| Background and purpose:Liver cancer is one of the most common malignant tumors in the world,and of which the most common type is hepatocellular carcinoma(HCC).HCC is one of the leading cause of cancer-related mortality in china,resulting in great economic burden for the country.As pathogenesis of HCC was concealed,the majority of patients had been diagnosed in the middle-late stages,thus missing the best opportunity for surgical treatment.Despite the great progress has been made in the diagnosis and treatment of HCC in recent years,there is still no effective therapeutic target and molecular targeted drugs could be chosen due to the unclear mechanism of pathogenesis of HCC,resulting in high disease mortality.Therefore,it is of great significance to seek novel therapeutic drugs for HCC treatment.Resveratrol,a natural polyphenolic compound,was considered as a promising drug for the treatment of liver cancer for its various antitumor biological activities.A large number of studies have found that resveratrol could inhibit the proliferation and metastasis of HCC tumor and induce the apoptosis of HCC cells.Resveratrol regulated the progression of HCC by regulating the expression of p53,SIRT1 and other tumorrelated genes.However,the underlying regulatory mechanism is yet to be defined.Glypican-3(GPC3)is a member of the heparan sulfate proteoglycan family,possessing an important function in the intracellular signal transduction.GPC3 was not expressed in normal adult tissues,but was highly expressed in most HCC tissues.Previous studies had found that the prognosis of patients with HCC was associated with the expression level of GPC3.Therefore,GPC3 is a promising biomarker for HCC diagnosis.Studies have reported that GPC3 was mainly involved in the occurrence and development of HCC by activating the wnt/β-catenin signaling pathway.In addition,there was a correlation between the expression of GPC3 and autophagy substrate p62,but its specific mechanism has not been clearly studied.Autophagy is the process that cytoplasm,organelles and proteins are wrapped in the double-membrane vesicles of the autophagosome,which is then targeted to the lysosome for degradation.Dysregulation of the autophagic process was related to many diseases,including neurodegenerative diseases,metabolic diseases and cancer.Studies have suggested that autophagy dysfunction could lead to malignant transformation of liver cells and the initiation of HCC.It has also been demonstrated that the level of basal autophagy in HCC tissues were significantly lower than normal tissues.It was reported that autophagy could be regulated by a variety of proteins and RNAs,including autophagy-related genes,lnc RNAs and so on.Studies have reported that there was a correlation between the expression of GPC3 and autophagy substrate p62,and resveratrol could also induce autophagy in HCC cells.However,whether GPC3 is involved in the regulation of resveratrol on HCC has not been reported.Therefore,this study is aiming at exploring the role of GPC3 and autophagy in resveratrol against HCC,to provide a novel theoretical basis for the mechanism of resveratrol against HCC.Methods: Huh7 cells were treated with different concentrations of resveratrol(0μmol/L,20μmol/L,40μmol/L,80μmol/L)for 24 hours,cell viability and apoptosis were assessed by MTT assay and flow cytometry,the expression of GPC3,β-catenin,cyclin D1,c-myc,caspase3,PARP and cleaved-caspase3 were detected by western blot,the expression of GPC3 was detected by immunofluorescence.Resveratrol alone or in combination with GPC3 plasmid was used to treat huh7 cells for 24 hours,cell viability was detected by MTT,the expression of PARP,caspase3 and cleaved-caspase3 were detected by western blot.Huh7 cells were treated with different concentrations of resveratrol(0μmol/L,20μmol/L,40μmol/L,80μmol/L)for 24 hours,the expression of Beclin1,LC3 and p62 were detected by western blot;huh7 cells were transfected with m Cherry-GFP-LC3 plasmid,then 80μmol/L resveratrol was added to huh7 cells for incubation for 24 hours,the changes of autophagy flow were observed by laser confocal microscopy;huh7 cells were transfected with GPC3 plasmid and si RNA targeting GPC3 for 24 hours respectively,the expression of Beclin1,LC3,p62,β-catenin,cyclin D1,and c-myc were detected by western blot;huh7 cells were treated with si RNA targeting β-catenin or wnt/β-catenin pathway inhibitor XAV-939 for 24 hours,western blot was used to detect the expression of Beclin1,p62,and LC3;the m Cherry-GFPLC3 plasmid and si RNA targeting GPC3 or β-catenin were co-transfection into huh7 cells for 24 hours,the changes of autophagy flow were observed by laser confocal microscopy;GPC3 plasmid alone or in combination with wnt/β-catenin pathway inhibitor XAV-939 were treated with huh7 cells for 24 hours,western blot was used to detect the expression of Beclin1,p62 and LC3.Results:(1)Resveratrol could inhibit the proliferation of huh7 cells and induce cells apoptosis in a dose-dependent manner(p <0.05).(2)Resveratrol could significantly down-regulate the expression levels of GPC3 in a dose-dependent manner(p <0.05).(3)Resveratrol could significantly reduce the expression levels of the targeting proteins of wnt/β-catenin pathway,such as β-catenin,c-myc,and cyclin D1 in a dose-dependent manner(p <0.05).(4)Overexpression of GPC3 could reverse the inhibition of cell proliferation and apoptosis induced by resveratrol(p <0.05).(5)Resveratrol could up-regulate the expression of autophagy-related proteins(p62,Beclin1 and LC3-II)and enhance the autophagy flow of cells(p <0.05).(6)Overexpression of GPC3 could down-regulate the expression of p62,Beclin1 and LC3-II in huh7 cells(p <0.05).(7)Knockdown of GPC3 could increase the expression of p62,Beclin1 and LC3-II in huh7 cells and enhance intracellular autophagy flow(p <0.05).(8)Knockdown of β-catenin could up-regulate the expression of p62,Beclin1 and LC3-II in huh7 cells and increase autophagy flow(p <0.05).(9)The expression of p62,Beclin1 and LC3-II were up-regulated after XAV-939 inhibited the wnt/β-catenin pathway in huh7 cells(p <0.05).(10)In huh7 cells,compared with the group of transfected with GPC3 alone,the expression of p62,Beclin1 and LC3-II in the XAV-939 combined with GPC3 plasmid group were up-regulated(p <0.05).Conclusion: Resveratrol could induce autophagy of hepatocellular carcinoma cells by down-regulating GPC3/wnt/β-catenin pathway,thus playing an anti-hepatocellular carcinoma role... |
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