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Exosomal circRNA-100338 promotes hepatocellular carcinoma metastasis via enhancing invasiveness and angiogenesis.

ABSTRACT: BACKGROUND:Exosomes play crucial roles in regulating the crosstalk between normal and cancer cells in the tumor microenvironment, and in regulating cancer proliferation, migration and invasion through their cargo molecules. METHODS:We analyzed the pro-invasiveness of exosomal circRNA-100,338 in HCC using the transwell invasion assay. The co-culture of human umbilical vein endothelial cells (HUVEC) and exosomes derived from HCC cell lines were used to evaluate the impact of HCC derived exosomes on HUVEC. Nude mice models were used to validate the findings in vitro. Clinically, quantitative RT-PCR was used to quantify the expression of serum exosomal circRNA-100,338 in HCC patients at both pre-surgery within one week and post-surgery within three weeks. RESULTS:We aim to investigate the pro-invasive role of exosomal circRNA-100,338 in HCC metastasis. We for the first time demonstrated that circRNA-100,338 was highly expressed in both highly metastatic HCC cells and their secreted exosomes. The transwell invasion assay showed that the overexpression or knockdown of exosomal circRNA-100,338 significantly enhanced or reduced the invasive abilities of HCC cells. Subsequently, in vitro and in vivo assays showed that exosomal circRNA-100,338 affected the cell proliferation, angiogenesis, permeability, and vasculogenic mimicry (VM) formation ability of human umbilical vein endothelial cells (HUVEC), and tumor metastasis. Furthermore, we also observed that the persistent high expression of exosomal circRNA-100,338 in serum of HCC patients who underwent curative hepatectomy may be a risk indicator of pulmonary metastasis and poor survival. CONCLUSIONS:Our findings indicated that metastatic ability of HCC cells could be enhanced by transferring exosomal circRNA-100,338 to recipient HUVECs, which could affect proangiogenic activity by regulating angiogenesis.

SUBMITTER: Huang XY

PROVIDER: S-EPMC6979009 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Rational: Patients with hepatocellular carcinoma (HCC) have a poor prognosis mostly due to intrahepatic as well as distal metastasis. Vasodilator-stimulated phosphoprotein (VASP), a regulator of actin cytoskeleton and cell migration, is overexpressed in HCC and correlated with its malignant features and poor prognosis. Very little is known about its function in HCC. Methods: qRT-PCR, Western blot and IHC were used to detect the VASP expression in tissues and cells. Transwell and wound healing assays were used to measure the migration and invasion of HCC cells. Immunoblotting and immunofluorescence were used for detection of epithelial-to-mesenchymal transition (EMT) progression in HCC cells. A lung metastasis mouse model was used to evaluate metastasis of HCC in vivo. The putative targets of miR-204 were disclosed by public databases and a dual-luciferase reporter assay. IP was used to show the interaction between VASP and CRKL. ChIP was used to analyze the binding of HIF-1α to VASP promoter region. Results: Our data involving both gain- and loss-of-function studies revealed that VASP activated AKT and ERK signaling and promoted HCC migration and invasion in vitro and in vivo by altering the EMT phenotype and expression of MMPs. We investigated the positive correlation between VASP and an adapter protein, CRKL. VASP dynamically co-localized at the SH3N domain of CRKL and mediated its function. Mechanistically, VASP overexpression at the transcriptional level was mediated by HIF-1α through direct binding to two hypoxia response elements (HRE) in the VASP promoter region. Furthermore, we identified hypoxia-induced down-regulation of miR-204, which functioned as the regulator of VASP overexpression at the post-transcriptional level. Also, hypoxia-activated p-Smad3 dependent TGF-β signaling indirectly promoted VASP expression. Conclusion: A variety of hypoxia-induced molecular mechanisms contributed to the upregulation of VASP at transcriptional and post-transcriptional levels. These mechanisms involved CRKL, HIF-1α, miR-204, and TGF-β activating the AKT and ERK signaling to promote EMT and expression of MMPs. Taken together, our results defined VASP as an oncogene of HCC pathogenesis and metastasis with the potential to serve as a prognostic biomarker.

Project description:Oncostatin M (OSM) is a pleiotropic cytokine of the interleukin (IL)-6 family that contributes to the progression of chronic liver disease. Here we investigated the role of OSM in the development and progression of hepatocellular carcinoma (HCC) in non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH). The role of OSM was investigated in (1) selected cohorts of NAFLD/NASH HCC patients, (2) liver cancer cells exposed to human recombinant OSM or stably transfected to overexpress human OSM, (3) murine HCC xenografts, and (4) a murine NASH-related model of hepatic carcinogenesis. OSM was found to be selectively overexpressed in HCC cells of NAFLD/NASH patients, depending on tumor grade. OSM serum levels, barely detectable in patients with simple steatosis or NASH, were increased in patients with cirrhosis and more evident in those carrying HCC. In this latter group, OSM serum levels were significantly higher in the subjects with intermediate/advanced HCCs and correlated with poor survival. Cell culture experiments indicated that OSM upregulation in hepatic cancer cells contributes to HCC progression by inducing epithelial-to-mesenchymal transition and increased invasiveness of cancer cells as well as by inducing angiogenesis, which is of critical relevance. In murine xenografts, OSM overexpression was associated with slower tumor growth but an increased rate of lung metastases. Overexpression of OSM and its positive correlation with the angiogenic switch were also confirmed in a murine model of NAFLD/NASH-related hepatocarcinogenesis. Consistent with this, analysis of liver specimens from human NASH-related HCCs with vascular invasion showed that OSM was expressed by liver cancer cells invading hepatic vessels. In conclusion, OSM upregulation appears to be a specific feature of HCC arising on a NAFLD/NASH background, and it correlates with clinical parameters and disease outcome. Our data highlight a novel pro-carcinogenic contribution for OSM in NAFLD/NASH, suggesting a role of this factor as a prognostic marker and a putative potential target for therapy. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

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Exosomal circRNA-100338 promotes hepatocellular carcinoma metastasis via enhancing invasiveness and angiogenesis.

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