| Purpose To investigate the activation of OCCs on OMVECs and the molecular mechanism,and to explore the effect of activated OMVECs on the progression of ovarian cancer.Methods 1.Immunohistochemistry was used to detect the differences in omental CD34 positive and ENG positive MVD among ovarian cancer patients with different omental metastatic situations.Immunofluorescence was used to detect VE-CAD and α-SMA in omental microvessels.2.HM-OCCs and parental cancer cells were used to establish ovarian orthotopic xenograft tumor models in nude mice.Immunohistochemistry was used to compare the difference in CD31+MVD between the two groups of nude mice.Non-tumorbearing nude mice were injected intraperitoneally with ovarian cancer cell TCM,and immunohistochemistry was used to analyze the changes in omental CD31+MVD.3.Edu proliferation test,scratch test,Transwell test and Matrigel tube formation test were used to detect the activation of OMVECs’ functions by TCM.4.WB and immunofluorescence were used to detect the effects of TCM on the expression of OMVECs’ protein markers.5.Transwell migration and invasion tests were used to test the induction effect of TCM treatment-activated OMVECs on the migration and invasion of OCCs.Nude mice subcutaneous tumor model was applied to test the effect of OMVECs on the growth of tumor.6.RNA-seq was used to analyze the differential expression of genes in HM-OCCs and parental cancer cells.Bioinformatics was used to search up-regulated genes,gene collections,and signal pathways in HM-OCCs and screen out the genes that may affect omental metastasis of ovarian cancer.7.RNA-seq results were validated by q RT-PCR and WB.ADAMTS2 gene,which was significantly up-regulated in HM-OCCs,was down-regulated by siRNA. OMVECs were co-cultured with OCCs indirectly.The expression of ENG in treated OMVECs were detected by WB.Edu proliferation test and Matrigel tube formation test were used to detect the proliferation ability and tube formation ability of OMVECs,respectively.Result 1.The omentum from ovarian cancer patients with omental metastasis have significantly higher MVD than those without metastasis,and the expression of α-SMA in omental microvessels was enhanced.2.Compared with parental cells,HM-OCCs orthotopic xenograft tumors have stronger ability to promote omental angiogenesis in nude mice.Intraperitoneal injection of TCM can also promote omental angiogenesis in tumor-free mice.3.Compared with parental cells,the TCM from HM-OCCs significantly enhanced the proliferation,migration,invasion and tube formation of OMVECs in vitro.4.Compared with parental cells,the TCM from HM-OCCs promoted the expression of α-SMA and ENG in OMVECs,while inhibiting the expression of VE-CAD.5.Activated OMVECs can promote the migration and invasion of OCCs in vitro.And OMVECs were able to promote the growth of subcutaneous tumors of ovarian cancer in nude mice.6.RNA-seq revealed that the signal pathways involving extracellular matrix and microenvironment were enriched in HM-OCCs and that the transcription level of ADAMTS2 gene in HM-OCCs was about 6 times that in parental cells.7.Down-regulating the expression of ADAMTS2 in HM-OCCs can inhibit its promotion to OMVECs proliferation and tube formation ability,and weaken its ability to promote the expression of ENG in OMVECs.Conclusion Ovarian cancer can promote omental vascular endothelial activation and angiogenesis through secreting proteins,forming a microenvironment that is conducive to tumor growth and metastasis,thereby promoting tumor progression.The role of ovarian cancer cells in activating omental endothelial cells may be achieved by secreting ADAMTS2.The deep molecular mechanism needs further study... |
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