沉默GPC-3基因转录对裸鼠肝癌移植瘤的抑制作用

摘要

目的：研究沉默磷脂酰肌醇蛋白多糖-3(glypican-3，GPC-3)基因转录抑制肝癌HepG2细胞裸鼠移植瘤生长与机制。方法：以人GPC-3基因序列设计并合成miRNA，构建GPC-3-miRNA干扰质粒。将其导入HepG2细胞，通过杀稻瘟菌素筛选出稳定株，移植裸鼠皮下成瘤。根据体积的均数值绘制肿瘤生长曲线，免疫组化法检测瘤组织内GPC-3、β-catenin、p-GSK3β及cyclinD1表达。结果：肝癌HepG2、Hep3B 和 MHCC-97H 细胞 GPC-3表达较强(50%～80%)， SMMC-7721和Bel-7402细胞中等(18%～25%)，PLC/PRF/5和Bel-7404细胞较弱(8%～10%)。以特异miRNA干预GPC-3基因转录，明显抑制 HepG2细胞增殖，呈时间依赖性；沉默 GPC-3能明显抑制裸鼠皮下移植瘤生长，且瘤组织β-catenin、p-GSK3β及cyclinD1等关键信号分子表达降低。结论：沉默GPC-3影响Wnt/β-catenin通路关键信号分子表达可能是抑制HepG2增殖的分子机制。%Objective: To silence glypican-3(GPC-3) gene transcription on effects of suppression of nude mice HepG2 cell xenograft growth and mechanism. Methods: miRNA targeting GPC-3 was designed, and plasmid GPC-3-miRNA was constructed and transfected into HepG2 cells. Stable cell clones were screened by Blasticidin S HCl, and transplanted into nude mice to establish xenograft, with monitored the tumor growth daily and the expression of GPC-3,β-catenin, p-GSK3βand cyclinD1 protein in tumor tissues detected by immunohistochemistry. Results:GPC-3 was the strongest expressions(50%-80%) in hepatoma HepG2, Hep3B, and MHCC-97H cells, the moderate expression (18%-25%) in SMMC-7721 and Bel-7402 cells, and the lowest expression (8%-10%) in PLC/PRF/5 and Bel-7404 cells. The GPC-3 gene transcription was significantly silenced by specific miRNA, HepG2 cell proliferation was inhibited with time dependence. The growth of nude mice HepG2 cell xenograft tumors was significantly suppressed by down-regulating GPC-3, accompanied the decreased ofβ-catenin, p-GSK3β, and cyclinD1 expression. Conclusion: Suppression of GPC-3 inhibits growth of HepG2 cell xenograft in nude mice through regulation of Wnt/β-catenin Signaling pathway.