Effects Of Hypoxia-induced Tongue Squamous Cell Carcinoma Cells Exosomes On Lymphatic Endothelial Cells And Detection Of MiRNA Expression Profile

Objective:By observing the effect of Exosomes from tongue squamous cell carcinoma（TSCC）cell line Tca8113 on proliferation,migration and tubegenesis of human lymphoendothelial cell line（HLECs）after hypoxia pretreatment,the mechanism of promoting lymphangiogenesis was initially explored.Methods:（1）Tca8113 hypoxia pretreatment:Tca8113 cells were cultured in a three-gas incubator（94%N₂,5%CO₂,1%O₂）to simulate hypoxia.The expression of Hypoxia Inducible factor-1（HIF-1α）was detected by Western Blot（WB）.（2）Extraction and identification of exosomes:Cell supernatant was collected to extract Normoxic exosomes（No-Exo）and Hypoxic exosomes（Hy-Exo）secreted under Normoxic conditions by differential centrifugation.Exosomes were identified by transmission electron microscopy,nanoparticle tracking analysis and flow cytometry.（3）Exosomes and HLECs co-culture:the concentration of exosomes was determined by BCA protein assay,and exosomes were added into the medium to make the concentration of exosomes in the medium suspension be 100μg/m L.The two groups of medium containing 100μg/m L exosomes were co-cultured with human lymphoendothelial cells for 12h,and exosomes uptake by HLECs was observed by laser confocal microscope.CCK-8 cell proliferation assay and clonogenesis assay were used to detect the effect of exosomes on HLECs proliferation.Transwell migration test and scratch healing test were used to detect the effect of exosomes on HLECs migration.The effect of exosomes on the tubule formation ability of HLECs was detected by tubule formation experiment.（4）Screening of differential mi RNAs from exosomes:The differential expression mi RNAs from Tca8113 exosomes under normoxia and hypoxia conditions were screened by high-throughput sequencing,and the target genes of differential expression mi RNAs were analyzed by GO enrichment and KEGG pathway.Results:（1）The positive expression of HIF-1αin Tca8113 cells after hypoxia preconditioning suggested that hypoxia preconditioning was successful.（2）The identification results of exosomes extracted by differential centrifugation were consistent with the characteristics of exosomes.（3）Confocal laser microscopy showed that exosomes in both groups could enter HLECs.CCK-8 cell proliferation assay and clonal formation assay confirmed that Hy-Exo could promote the proliferation of HLECs more than No-Exo（P<0.05）.Transwell migration experiment and scratch healing experiment showed that HY-Exo promoted the migration ability of HLECs more than No-Exo（P<0.05）.The tubule formation experiment showed that Hy-Exo promoted the tubule formation ability of HLECs more than No-Exo（P<0.05）.（4）High-throughput sequencing results showed that there were 123 differentially expressed mi RNAs between Hy-Exo and No-Exo,and bioinformatics methods were used to preliminarily analyze the target genes and signaling pathways that might be involved in regulation.Conclusion:Hy-Exo can promote the proliferation,migration and duct formation of HLECs more than No-Exo,and the differential mi RNAs carried by Hy-Exo may play an important role in promoting lymphangiogenesis.