| Background and Objectives: Liver cancer is one of the most common malignant tumors in the world,with high morbidity and mortality.Seventy to ninety percent of liver cancer is Hepatocellular carcinoma(HCC).The incidence and fatality of HCC has been increased year by year.The occurrence and development of HCC is an extremely complex pathological process,and its underlying molecular mechanism is still unclear.Currently,limited measures can be chosen to the treatment of HCC,and most patients have lost the opportunity for surgery because they are already in the middle or advanced stages of HCC when diagnosed.Drugs are still the main treatment for advanced HCC.However,the commonly used chemotherapy drugs are expensive and have significant side effects,so it is of great importance to find novel drugs for the treatment of HCC.Resveratrol,a phytochemical constituent,is a natural antioxidant and anti-inflammatory agent that may prevent or slow the progression of inflammation-related diseases,including tumors.Studies have shown that resveratrol can regulate the expression of various HCC related genes and the activity of signaling pathways to inhibit the progression of HCC,and thus it is a promising therapeutic drugs for HCC.However,the mechanism by which resveratrol regulates HCC has not been fully elucidated.Exosomes are small membrane-bound vesicles with a diameter of approximately30-150 nm,and can be released into the extracellular space by many types of cells.These vesicles could carry a variety of biologically active cellular components,such as proteins,mRNA,and miRNAs,and are involved in cell waste management and intercellular communication.Almost all cells can produce exosomes.Studies have shown that HCC cell-derived exosomes contain a variety of active factors that may promote tumor growth and metastasis,such as LncRNA,which could promote HCC cells proliferation,tumor angiogenesis and induce drug resistance by modulating the expression of a variety of HCC-related genes.Therefore,inhibiting the formation and secretion of tumor exosomes is another important way to block the progression of HCC.The present study aimed to investigate the effects of resveratrol on secretion of HCC exosomes and the role of exosomes induced by resveratrol in HCC cell proliferation,migration and epithelial-mesenchymal transformation,and the further mechanism was explored to provide a novel theoretical basis of anti-HCC effect of resveratrol.Methods:(1)Huh7 cells were stimulated with different concentrations of resveratrol(0μmol/L,80μmol/L).Exosomes were extracted by ultra-high speed centrifugal method.The concentration and particle size of exosomes were detected by Nanoparticle Tracking Analysis(NTA),and the expressions of exosomal-labeled proteins Hsp70 and Alix were detected by Western blot.Rab27 a expression was detected by Western blot and immunofluorescence after Huh7 cells were stimulated with resveratrol at different concentrations(0μmol/L,20μmol/L,40μmol/L,80μmol/L).(2)Huh7 cells were treated with 20μmol/L GW4869 and 80μmol/L resveratrol alone or combined with them for 24 h.Exosomes were extracted by ultra-high speed centrifugation method,and the expressions of exosomal-labeled proteins Hsp70 and Alix were detected by Western blot.Cell proliferation was detected by confocal high-content imaging system,cell migration was detected by cell scratch assay,and expression of cyclin D1,PCNA,E-cadherin,N-cadherin and Vimentin was detected by Western blot.(3)Exosomes derived from Huh7 cells were extracted by ultra-high speed centrifugation method,and Huh7 cells were incubated with PKH67 fluorescent dye labeled exosomes.The fluorescence intensity in the cells was detected by high-resolution confocal laser fluorescence microscopy.Huh7 cells were incubated with exosomes derived from Huh7 cells and resveratrol induced Huh7 cells at 20μg/m L for24 h.Cell proliferation was detected by confocal high-content imaging system,cell migration was detected by cell scar assay,and expression of cyclin D1,PCNA,E-cadherin,N-cadherin,Vimentin,p62,LC3,Beclin1,GSK-3β,β-catenin and c-myc was detected by Western blot.Huh7 cells were transfected with m Cherry-GFP-LC3 double fluorescent plasmid for 6 hours,and the exosomes derived from Huh7 cells and those induced by resveratrol were incubated.After 12 hours,the changes of autophagy flow were observed by high-resolution confocal laser fluorescence microscopy.(4)Huh7 cells were stimulated with 0 and 80μmol/L resveratrol for 24 h,and exosomes were extracted by ultra-high speed centrifugal method.The composition differences of LncRNA in hepatocellular carcinoma exosomes induced by resveratrol were analyzed by high-throughput sequencing.Huh7 cells were treated with different concentrations of resveratrol(0μmol/L,20μmol/L,40μmol/L,80μmol/L)for 24 h,and the expression of LncRNA SNHG29 in cells and exosomes was detected by real-time quantitative PCR.(5)SiRNA targeting LncRNA SNHG29 was synthesized and transfected into Huh7 cells for 24 h.The expressions of LncRNA SNHG29 in Huh7 cells were detected by real-time quantitative PCR and the expressions of p62,LC3,Beclin1,GSK-3β,β-catenin and c-myc were detected by Western blot.Huh7 cells were transfected with m Cherry-GFP-LC3 double fluorescent plasmid for 6h,and then the siRNA of LncRNA SNHG29 was transfected.After 12 h,the changes of autophagy flow were observed by high-resolution confocal laser fluorescence microscopy.Results:(1)Resveratrol inhibited the secretion of exosomes in Huh7 cells and down-regulated the expression of Rab27a(p<0.05).(2)Compared with the control group,both resveratrol and GW4869 inhibited the proliferation and migration of Huh7 cells,decreased the expression of cyclin D1,PCNA,N-cadherin and Vimentin,increased the expression of E-cadherin(p<0.05).(3)Compared with the control group,HCC cell-derived exosomes promoted the proliferation and migration of Huh7 cells,up-regulated the expression of cyclin D1 and PCNA,down-regulated the expression of E-cadherin,and up-regulated the expression of N-cadherin and Vimentin(p<0.05);Compared with the hepatoma cell exosome group,resveratrol induced hepatoma cell exosomes inhibited the proliferation of Huh7 cells,down-regulated the expression of cyclin D1,PCNA,N-cadherin and Vimentin,up-regulated the expression of E-cadherin(p<0.05).(4)Compared with the control group,HCC cell-derived exosomes up-regulated the expression of LC3Ⅱ and Beclin1,down-regulated the expression of p62,and enhanced intracellular autophagy flow(p<0.05);Compared with the hepatoma cell exosome group,resveratrol induced hepatoma cell exosomes down-regulated LC3Ⅱ and Beclin1 expressions,up-regulated p62 expression and inhibited autophagy flow(p<0.05).(5)Compared with the control group,HCC cell-derived exosomes upregulated the expression of Wnt/β-catenin target protein c-myc in Huh7 cells,down-regulated the expression of GSK-3β,and induced the translocation of β-catenin from cytoplasm to nucleus(p<0.05);Compared with the hepatoma cell exosome group,resveratrol induced hepatoma cell exosomes down-regulated the expression of Wnt/β-catenin target protein c-myc in Huh7 cells,up-regulated the expression of GSK-3β,and inhibited the translocation of β-catenin from cytoplasm to nucleus(p<0.05).(6)Resveratrol upregulated the expression of LncRNA SNHG29 in Huh7 cells and induced increased secretion of LncRNA SNHG29 in exosomes of Huh7 cells(p<0.05).(7)Interventing LncRNA SNHG29 in Huh7 cells up-regulated the expression of LC3Ⅱ and Beclin1,down-regulated the expression of p62,and enhanced intracellular autophagy flow(p<0.05),but had no effect on the Wnt/β-catenin pathway(p>0.05).Conclusion: On the one hand,resveratrol inhibits the secretion of exosomes of liver cancer by down-regulating Rab27 a,thus inhibiting the progression of liver cancer.On the other hand,resveratrol induced exosomes inhibit the progression of liver cancer by inhibiting autophagy and activation of the Wnt/β-catenin pathway,and its inhibition of autophagy may be mediated by the exosome LncRNA SNHG29. |
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