| Background: Metastasis is the leading cause of death in most malignancies.Migration and invasion are the prerequisites of tumor cell metastasis.The interaction between chemokine receptor and its ligand plays a very important role in tumor cell migration and invasion.The chemokines specific expression by target organ and corresponding receptors expressed by tumor cells are involved in the selection of metastatic organs.The role of the chemokine receptor 4(CXCR4)and its ligand,stromal cell-derived factor-1a(SDF-1a),in tumor metastasis has been demonstrated in breast,ovarian,prostate,pancreatic cancer and liver cancer were reported.Exosomes are recently discovered nanoscale(30-150 nm in diameter)lipid bilayer membrane vesicles containing proteins and nucleic acid materials(e.g.m RNAs,noncoding RNAs and DNA).Exosomes can be taken up by the recipient cells and affect the biological behavior of the recipient cells by transferring their contents.Studies have shown that exosome secreted by tumor cells can play an important role in tumor cell proliferation,angiogenesis and tumor invasion and metastasis.Hca-F and Hca-P are a pair of murine ascites hepatocarcinoma cell lines derived from the same parental cell with different lymphatic metastasis.Hca-F cells lysed more than 70%,while the Hca-P cell lymph node metastasis rate was less than 30%,but the mechanism is not yet clear.Our previous studies showed that CXCR4 expressed on the surface of Hca-F and Hca-P cells can bind to SDF-1a in 615 mouse lymph node homogenates,inducing the secretion of and matrix metalloproteinase-9(MMP-9)andmatrix metalloproteinase-2(MMP-2)from Hca-F and Hca-P cells,so as to promote cell migration and invasion,and the level of CXCR4 expressed by Hca-F cells was significantly higher than that of Hca-P cells.However,the role and mechanism of exogenous endogenous CXCR4 in the invasion and metastasis of mouse hepatoma cells have not been reported yet.Objectives:(1)To explore the roles of exosomes derived from highly metastatic murine hepatocarcinoma Hca-F cells promote Hca-P cells migration and invasion and the relationship with CXCR4;(2)To investigate the relationship between CXCR4 and Hca-F cell-derived exosomes in promoting lymphatic endothelial cell proliferation and lymphangiogenesis..Methods:(1)Hca-F and Hca-P cells were cultured in media supplemented with exosome-depleted FBS,exosomes(F-Exo and P-Exo respectively)were extracted by ultracentrifugation(100000 g/min),and transmission electron microscopy was used to observe the diameter and morphology of extracellular vesicles,Western blot was used to detect the specific expression of CD63 and HSP70 on exosome membrane surface to verify the reliability of the exosomes.(2)F-Exo was dyed red with fluorescent dye PKH26,co-incubated with recipient cell Hca-P(previously stained blue with DAPI).The uptake of exosomes with red fluorescence in the recipient cells was observed by confocal microscopy.(3)Lipofectamine 2000 was used to transfect small interfering RNA(si RNA)of CXCR4(si CXCR4),the si RNA unrelated sequence(NC)and Hca-F cell itself(Control)served as negative controls.(4)Transwell chamber(8.0 mm pore size)migration and invasion assays were used to examine the effects of exosome CXCR4,SDF-1a and LEC on migration and invasion of mouse hepatocellular carcinoma cells.(5)Hca-F cells were treated with GW4869,an inhibitor for the exosome formation and release,co-incubated with Hca-P cells use transwell chamber(0.4 mm pore size to block the passage of particles larger than the exosomes)to detect the effect of inhibitingsecretion of exosomes on the migration and invasion ability of mouse hepatoma cells.(6)The effects of exosomes on the proliferation and lymphatic vessel formation of lymphatic endothelial cells were examined by lymphangiogenesis assay and CCK-8 cell proliferation assay.(7)The expression of CXCR4 m RNA and protein in cells and exosomes were detected by q RT-PCR and Western blot.Western blot was used to detect the expression of MMP-9,MMP-2 and VEGF-C.Results:(1)Transmission electron microscopy showed that the extracted vesicles were spherical with a diameter of about 30-100 nm and contained bilayer membrane,which was in accordance with the morphological features of exosomes.Western blot showed that exosomes markers CD63 and HSP70 obvious expression in the extracted vesicles,β-tubulin was significantly expressed in the cells,indicating that the exosomes were extracted successfully.(2)Confocal microscopy showed that Hca-P cells labeled with blue fluorescent dye DAPI contained a fluorescent dye PKH26-labeled exosome,indicating that F-Exo could be taken up by Hca-P cells.(3)Transwell migration assay showed that the number of transmembrane cells(400±2.6)in Hca-P + F-Exo group was significantly higher than that in Hca-P group(229±9.3),P <0.05.Transwell invasion assay(matrigel coating)showed that the number of transmembrane cells(229±3.8)in Hca-P + F-Exo group was significantly higher than that in Hca-P group(98±5.1),P < 0.05.Western blot results showed that the CXCR4 protein level in F-Exo was significantly higher than that in P-Exo,and the expression of CXCR4 in Hca-P cells incubated with F-Exo was also significantly higher than that of Hca-P cells,but still slightly less than Hca-F cells.(4)Western blot results showed that CXCR4 protein level in si CXCR4 group was significantly lower than that in Control group and NC group(P < 0.05),indicating that si RNA transfection was successful.Exosomes secreted by the above three groups of cells were extracted,western blot showed that the CXCR4 protein level in si CXCR4exosomes(si CXCR4-Exo)was significantly lower than that in the negative control(Control-Exo and NC-Exo).Hca-P cells were incubated with CXCR4 knockdown Hca-F exosomes(Hca-P+si CXCR4-Exo),CXCR4 protein expression was significantly lower than that of the negative control(Hca-P+NC-Exo).The transwell assay showed that the number of transmembrane cells in Hca-P+si CXCR4-Exo group(365±3.5)was significantly lower than that in Hca-P+NC-Exo group(503±5.3)(P < 0.05).Transwell chamber invasion assay(matrigel coating)showed that the number of transmembrane cells in Hca-P+si CXCR4-Exo group(147±4.2)was significantly lower than that in Hca-P+NC-Exo group(389±5.7)(P < 0.05).The Hca-F cells in the upper chamber of transwell chamber(0.4 mm pore size)were treated with the exosome inhibitor GW4869 to collect the Hca-P cells in the lower chamber.Western blot results showed that the expression of CXCR4 in Hca-P+F-GW4869 group was significantly lower than that Hca-P+F-DMSO group.The Hca-P cells in the lower chamber were transferred to tanswell chamber with a pore size of 8.0 mm and the results of transwell chamber migration assay(without matrigel coating)showed that the number of transmembrane cells in Hca-P+F-GW4869 group(196±2.5)was significantly lower than that in Hca-P+F-DMSO group(524±4.3).Transwell chamber invasion assay(matrigel coating)showed that the number of transmembrane cells in Hca-P+F-GW4869 group(173±2.2)was significantly lower than that in Hca-P+F-DMSO group(375±5.4)(P < 0.01).(5)Transwell chamber migration assay(without matrigel coating)showed that the number of transmembrane cells(92±3.3)after CXCR4 knockdown Hca-F cells incubated with SDF-1a was significantly lower than that in Control+SDF-1a group(286±2.1)and NC+SDF-1a group(319±4.1),but comparable to Hca-F cell line(119±4.5).Transwell chamber invasion assay(matrigel coating)showed that the number of transmembrane cells in si CXCR4+SDF-1a group(52±2.3)was significantly lower than that in Control+SDF-1a group(201±5.1)and NC+SDF-1a group(157±4.9)(P < 0.05),but equivalent with Hca-F group(69±2.5).Western blot results showed that theexpression of MMP-9,MMP-2 and VEGF-C in si CXCR4+SDF-1a group was significantly lower than that in Control+SDF-1a group and NC+SDF-1a group,equivalent to the Hca-F group.After using lymphatic endothelial cells instead of SDF-1a,transwell chamber migration assay(without matrigel coating)showed that the number of transmembrane cells in si CXCR4+LEC group(10±2.3)was significantly lower than that in Control+LEC group(307±4.1)and NC+LEC group(319±5.1)(P < 0.05),but no significant difference with Hca-F group(132±4.4).Transwell chamber invasion assay(matrigel coating)showed that the number of transmembrane cells in si CXCR4+LEC group(52±2.3)was significantly lower than that in Control+LEC group(216 ± 5.1)and NC+LEC group(219±4.6)(P < 0.05)and comparable to Hca-F group(89±2.7).Western blot results showed that the expression of MMP-9,MMP-2 and VEGF-C in si CXCR4+LEC group was significantly lower than that in Control+LEC group and NC+LEC group,which was comparable to Hca-F group.(6)The results of lymphangiogenesis showed that after incubation with CXCR4-knockdown Hca-F cells the number of LEC-forming tubes was significantly lower than that of the Control-Exo+LEC and NC-Exo+LEC groups(P < 0.01).The results of CCK-8 cell proliferation showed that incubation with CXCR4 knockdown Hca-F cell-derived exosomes the proliferation rate of LECs was lower than that of Control-Exo+LEC group and NC-Exo+LEC group C(P < 0.05).Exosomes from Hca-F cells were co-cultured with CXCR4-silenced lymphatic endothelial cells(LEC-si CXCR4+F-Exo).The number of lymphangiogenesis was not significantly different from that of the negative control group(P > 0.05).CCK-8 experiments showed that the LEC-si CXCR4+F-Exo group grew at a similar rate to the negative control group.Conclusions:(1)Hca-P cells motility and invasion are enhanced via internalized Hca-F cells derived exosomes.(2)Hca-F cells derived exosomes increase Hca-P cells migration and invasion throughtransferring CXCR4.(3)Hca-F cells can transport CXCR4 through exosomes to promote lymphatic endothelial cell proliferation and lymphangiogenesis,which may interact with CXCR4 and SDF-1a on the surface of lymphatic endothelial cells,then increase MMP-9,MMP-2 and VEGF-C secretion.Horizontal transfer of exosomal CXCR4 may promote the migration and invasion of tumor cells and the ability of lymphangiogenesis.The CXCR4 on exosome may be a candidate target for the inhibition of tumor invasion and lymphatic metastasis. |
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