基于HIF-1α/VEGF/NF-κB信号通路探讨天冬多糖调控COMMD3抑制肝癌血管生成的作用机制

Antiangiogenic mechanism of asparagus polysaccharide in hepatocellular carcinoma by regulating COMMD3 via the HIF-1α/VEGF/NF-κB signaling pathway

目的:研究天冬多糖通过HIF-1α/VEGF/NF-κB信号通路调控COMMD3抑制肝癌血管生成的作用机制。方法:体外实验:采用平板克隆实验、流式细胞凋亡检测、划痕实验、Transwell小室实验和血管形成实验检测天冬多糖干预COMMD3过表达的SK-Hep1和Hep-3B细胞的增殖、凋亡、迁移、侵袭和血管生成。Western Blot测定COMMD3、HIF1-α、VEGF和NF-κB蛋白表达水平。体内实验:通过构建裸鼠皮下移植瘤模型,观察天冬多糖灌胃干预后裸鼠瘤体的变化和免疫组化染色检测各组裸鼠皮下移植瘤的HIF-1α、VEGF、NF-κB和CD34的表达变化。结果:体外实验:与Control组的肝癌细胞SK-Hepl和Hep-3B比较,OvCOMMD3组的增殖(P<0.01)、迁移(P<0.05)、侵袭(P<0.05)和血管生成(P<0.01)能力显著增强,Control+ASP组的增殖(P<0.05,P<0.01)、迁移(P<0.01)、侵袭(P<0.01)和血管生成(P<0.01)能力显著降低,促凋亡(P<0.01)能力显著增强。与Control组比较,ASP对肝癌细胞的增殖、迁移、侵袭和血管生成的抑制作用和对COMMD3、HIF-1α、VEGF、NF-κB蛋白表达的抑制作用都被OvCOMMD3所回补。体内实验:ASP对裸鼠瘤体生长和血管生成的抑制作用能够被COMMD3过表达所回补,ASP对瘤体中HIF-1α、VEGF、NF-κB和CD34表达的抑制作用也被COMMD3过表达所回补。结论:天冬多糖可以通过下调COMMD3的表达抑制肝癌细胞的增殖、迁移、侵袭和血管生成,其机制可能与调节HIF-1α/VEGF/NF-κB信号通路有关。

Objective:To explore the antiangiogenic mechanism of asparagus polysaccharide in hepatocellular carcinoma by regulating COMMD3 via the HIF-1α/VEGF/NF-κB signaling pathway.Methods:Experiments in vitro:The clonogenic assay,apoptosis assay,cell wound healing assay,transwell chamber invasion assay and in vitro tube formation assay were used to detect the proliferation,apoptosis,migration,invasion,and angiogenesis of hepatocellular carcinoma cells by regulating COMMD3.The expression levels of COMMD3,HIF-1α,VEGF and NF-κB protein were determined by Western Blot.Experiments in vivo:The changes of subcutaneous transplanted tumors in nude mice were observed after giving asparagus polysaccharide,and the expression of HIF-1α,VEGF,NF-κB and CD34 in subcutaneous transplanted tumors in nude mice were detected by immunohistochemical staining.Results:Experiments in vitro:Compared with the hepatoma carcinoma cell SK-Hepl and Hep-3B in the control group,the proliferation(P<0.01),migration(P<0.05),invasion(P<0.05)and angiogenesis(P<0.01)abilities of the OvCOMMD3 group were significantly enhanced,the proliferation(P<0.05,P<0.01),migration(P<0.01),invasion(P<0.01)and angiogenesis(P<0.01)abilities of the Control+ASP group were significantly decreased,and the pro-apoptotic(P<0.01)abilities were significantly enhanced.Compared with the control group,the inhibitory effects of ASP on the proliferation,migration,invasion and angiogenesis of hepatocellular carcinoma cells and on the expressions of COMMD3,HIF-1α,VEGF,and NF-κB were all compensated by OvCOMMD3.Experiments in vivo:The inhibitory effects of ASP on tumors growth and angiogenesis in nude mice can be compensated by the overexpression of COMMD3,and the inhibitory effects of ASP on the expressions of HIF-1α,VEGF,NF-κB and CD34 in tumors were also compensated by the overexpression of COMMD3.Conclusion:Asparagus polysaccharide can inhibit the proliferation,migration,invasion and angiogenesis of hepatocellular carcinoma cells by downregulating the expression of COMMD3,which may be related to the regulation of HIF-1α/VEGF/NF-κB signaling pathway.