Effects Of Hypoxia-induced Exosomes On Biological Behavior And Sorafenib Sensitivity Of Hepatocellular Carcinoma

Objective To investigate the effects of hypoxia-induced exosomes on biological behavior and sorafenib sensitivity of normoxic heptocellular carcinoma cells.Methods Hypoxic exosomes and normoxic exosomes derived from Huh7 cells and MHCC-97 H cells were isolated from the supernatant by differential ultracentrifugations.The morphological feature of exosomes were observed by transmission electron microscope(TEM);Nanoparticle tracking analysis(NTA)was uesd to analyse the size distribution and concentration of exosomes;the surface markers of exosomes were identified by western blotting.After co-cultured with hypoxic exosomes or normoxic exosomes,The abilities of cell proliferation,migration and invasion of Huh7 cells were mesasured by CCK-8 assay,cell scratch assay and Transwell chamber experiment,respectively.The half maximal inhibitory concentration(IC50)of sorafenib on Huh7 cells and MHCC-97 H cells was measured by CCK-8 assay.Western blot was used to dectect the expression of EMT protein makers E-cadherin,N-cadherin,Vimentin in HCC cells co-cultured with exsomes.MiRNAs microarray analysis was performed to identified differentially expressed exosomal miRNAs between Huh7-derived hypoxic exosomes and nomoxic exosomes.Results Hypoxic exosomes and normoxic exosomes derived from Huh7 cells and MHCC-97 H cells presented a round or oval vesicular structures under transmission electron microscope with a size between 30-150 nm,and the presence exosomal surface markers CD63 and CD81 were detected in the lysate.Nanoparticle tracking analysis showed that there was no significant difference in particles size between Huh7 derived hypoxic exosomes and normoxic exosomes,but hypoxia stimulated the secretion of exosomes.Compared with the control group(PBS and normoxic exosomes),hypoxia exosomes significantly promoted cell proliferation,migration and invasion of the normoxic HCC cells,and reduced the sensitivity to sorafenib.After co-cultured with hypoxic exosomes,HCC cells showed significantly decreased expression of E-cadherin,and increased expression of N-cadherin and Vimentin.The result of miRNA-seq analysis showed that there were 151 miRNAs significantly dysregulated bwteeen Huh7-derived normoxic and hypoxic exosomes.Bioinformatics analysis was conducted on the top 10 up-regulated hypoxic exosomal miRNAs.Conclusions We successfully isolated and identified exosomes derived from normoxic and hypoxic Huh7 cells and MHCC-97 H cells,and discovered that hypoxia could stimulate Huh7 cells secrete exoxomes.Forthermore,Hypoxic exosomes derived from hepatocellular carcinoma cells enhance cell proliferation,migration and invasion of normoxic HCC cells,and reduce the sensitivity to sorafenib.We also found that hypoxic exosomes could induce HCC cells to undergo Epithelial-mesenchymal transition(EMT).Moreover,the results of exosomal miRNA sequencing and its related bioinformatic analysis indicated that miRNA carried by hypoxic exosomes may play an important role in that process.In summary,We concluded that the intercellular communication mediated by hypoxic exosomes may be one of the key mechanisms for increased malignancy of HCC cells in the hypoxic microenvironment of hepatocellular carcinoma.