The Mechanism of Exosomal Delivery of the Vasohibin 2 in Post-hepatitis B Liver Cancer Development
Objective To analyze mi RNA-200 levels in serum and exosomes from HepG2 cells infected with the HBx protein of the hepatitis B virus,to investigate the expression of Vasohibin 2(VASH2)in HepG2 cells,and to elucidate the impact of miRNA-200 and VASH2 on the invasion and migration capabilities of HepG2.2.15 cells.Methods 15 patients with hepatocellular carcinoma(HCC)(study group)and 15 healthy subjects(control group)were selected for the study;the serum Exo miRNA-200 expression levels were compared between the 2 groups.Exo was isolated from HepG2.2.15 cells,which were subsequently divided into an overexpression group(transfected with miRNA-200 mimic)and a negative control group(transfected with miRNA-NC);TEM,Western blot,and tracking of nanoparticles were used to characterize exosomes;Western blot and qRT-PCR were used to detect VASH2 expression in each group of HepG2.2.15 cells.The proliferation,migration and invasion of cells as well as cell cycle status in the overexpressed Exo group(Exo from the miRNA-200 overexpressed group)and the control group(Exo from the untransfected group)were analyzed in HepG2 cells supplemented with Exo.The proliferation rate of HepG2 cells was evaluated using the CCK-8 method at 12,24,48,and 72 hours after transfection.The number of migrating and invading HepG2 cells was assessed using the Transwell method at 24 hours after transfection.Cell cycle analysis was performed using flow cytometry at 48 hours after transfection,and the expression level of VASH2 protein was detected by Western blot.Luciferase reporter gene assay was used to evaluate whether miRNA-200 targeted VASH2.Results Serum Exo miRNA-200 expression level was significantly reduced in the study group compared to the control group(P＜0.01).Both VASH2 mRNA and protein expression levels were significantly reduced in HepG2.2.15 cells in the overexpression group compared with the negative control group(P＜0.01).There was no statistically significant difference in cell proliferation rate between the overexpression Exo group and the control group(P＞0.05);the number of migrating and invading cells in the overexpression Exo group was significantly reduced compared with that of the control group(P＜0.01);the difference in the distribution of HepG2 cells in G1,S and G2 phases before and after overexpression of Exo was not statistically significant(P＞0.05);the relative expression level of VASH2 protein in the overexpression Exo group was significantly lower than that of the control group(P＜0.01).The miRNA-200 mimics+VASH2 WT group displayed significantly lower luciferase activity compared to the miR-NC+VASH2 WT group(P＜0.01),whereas MiRNA-200 mimics+VASH2 MUT and miR-NC+VASH2 MUT did not differ statistically significantly in luciferase activity(P＞0.05),which indicated that miRNA-200 targeted VASH2.Conclusion The expression of miRNA-200 is significantly reduced in serum exosomes of patients with HCC,while its up-regulation has been shown to suppress VASH2 expression in HepG2.2.15 cells.The overexpression of miRNA-200 in exosomes derived from HepG2.2.15 cells can inhibit the invasion and migration of HepG2 cells.
hepatocellular carcinomahepatitis B virus X proteinexosomemiR-200Vasohibin 2
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