沉默B7-H3基因表达对人肝癌细胞侵袭能力的影响

康富标; 王玲; 王燕; 李东; 孙殿兴

doi:10.3971/j.issn.1000-8578.2016.12.007

沉默B7-H3基因表达对人肝癌细胞侵袭能力的影响

Effects of Silencing B7-H3 Gene on Invasion of Human Hepatocellular Carcinoma Cells

摘要

摘要:
目的研究靶向沉默B7-H3基因表达对HepG2细胞侵袭能力的影响及可能机制。
方法设计针对B7-H3基因的shRNA沉默质粒，转染HepG2细胞，下调B7-H3的表达。划痕修复实验检测转染前后HepG2细胞移行能力的变化，Transwell实验检测基因沉默对细胞侵袭能力的影响，MST-1法和ELISA凋亡试剂盒分别检测细胞增殖和凋亡水平变化。通过Western blot实验和明胶酶谱实验检测侵袭相关分子MMP-2、MMP-9的表达和活性变化。
结果成功构建B7-H3 shRNA沉默质粒并转染HepG2细胞。与对照质粒转染组和未转染组比较，沉默B7-H3基因表达后，B7-H3 shRNA转染组HepG2细胞的移行（24 h: P=0.001; 48 h: P<0.001; 72 h: P<0.001）和侵袭（P<0.001）能力受到显著抑制，细胞的增殖和凋亡水平没有明显变化（P>0.05）。MMP-2、MMP-9的蛋白表达（MMP-2: P<0.001; MMP-9: P=0.007）和活性（MMP-2: P<0.001; MMP-9: P<0.001）均显著下降。
结论通过B7-H3 shRNA沉默质粒靶向沉默B7-H3的基因表达能抑制HepG2细胞的侵袭能力，其机制可能与抑制MMP-2、MMP-9的表达和活性有关。

Abstract:
Objective To investigate the effects of silencing B7-H3 gene on invasion of human hepatocellular carcinoma (HCC) cell line HepG2 in vitro and its possible mechanism.
Methods The shRNA silencing plasmid was designed and transfected into HepG2 cells to down-regulate B7-H3 gene expression. The movement abilities before and after transfection were measured by scratch wound healing assay. The invasive abilities were measured using transwell invasion model. Cell proliferation and apoptosis were measured by WST-1 assay and ELISA apoptosis detection kit, respectively. Western blot and gelatin zymography experiment were used to detect the expressions and activities of invasion related molecules MMP-2, MMP-9.
Results The shRNA silencing plasmid was designed and transfected into HepG2 cells successfully. B7-H3 depletion suppressed scratch wound healing ability(24h: P=0.001; 48h: P<0.001; 72h: P<0.001) and invasive ability(P<0.001) of HepG2 cells significantly, whereas there was no obvious effects on cells’ proliferation and apoptosis(P>0.05). B7-H3 depletion downregulated the expressions and activities of MMP-2, MMP-9(P＜0.05).
Conclusion Targeting silence B7-H3 expression can inhibit the invasion ability of HepG2 cells in vitro, which may be related with down-regulation of expressions and activities of invasion related molecules MMP-2, MMP-9.

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