylation of PI3 K and AKT.Overexpression of BMP9 and NICD1 significantly up-regulated the expression of c-Myc,MMP9 and Cyclin A2,which were blocked by LY294002.(4)Metabolomic sequencing tested 235 up-regulated differential metabolites and13 down-regulated differential metabolites of BMP9 overexpressing in SKOV3 cells.BMP9/Notch1/ID2 signal axis was closely related to 3-phosphoglycerate acid and other metabolites.Conclusions: BMP9 up-regulates the expression of ID2,c-Myc,Cyclin A2,MMP9 target genes by activating Notch1/PI3K/AKT signaling axis,which promotes the proliferation,migration and invasion of ovarian cancer cells,and then participates in malignant progression of ovarian cancer.BMP9/Notch1/ID2 signaling axis may be involved in biological roles of ovarian cancer by regulating 3-phosphoglycerate acid and other metabolites. |
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