Role And Mechanism Of HIF-1Α-mediated GPC3 Secretion In Promoting Hepatocellular Carcinoma Progression By Hypoxia

Background and objective: Liver cancer is the sixth most malignant tumor in the world,of which hepatocellular carcinoma（HCC）accounts for about 90%.At present,the incidence and mortality of HCC are in the forefront of tumors,and the treatment effect is not good,and the recurrence rate is high.The pathogenesis of HCC is complex and has not been clearly studied,and effective targeted drugs and treatments are also lacking.Therefore,in-depth research on the molecular mechanism of hepatocellular carcinoma development and identification of appropriate target molecules and signaling pathways related to tumor phenotypes are the key to develop effective targeted drugs for HCC.Phosphatidylinositol proteoglycan 3（GLypican-3,GPC3）,a member of the Glypican family,is anchored to cell membranes via GPI.GPC3 is not expressed or low expressed in normal tissues,but is highly expressed in HCC.The highly expressed GPC3 promotes the growth,proliferation,adhesion and migration of HCC cells by stimulating the typical Wnt/β-catenin signaling pathway.However,the expression and regulation mechanism of GPC3 under hypoxia has not been reported.Hypoxia is a normal process in tumor development.It mainly regulates hypoxia-inducible Factor 1-alpha（HIF-1α）to activate the expression of downstream genes,inhibit the activity of anti-tumor immune cells,and promote the immune escape of cancer cells,thereby promoting the growth and metastasis of tumor cells.In particular,it plays an important role in the development of liver cancer.However,the regulation mechanism of hypoxia on HCC is complex and has not been fully studied.Recent studies have found that exosomes are also involved in the occurrence and development of liver cancer.Exosomes,through the bioactive substances carried by exosomes,activate the signaling pathway of target cells and promote tumor progression.The formation and secretion of exosomes is regulated by a series of small proteins,The soluble N-ethylmaleimide sensitive factor Attachment protein（Rab）and N-ethylmaleimide sensitive factor attachment protein（SNARE）complexes were found.It has been reported that GPC3 also exists in exosomes,but the secretion mechanism of GPC3 and the role of exosome GPC3 in HCC are not clear.The purpose of this study was to investigate the effect and mechanism of hypoxia on the expression and secretion of GPC3 and the mechanism of exosome GPC3 in promoting the proliferation and migration of hepatocellular carcinoma,so as to provide a theoretical basis for further clarifying the development of HCC.Methods:（1）Huh7 cells were treated with 1% O₂ for different time: the expressions of GPC3 and HIF-1α in Huh7 cells were detected by RT-QPCR,and the expressions of GPC3,HIF-1α,STX11 and SYT7 were detected by Western Blot and immunofluorescence.Exosomes were extracted from cell supernatant by ultra-high speed centrifugation method.The morphology of exosomes was observed by transmission electron microscopy,the concentration and particle size of exosomes were detected by nano-particle size tracing analysis（NTA）,and the expressions of exosome labeled proteins HSP70,Alix,CD63 and GPC3 were detected by Western Blot.（2）si RNA targeting HIF-1α was synthesized.Huh7 cells were transfected with50 n M si RNA by liposome transfection method for 6h and treated with 1% O₂ for 24 h.The expressions of HIF-1α,GPC3,STX11 and SYT7 were detected by Western Blot and immunofluorescence.The stable cell lines with HIF-1α knockdown were constructed by Crispr Cas9 gene knockout technology.After treated with 1% O₂ for 24 h,the supernatant exosomes were extracted by ultra-high speed centrifugation method,and the expressions of exosome marker proteins Alix,HSP70 and CD63 were detected by Western Blot.NTA was used to detect the particle size and concentration of exosomes.（3）Huh7 cells were incubated with GW4869（0μmol/L,10μmol/L,20μmol/L）for2h,then treated with 1% O₂ for 24 h.Western Blot was used to detect GPC3 expression level in Huh7 cells.Huh7 cells were treated with 20μmol/L GW4869 for 2h and then treated with 1% O₂ for 24 h.The fluorescence intensity of GPC3 in cells was detected by immunofluorescence.（4）GPC3 knockdown stable cell lines were constructed by Crispr Cas9 gene knockout technology,and exosomes were extracted by ultra-high speed centrifugation method.After exosomes were labeled with PKH67 dye,Huh7 cells were re-incubated with 20μg/m L for 24 h,and exosomes absorption was observed by high resolution confocal fluorescence microscope.Exosomes were extracted from GPC3 knockdown cell lines and control cell lines,and incubated with 20μg/m L Huh7 cells for24h-48 h.Western Blot was used to detect the protein expression levels of PCNA,Cyclin D1,E-cadherin,N-cadherin,Vimentin,C-my C,β-catenin,GSK-3β,Beclin1,LC3Ⅱ、p62.Confocal high-intension imaging analysis system was used to detect cell proliferation.The migration ability of cells was detected by scratch test.After transfection with m Cherry GFP-LC3 dual fluorescent plasmid for 6h,Huh7 cells were incubated with 20μg/m L GPC3 normal cell line exosomes and GPC3 knockdown cell line exosomes for 12 h,respectively.Autophagosomes were observed under high resolution confocal fluorescence microscope.（5）Four-week-old nude mice were randomly divided into control group,GPC3 normal exosome group and GPC3 knockdown exosome group.1×107/m L Huh7 cells and 250μg exosomes were mixed,and 200 u L tumor was formed subcutaneously in each nude mouse.After 25 days,the mice were euthanized with 0.1% pentobarbital sodium and the tumor size of nude mice was observed.The tumor tissues of nude mice were taken out,and the positive expression rates of Vimentin,PCNA and C-My C were detected by immunohistochemical staining.Results:（1）Compared with the control group,1% O₂ up-regulated the m RNA level of GPC3 in cells,but down-regulated the protein level in a time-dependent manner（P<0.05）.Meanwhile,hypoxia promoted the secretion of exosome and exosome GPC3,up-regulated the expression of exosome marker proteins Alix,HSP70 and CD63,and down-regulated the expression of exosome secretion-regulating proteins STX11 and SYT7（P<0.05）.（2）Compared with the control group,interference with HIF-1α up-regulated GPC3 protein level,inhibited exosome secretion,down-regulated the expression of exosome marker proteins Alix,HSP70 and CD63,but up-regulated the expression of exosome secretion-related proteins STX11 and SYT7（P<0.05）.（3）Compared with the control group,the protein level of GPC3 was up-regulated with the concentration of GW4869;Compared with the control group,the protein level of GPC3 in cells was down-regulated by hypoxia,but the protein level of GPC3 in cells recovered after pretreatment with GW4869（P<0.05）.（4）PKH67 staining showed that exosomes could be self-absorbed by HCC cells.Compared with the control group,exosomes in GPC3 normal group promoted the proliferation and migration of Huh7 cells,promoted the tumor growth of mice,up-regulated the expression of N-cadherin,Vimentin,PCNA,Cyclin D1,down-regulated the expression of E-cadherin（P<0.05）;Compared with GPC3 normal group,GPC3 knockdown group exosomes down-regulated the expression of N-cadherin,Vimentin,PCNA,Cyclin D1 protein,and up-regulated the expression of E-cadherin protein（P<0.05）.（5）Compared with the control group,the expression of Wnt/β-catenin signaling pathway proteins C-myc and β-catenin were up-regulated by exosomes in GPC3 normal group,and the expression of GSK-3β was down-regulated（P<0.05）;Compared with GPC3 normal group,GPC3 knockdown group exosomes down-regulated the expression of Wnt/β-catenin signaling pathway proteins C-myc and β-catenin,and up-regulated the expression of GSK-3β（P<0.05）.（6）Compared with the control group,exosomes in GPC3 normal group up-regulated the expression of autophagy related proteins Beclin1 and LC3Ⅱ,down-regulated the expression of p62,and enhanced the intracellular autophagy flow（P<0.05）;Compared with normal GPC3 group,GPC3 knockdown group exosomes down-regulated the expression of autophagy related proteins Beclin1 and LC3Ⅱ,up-regulated the expression of p62,and decreased intracellular autophagy flow（P<0.05）.Conclusion: Hypoxia promotes the secretion of exosome GPC3 through HIF-1α.Exosome GPC3 affects the proliferation and EMT of HCC cells through Wnt/β-catenin signaling pathway and autophagy.