Mechanism Study On The Of Asparagus Polysaccharides Inhibiting Migration,invasion And Angiogenesis Of Hepatocellular Carcinoma Cells Based On HIF1α/VEGF Signaling Pathway

Objective:To investigate the differential expression of HIF1α mRNA between hepatocellular carcinoma(HCC)tissues and paracancerous tissues or normal liver tissues,and its correlation with survival prognosis and clinicopathological features of HCC patients,and further clarify the function of HIF1α in HCC cells(SK-Hep1 and Hep-3B).Later,in vitro experiments were conducted to study the effects of asparagus polysaccharides(ASP)on proliferation,migration,invasion and angiogenesis of HCC cells(SK-Hep1 and Hep-3B)as well as HIF1α/VEGF signaling pathway related molecular under normoxia and hypoxia.Finally,in vivo experiments were carried out to verify the effects of ASP on the growth of subcutaneous HCC cells xenograft tumor in nude mice and the expression of HIF1α/VEGF signaling pathway related molecular.Methods:The Oncomine and GSE14520 databases were used to analyze the differential expression of HIF1α mRNA between HCC tissues and paracancerous tissues or normal liver tissues,which was further verified by immunohistochemistry of the Human Protein Atlas database and western blotting of 12 pairs of HCC tissues and paracancerous tissues.The GSE14520 database was used to analyze the effects of differential expression of HIF1α mRNA on overral survival(OS)and recurrence-free survival(RFS)in HCC patients,and its correlation with clinicopathological features.HIF1α overexpression and RNA interference lentiviral stable transformation strains were constructed to study the effects of HIF1α on proliferation,migration,invasion and angiogenesis of HCC cell lines(SK-Hep1 and Hep-3B).ASP was used to treat HCC cells(SK-Hep1 and Hep-3B)under normoxia and hypoxia in vitro,and then the effects of ASP on proliferation,migration,invasion and angiogenesis of HCC cells(SK-Hep1 and Hep-3B)were observed.The effects of ASP on the expression of HIF1α/VEGF signaling pathways related molecular in HCC cells(SK-Hep1 and Hep-3B)were detected by ELISA,QRT-PCR,Western blotting and immunofluorescence under normoxia and hypoxia in vitro.The transplanted tumor model of subcutaneous SK-Hep1 in nude mice was constructed to observe the effect of ASP on the growth of transplanted tumors.The effect of ASP on the expression of HIF1α/VEGF signaling pathway related molecules in transplanted tumors was confirmed by immunohistochemistry and western blotting.Results: 1.The expression of HIF1α mRNA in HCC tissues was significantly up-regulated compared with paracancerous tissues or normal liver tissues,which were further surpporteded by immunohistochemistry and western blotting.HCC patients with high HIF1α mRNA expression had shorter OS than these HCC patients with low HIF1α mRNA expression(P=0.048),but there was no significant difference for recurrence-free survival(P=0.066).HCC Patients with high expression of HIF1α mRNA are more likely to develop TNM stage III and BCLC stage C stage.HIF1α overexpression and RNA interference lentiviral stable transformation strains were successfully constructed,which further confirmed that HIF1α could promote the proliferation,migration,invasion and angiogenesis of HCC cells(SK-Hep1 and Hep-3B).2.ASP could significantly inhibit the proliferation,migration,invasion and angiogenesis of HCC cells(SK-Hep1 and Hep-3B)with a dose-dependent manner under normoxia and hypoxia in vitro.The results of ELISA,QRT-PCR,western blotting and immunofluorescence showed that ASP could inhibit the expression of HIF1α and VEGF in HCC cells(SK-Hep1 and Hep-3B)under normoxia and hypoxia.The results of western blotting further indicated that ASP could inhibit the expression of p-AKT,p-m TOR and p-ERK protein,but had no significant effects on AKT,m TOR and ERK proteins,under normoxia and hypoxia in vitro.3.ASP could significantly inhibit the growth of subcutaneous SK-hep1 xenografts in nude mice.Results of immunohistochemistry showed that ASP could inhibit the expression of HIF1α,VEGF and CD34 protein in transplanted tumors.The results of western blotting further indicated that ASP could inhibit the expression of HIF1α,VEGF,p-AKT and p-ERK proteins in transplanted tumors,but had no significant effects on AKT and ERK protein expression.Conclusion: 1.Expression of HIF1α mRNA was significant up-regulated in HCC tissues,and HCC patients with high HIF1α mRNA expression had poor prognosis.HIF1α could significantly promote the proliferation,migration,invasion and angiogenesis of HCC cells(SK-Hep1 and Hep-3B).2.ASP could inhibit the proliferation,migration,invasion and angiogenesis of HCC cells(SK-Hep1 and Hep-3B)under normoxia and hypoxia in vitro.Mechanistic studies had shown that ASP could inhibit the expression of HIF1α and VEGF in HCC cells(SK-Hep1 and Hep-3B)under normoxia and hypoxia in vitro.Signal pathway studies showed that ASP could suppress the expression of p-m TOR,p-AKT and p-ERK proteins in HCC cells(SK-Hep1 and Hep-3B)under normoxia and hypoxia,but had no significant effects on the expression of m TOR,AKT and ERK proteins.3.ASP could inhibit the growth of subcutaneous SK-Hep1 xenografts in nude mice.Mechanistic studies have shown that ASP could inhibit the expression of HIF1α,VEGF,CD34,p-AKT and p-ERK proteins in transplanted tumors,but has no obvious effects on AKT and ERK proteins.