Primary Studieson Interaction Between Exosomes From Hepatocellular Carcinoma Cells And TLR8

ObjectiveExosomes play an important role in the occurrence and development of tumors,and a variety of contents can also play different roles in cells and regulate various signaling pathways.As the first line to defend pathogens and tumors,toll-like receptors can mediate activation of down-stream signal pathway and then regulate related cytokines production,finally function in the tumor development.In this study,exosomes derived hepatocellular carcinoma cells(TEXs),were used as an external stimulus to stimulate the proliferation,invasion and migration of hepatocellular carcinoma cells.These were performed to discuss the expression of cell receptor TLR8 and its signaling pathway related molecules and downstream cytokines;to investigate the effect of TLR8 agonists ssRNA40 on exosomes secretion of HCC cells,in order to explore the interaction between TEXs and TLR8 in HCC cells,and reveal this interaction on HCC migration and metastasis.Methods1.Cell scratch assay for test of the wound-healing rates of Hep G2 and SMMC-7721 cellsThe HepG2 and SMMC-7721 cells were seeded in 6-well plate until the cells grew to 90–95%confluency.The scratch wound was generated in the surface of the plates with a 20μl pipette tip.Different concentrations of exosomes(10、20、30 and 40μg/mL)were co-cultured with cells for 24 h.The scratch area was photographed by a microscope at 0 and 12 h.Using Image J software,the changes of wound healing rate of HepG2 and SMMC-7721 cells were analyzed.2.Cell proliferation assaysThe Hep G2 and SMMC-7721 cells(5×10~3)were seeded at equal densities into a 96-well plate for overnight incubation.Then,the cells were treated with different concentrations of exosomes(10、20、30 and 40μg/mL)for 24 h.cell proliferation assay(CCK-8 assay)were performed and detected optical density(OD)at 490 nm wave length.3.Cell migration and invasion assaysCell migration and invasion assays were carried out using 24-well transwell chambers with an 8μm pore polycarbonate membrane insert.For the migration assays,the cells were seeded into the upper chambers(without Matrigel)at a density of 1×10~5.200μL RPMI 1640 medium supplemented without FBS was added to the upper chambers and 600μL 10%FBS-1640 was added to the lower wells.The assays were carried out for 24 h at 37°C.Then cells were fixed,stained and photographed under microscopy.Migration or invasion efficiency was determined by calculation of transferred cells number from five fields randomly picked.Each experiment was performed in triplicate.4.Expression determination of TLR8 and downstream signaling pathways related molecules and cytokinesThe Real-time fluorescence quantitative RT-PCR and Westeron blot assay were used to detect the mRNA and protein expression levels of TLR8,MyD88and NF-κB,and ELISA was performed to detect the related downstream cytokines expression.5.Effects of TLR8 specificity agonists ssRNA40 on exosomes expression of HepG2 and SMMC-7721 cellsAfter different concentrations of ssRNA40(0.5,1,2 and 4μg/mL)were co-cultured with HepG2 and SMMC-7721 cells,then the Bradford assay and CHE activity were used to assess the amount of exosomes.Results1.Different concentrations of exosomes(10,20,30,40μg/mL)acted on Hep G2 and SMMC-7721 cells after 24 h,wound-healing rate of HepG2 and SMMC-7721 cells were obviously increased(P<0.05),and increase with the increase of the concentration of the TEXs.2.Compared with the blank control group,the number of HepG2 cell migration increased significantly after the addition of exosomes(P<0.05).At the same time,the invasive ability of cells was also significantly enhanced(P<0.05).However,under the same conditions,smmc-7721 cells did not pass through pore polycarbonate membrane.3.The proliferation of Hep G2 and smmc-7721 hepatocellular carcinoma cells showed no significant change after exosomes of different concentrations of hepatocellular carcinoma cells on HepG2 and SMMC-7721 cells(P>0.05).4.The real-time fluorescent quantitative PCR results showed that the mRNA expression levels of TLR8,MyD88 and NF-κB were significantly increased after TEXs co-culture(P<0.05).5.The results of Western blot showed that the protein expression levels of TLR8,MyD88 and NF-KB were obviously increased.6.The results of ELISA assay showed that the expression level TNF-αand IL-6 were increased significantly.7.ssRNA40 reduced the secretion amount of TEXs,especially in a concentration-dependent manner..Conclusions1.Exosomes from hepatocellular carcinoma cells can induce the migration of tumor cells,but not cell proliferation,up-regulated the expression levels of factors of TLR8/MyD88/NF-KB signaling pathway and the down-stream cytokine IL-6 and TNF-α.2.TLR8 activation in HCCs could suppress the secretion of the exosomes of hepatoma cells.3.We could speculate TEXs may interact with TLR8 in HCCs,to regulate migration of HCCs,which would contribute to further understand development of hepatocellular carcinoma.