Exosomal MiR-494 Shed From Hypoxic HCC Cells Induced Endothelial-to-mesenchymal Transition

Backgroud and objectiveLiver cancer is a common malignant tumor that seriously threatens life and health.China counts a large amount of liver cancer patients.About 50%of liver cancer patients worldwide come from China,and about 55%of liver cancer-related deaths occur in China.Because of high malignancy,low early diagnosis rate and poor prognosis,it is urgent to find effective prognostic factors.Liquid biopsy which has the feature of minimally invasive and repeatable can act as a marker for cancer detection and prognosis evaluation.Exosomal detection is a emerging liquid biopsy approach.Cancer cells can derive exosomes containing RNA and protein to modulate the function and phenotype of accepted cells.Exosomes have information,material,function and carrier characteristics.Hypoxia,which can induce angiogenesis as well as promote cancer metastasis,is a main element of tumor microenvironment(TME)and poor prognosis.This study aims to select gene that regulates angiogenesis of liver cancer in hypoxia-derived exosomes,and explore its role and mechanism in cancer angiogenesis.MethodsExosomes were collected and identified from hypoxic HCC cells and were used to stimulate HUVEC.The regulated function of hypoxic cancer-derived exosomes in HUVEC was analyzed by EdU,transwell,tube formation,permeability test and morphological observation.Gene microarray and database analysis was utilized to select key microRNA in hypoxic cancer-derived exosomes and intravascular cancer thrombosis(miR-494).In vivo angiogenesis experiments were used to explore the effects of miR-494 on blood vessels.We investigated the mechanism of miR-494 in regulating the target gene Cav-2,and affecting the function of vascular endothelial cells and endothelial-mesenchymal transition(EndMT)by microRNA database,qRT-PCR,western blot,luciferase reporter assay and rescue experiments.Finally,the plasma specimen of patiens,database and immunohistochemistry analysis were used to explore the expression and clinical significance of miR-494 and its target gene Cav-2 in different stage of patients.ResultsExosomes derived from hypoxic HCC cells can increase the ability of migration,tube formation and permeability and EndMT of HUVEC.miR-494 was significantly upregulated in Exosomes derived from hypoxic HCC cells.Exosomal miR-494 can be taken in HUVEC to increase the ability of migration,tube formation,permeability and EndMT of HUVEC.miR-494 can regulate the expression of key gene of EndMT.In vivo angiogenesis experiments further confirmed that miR-494 can regulate endothelial cell function.miR-494 can combine with the 3’UTR of Cav-2 and down-regulate the expression of Cav-2 by database analysis and verification experiments.Overexpression of Cav-2 can reverse the promotion of miR-494 on HUVEC migration,tube formation,permeability and EndMT.The plasma specimen of patiens,database analysis combined with immunohistochemistry analysis found that patients with high expression of miR-494 had a worse prognosis,and Cav-2 was down-regulated in internal tumor endothelial cells.Conclusion1、Exosomes derived from hypoxic HCC cells can enhance vascular endothelial cell migration,permeability,tube formation and EndMT.2、Exosomal miR-494 shed from hypoxic HCC cells regulates the function of vascular endothelial cells.3、miR-494 affects endothelial cell function by regulating the expression of the target gene Cav-2.4、The abnormal expression of miR-494 and Cav-2 in patients with liver cancer is related to the prognosis of patients.