PBLD Regulates Angiogenesis Via Modulating The "Microenvironmental Cross-talk" Between Hepatocellular Carcinoma Cells And Vascular Endothelial Cells

ObjectiveHepatocellular carcinoma(HCC)ranks in the second of malignant tumor mortality owing to the high rate of recurrence and metastasis in China.Since HCC is a solid tumor with abundant blood supply,angiogenesis plays an extremely significant role in tumorigenesis and progression.Thereby,the tyrosine-kinase inhibitors(TKIs)including sorafenib,lenvatinib and regorafenib,which have antiangiogenic effects,are proved to exerts a survival benefit.However,relatively limited benefits and development of resistance restrict its extensive application.That’s mainly due to the complexity and heterogeneity of tumor microenvironment(TME),targeting single specific molecular or signaling pathway is hard to completely eliminate the positive effects of TME on tumor angiogenesis.In our previous study,PBLD were proved to play an inhibitory role in the progression of HCC.Here,by taking the cancer cells and the whole TME into consideration,and focusing on the VEGF/VEGFR2 signaling pathway which has a central regulatory effect on tumor angiogenesis,our present research uncovers the important role of PBLD in HCC angiogenesis and further reveals the molecular regulatory network involved.Thereby this study will provide a scientific and theoretical basis for new anti-tumor angiogenesis strategies and combination therapy.Methods1.Negative effects of PBLD on HCC angiogenesis1)The expression of PBLD in HCC tissues were analysed using 90 pairs HCC tissues,TCGA and GEO datasets.TCGA datasets and clinical HCC samples were used to verify the negative correlation between PBLD and angiogenesis-related genes expression.2)The tumor conditional mediums(TCM)from HCC cells with PBLD up-regulated,down-regulated and their controls were collected and applied to culture HUVECs,then CCK8,transwell,wound-healing and tube-formation assay were used to evaluate the proliferation,migration and tube formation ability of HUVECs2.Mechanism of PBLD suppression on angiogenesis viaDUSP6-ERK-HIF-la-VEGF axis in HCC cells1)Function tests were used to evaluate PBLD suppression on angiogenesis under hypoxia condition.Western blot verification in HCC cells and IHC staining in HCC tissues were performed to confirmed PBLD regulatory role in HIF-1a/VEGF axis.2)KEGG signaling pathways enrichment analysis of our preciously microarray and TCGA dataset were used to screen out signal pathway that contributing to PBLD regulation on HIF-la/VEGF,and western blot,signaling pathway inhibitors and agonists assay were carried out for further verification.3)Functional rescue assay was performed to detect whether DUSP6 participated in PBLD inhibition on ERK/MAPK signaling pathway.4)Protein half-life detection,co-immunoprecipitation assay,ubiquitination level detection and ubiquitination site mutation were carried out to figure out the mechanism underlying the PBLD regulation on DUSP6 expression.3.Mechanism of PBLD suppression on angiogenesis through exosome-miR-940-EST2-VEGFR2 axis in HUVECs1)Transmission electron microscope(TEM)was used to analyze the size and morphology of exosomes from HCC cells.Expression of specific exosome markers,CD63,CD9 and HSP70,were also identified by western blot assay.Uptake of HCC cells-derived exosomes by HUVECs were detected by cell immunofluorescence.2)To investigate the effects of HCC secreted exosomes on angiogenesis by co-culturing with HUVECs,and subsequently function test on HUVECs were performed.Differential microRNA expression profile in HCC secreted exosomes were captured through microRNA microarray.Further cell lines and HCC tissues verification,gain-of-function and loss-of-function studies were carried out to investigate miR-940 effects on angiogenesis.Western blot assay was used to detect miR-940 effects on expression of VEGFR2 and signaling pathways downstream of VEGF/VEGFR2.3)Online microRNA target prediction software,qRT-PCR、western blot,dual-luciferase reporter and functional rescue assay were used to identify the target gene of miR-940.4)In vivo and in vitro functional test were carried out to evaluate the effects of exosomes/miR-940/ETS1 axis on PBLD-regulated angiogenesis5)Online bioinformatics analysis was used to identify potential transcription factors for miR-940,further CO-IP,qRT-PCR and CHIP were performed to explored the mechanism in regulation of miR-940 expression by PBLD.Results1.GSEA analysis of TCGA and GEO datasets showed that PBLD expression is associated with liver cancer metastasis and survival.Decreased expression levels of PBLD in HCC was reconfirmed by immunohistochemistry on tissue microarray.Negative correlation between PBLD and angiogenesis-related genes and CD31 were verified using TCGA dataset and clinical samples of our hospital cohorts.2.Compared with control group,TCM from PBLD-overexpressed HCC cells inhibited proliferation,migration and tube-formation abilities of HUVECs.3.PBLD suppressed HIF1-a/VEGF axis via promoting dephosphorylation of ERK,resulting in downregulation of VEGF expression and secretion in HCC cells.4.PBLD decreased ERK1/2 phosphorylation by protecting DUSP6 from ubiquitin-proteasome degradation.5.PBLD regulates angiogenesis through exosomes/miR-940/ETS1 axis.6.PBLD activated TCF4 transcriptional promotion effects on miR-940 by directly interacting with it.ConclusionPBLD exerted an inhibitory effect on angiogenesis of HCC not only by the ERK/HIF-lα/VEGFA signaling pathway,but also through the HCC cells derived exosomes-mediated miR-940/ETS1/VEGFR-2 axis.These explorations may provide a theoretical basis for exploring new anti-tumor angiogenesis strategies.