Anti-hepatocellular Carcinoma Effects Of RAj-Tspin,a Novel Peptide From Apostichopus Japoicus,in Huh7 Cells

Objective:Hepatocellular carcinoma(HCC)is the third leading cause of cancer-related mortality worldwide.Patients with HCC have unfavourable prognosis,although significant advances have been made in its diagnosis and treatment.Intrahepatic and extrahepatic metastases are major challenges for unfavourable prognosis of HCC.Improving the survival rate of HCC has great clinical significance with focusing on exploring the underlying mechanisms of HCC metastasis and seeking new therapeutic targets.rAj-Tspin,a soluble gene recombinant peptide from Apostichopus japoicus,containing a thrombin-sensitive protein-1(TSP-1)structural domain and an arginine-glycine-aspartate(Arg-Gly-Asp,RGD)motif,can bind to integrin receptors on the cell surface,inhibit the invasion and adhesion of tumor cells,and promote the apoptosis of tumor cells.ZYX(zyxin)is a member of the focal adhesion protein family.Its function is mainly involved in cell adhesion,cell invasion and cell proliferation through the interaction of multiple proteins.ZYX has been reported to be abnormally overexpressed in a variety of tumor tissues,which plays an important role in the process of tumor cell invasion and metastasis,and is closely related to the poor prognosis of cancer patients.However,the expression and role of ZYX in HCC remain unclear.Previous studies of our research group have shown that rAj-Tspin can inhibit ITGB1/FAK/AKT signaling pathway,but the specific inhibition mechanism is not clear.Therefore,this study combined with existing studies,focusing on ITGB1/ZYX,to explore the role of rAj-Tspin and ITGB1/ZYX in the progression of HCC and the interaction between ITGB1 and ZYX,so as to provide new strategies for the prevention and treatment of HCC.Methods:1.The effects of different concentrations of rAj-Tspin on the proliferation,migration,invasion and adhesion of Huh7 human hepatoma cells in vitro were investigated by CCK-8 assay,scratch assay,Transwell assay and adhesion assay.TUNEL assay was used to detect the effect of rAj-Tspin on apoptosis of Huh7 cells.The effects of rAj-Tspin on the protein expression levels of apoptosis and EMT process-related proteins were observed by Western blot.2.The effects of rAj-Tspin at different dosages on tumor growth in vivo were observed by establishing subcutaneous transplantation tumor model and in-situ tumor transplantation model of Huh7 cells in nude mice.The effects of rAj-Tspin on liver histopathology,and expression of EMT-related proteins were detected by H&E staining and immunohistochemical staining experiments.3.The expression and prognosis of ZYX in HCC were analyzed by GEPIA,UALCAN public information databases.4.The expression levels of ITGB1 and ZYX in normal liver tissues and HCC tissues and in LO2 normal hepatocytes and Huh7 human HCC cells were detected by immunoblotting.The effects of rAj-Tspin on the protein expression of ITGB1 and ZYX and the level of FAK/AKT phosphorylation were detected in vivo and in vitro.5.The specific mechanism of rAj-Tspin was investigated in vitro.5.1 The transfection of Huh7 cells was performed by using ZYX siRNA,and the experimental groups were as follows: negative control group(si-Control),knockdown group(si-ZYX),administration group(si-Control+rAj-Tspin)and knockdown administration group(si-ZYX+rAj-Tspin).The expression of ZYX was examined by Western blot assay was used to detect the expression of ZYX and verify the transfection effect.Wound healing test,Transwell test and adhesion test were used to investigate the effects of ZYX expression down-regulated on the proliferation,migration,invasion and adhesion of Huh7 cells in vitro.TUNEL assay was performed to detect the number of apoptosis-positive cells.The effects of down-regulation ZYX expression on ITGB1,ZYX,FAK/AKT pathway related proteins,apoptosis and EMT process related proteins were observed by Western blot assay.5.2 The transfection of Huh7 cells was performed by using ITGB1 siRNA,and the experimental groups were as follows: negative control group(si-Control),knockdown group(si-ITGB1),administration group(si-Control+rAj-Tspin)and knockdown administration group(si-ITGB1 + rAj-Tspin).The expression of ITGB1 was examined by Western blot assay to verify the transfection effect.The related cell phenotype and protein detection experiments were as the same as in 5.15.3 Huh7 cells were transfected with ITGB1 overexpression plasmid,and the expression of ITGB1 was detected by Western blot assay to verify the transfection effect.The experimental groups were as follows: negative control group(pcDNA 3.1),overexpression group(pcDNA-ITGB1),and overexpression administration group(pcDNA-ITGB1+rAj-Tspin).The related cell phenotype and protein detection experiments were as the same as in 5.15.4 Huh7 cells were co-transfected with ITGB1 overexpression plasmid and ZYX siRNA,and the protein expression levels of ITGB1 and ZYX were detected by Western blot assay to verify the transfection effect.The experimental groups were as follows:negative control group(pcDNA 3.1),overexpression group(pcDNA-ITGB1),overexpression null transfection group(pcDNA-ITGB1+si-Control),and overexpression co-transfection group(pcDNA-ITGB1+siZYX).The related cell phenotype and protein detection experiments were as the same as in 5.15.5 The interaction and localization between ITGB1 and ZYX were verified by Co-IP assay and immunofluorescence co-localization staining.Results:1.rAj-Tspin inhibited the growth of Huh7 cells in vitro.It exhibited inhibition of cell viability and the ability of migration,invasion and adhesion in tumor cells,promoted apoptosis and regulated the expression of apoptosis and EMT-related proteins in tumor cells.2.In vivo experiments showed that rAj-Tspin inhibited the growth of subcutaneous transplanted tumors and in-situ transplanted tumors in nude mice,and had no significant effect on the body weight of nude mice,while slowing down the EMT process.3.The search through GEPIA,UALCAN cancer database indicated that ZYX was overexpressed in liver cancer tissues and patients with high ZYX expression had a worse prognosis than those with low expression.4.Western blot analysis showed that compared with normal liver tissues and normal LO2 hepatocytes,the protein expression levels of ITGB1 and ZYX were significantly increased in HCC tissues and Huh7 cells.Moreover,rAj-Tspin could inhibit the protein expression levels of ITGB1 and ZYX and the phosphorylation levels of FAK/AKT pathway in tissues and cells.5.ZYX/ITGB1 siRNA transfection inhibited the ability of migration,invasion and adhesion in Huh7 cells,promoted the apoptosis of tumor cells and regulated the expression of apoptosis and EMT-related proteins in tumor cells.ZYX siRNA transfection had no significant effect on the expression level of ITGB1.6.Overexpression of ITGB1 could activate ZYX/FAK/AKT pathway and promote the ability of migration,invasion and adhesion in Huh7 cells,but this trend could be blocked by rAj-Tspin and ZYX siRNA.Co-IP assay and immunofluorescence co-localization staining assay showed that ITGB1 and ZYX had interaction.Conclusion:1.rAj-Tspin can inhibit the growth of hepatocellular carcinoma in vivo,promote the apoptosis of hepatocellular carcinoma cells and inhibit the migration,invasion and adhesion of tumor cells.2.ZYXIN is significantly increased in hepatocellular carcinoma cells.ITGB1 interacts with ZYX.Knockdown of ITGB1/ZYX can inhibit tumor cell invasion and promote tumor cell apoptosis.ITGB1/ZYX signal pathway can become a potential therapeutic target for hepatocellular carcinoma.3.rAj-Tspin inhibits the migration,invasion and adhesion of hepatocellular carcinoma cells by inhibiting ITGB1/ZYX/FAK/AKT signaling pathway.