磷脂酰肌醇蛋白聚糖3促进肝细胞癌侵袭和转移的初步研究

doi:10.3877/cma.j.issn.1674-0793.2019.02.001

中华普通外科学文献(电子版) ›› 2019, Vol. 13 ›› Issue (02) : 85 -89. doi: 10.3877/cma.j.issn.1674-0793.2019.02.001

所属专题：文献；

论著

磷脂酰肌醇蛋白聚糖3促进肝细胞癌侵袭和转移的初步研究

岳潇¹, 张国培¹, 李绍强¹^,^†(

)

^1. 510080　广州，中山大学附属第一医院肝外科

收稿日期:2018-12-07 出版日期:2019-04-01
通信作者: 李绍强
基金资助:
国家自然科学基金面上项目(81472254); 广东省科技计划社会发展基金项目(2016A020215064)

Initial study of invasion and metastasis in hepatocellular carcinoma regulated by glypican 3

Xiao Yue¹, Guopei Zhang¹, Shaoqiang Li¹^,^†()

^1. Department of Liver Surgery, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China

Received:2018-12-07 Published:2019-04-01
Corresponding author: Shaoqiang Li
About author:
Corresponding author: Li Shaoqiang, Email: lisqsums@126.com

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目的

探讨磷脂酰肌醇蛋白聚糖3（GPC3）是否能促进肝细胞癌（HCC）侵袭、转移。

方法

通过siRNA靶向沉默GPC3在人肝癌细胞Hep3B、HepG2中的表达，慢病毒介导GPC3在人肝癌细胞SMMC-7721和SK-HEP-1中过表达，用于体外细胞功能试验和建立体内裸鼠原位种植瘤模型。Western blotting、qPCR检测GPC3、上皮间质转化（EMT）相关分子N-Cadherin、E-Cadherin、SNAIL、Vimentin和基质金属蛋白酶（MMP）-2、MMP-3、MMP-7的表达。

结果

过表达GPC3的Hep3B、HepG2细胞株的迁移和侵袭能力显著大于GPC3低表达的SMMC-7721、SK-HEP-1细胞株（P<0.05）；小鼠肝脏原位癌模型显示，过表达GPC3的HCC肝脏转移发生率明显高于GPC3低表达者（P<0.05）。过表达GPC3可以降低E-Cadherin，上调N-Cadherin的表达；干扰GPC3表达后，E-Cadherin表达上调，N-Cadherin表达下调。GPC3过表达可显著上调MMP-2、MMP-3、MMP-7的表达（P<0.05）。

结论

GPC3可通过诱导HCC发生EMT和分泌MMPs，促进HCC细胞迁移和侵袭。

关键词: 癌,肝细胞, 磷脂酰肌醇蛋白聚糖类, 肿瘤侵润, 上皮间质转化

Objective

To investigate the role of GPC3 in invasion and metastasis of hepatocelluar carcinoma (HCC).

Methods

SiRNA targeting GPC3 mRNA was transfected into Hep3B and HepG2 cell lines. Recombinant lentiviral vector containing GPC3 gene was constructed and transfected into SMMC-7721 and SK-HEP-1. The expressions of epithelial-mesenchymal transition (EMT) markers (N-Cadherin, E-Cadherin, SNAIL, Vimentin) and metaproteinases (MMP)-2, MMP-3, MMP-7 were detected by Western blotting and qPCR.

Results

The ability of migration and invasion of those with GPC3 high expression was higher than those with low GPC3 expression (P<0.05). The occurrence of metastasis was higher in those with GPC3 high expression in mice orthotopic HCC model compared with low GPC3 expression (P<0.05). Over-expression of GPC3 up-regulated N-cadherin expression and inhibited E-cadherin, whereas, interference GPC3 expression by siRNA targeted GPC3 inhibited N-cadherin expression and upregulated E-cadherin expression. Furthermore, over-expression of GPC3 up-regulated MMP-2, MMP-3, MMP-7 expression in SMMC-7721 and SK-HEP-1 cell lines (P<0.05).

Conclusion

GPC3 promotes HCC cell migration and invasion through inducing EMT and up-regulation of MMPs expression.

Key words: Carcinoma, hepatocellular, Glypicans, Neoplasm invasiveness, Neoplasm metastasis, Epithelial-mesenchymal transition

图3 慢病毒载体转染GPC3的表达　A为慢病毒载体转染GPC3在SMMC-7721、SK-HEP-1细胞株中的蛋白及mRNA表达；B为慢病毒载体的转染效率；NC为对照组，GPC3为GPC3过表达组，^*** P<0.001

图4 GPC3敲除组和过表达组肝癌细胞体外迁移和侵袭实验　A为两组肝癌细胞迁移能力比较；B为两组肝癌细胞侵袭能力比较

图5 体内原位成瘤种植瘤模型　箭头所示为种植瘤（最右侧）和转移瘤体位置

图6 Hep3B、HepG2的GPC3敲除组和SMMC-7721、SK-HEP-1的过表达组中EMT相关分子表达水平　A为EMT相关分子蛋白水平表达；B为EMT相关分子mRNA水平表达；N-Cadherin（N-Ca），E-Cadherin（E-Ca）；* P<0.05，** P<0.01

图7 GPC3敲除组和过表达组MMPs相关分子表达　A为Western blotting检测蛋白水平；B为qPCR检测mRNA相对表达量；^*P<0.05，^**P<0.01，^***P<0.001

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