Department of Pathology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China.
World J Gastroenterol. 2009 Nov 21;15(43):5442-8. doi: 10.3748/wjg.15.5442.
To develop short hairpin RNA (shRNA) against heparanase, and to determine its effects on heparanase expression and the malignant characteristics of gastric cancer cells.
Heparanase-specific shRNA was constructed and transferred into cultured the gastric cancer cell line SGC-7901. Stable subclonal cells were screened by G418 selection. Heparanase expression was measured by reverse transcriptase-polymerase chain reaction (RT-PCR), real-time quantitative PCR and Western blotting. Cell proliferation was detected by 2-(4, 5-dimethyltriazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetry and colony formation assay. The in vitro invasiveness and metastasis of cancer cells were measured by cell adhesion assay, wound healing assay and matrigel invasion assay. The angiogenesis capabilities of cancer cells were measured by tube formation of endothelial cells.
Stable transfection of heparanase-specific shRNA, but not of scrambled shRNA and mock vector, resulted in reduced mRNA and protein levels of heparanase. The shRNA-mediated knockdown of heparanase did not affect the cellular proliferation of SGC-7901 cells. However, the in vitro invasiveness and metastasis of cancer cells were decreased after knockdown of heparanase. Moreover, transfection of heparanase-specific shRNA decreased the in vitro angiogenesis capabilities of SGC-7901 cells.
Stable knockdown of heparanase can efficiently decrease the invasiveness, metastasis and angiogenesis of human gastric cancer cells. In contrast, stable knockdown of heparanase does not affect the cell proliferation.
构建针对乙酰肝素酶的短发夹 RNA（shRNA），并观察其对乙酰肝素酶表达及胃癌细胞恶性生物学行为的影响。
构建乙酰肝素酶特异性 shRNA 并转染胃癌细胞系 SGC-7901，G418 筛选稳定的亚克隆细胞株。采用逆转录-聚合酶链反应（RT-PCR）、实时定量 PCR 和 Western blot 检测乙酰肝素酶的表达。噻唑蓝（MTT）比色法和集落形成实验检测细胞增殖能力，细胞黏附实验、划痕愈合实验和基质胶侵袭实验检测细胞体外侵袭和转移能力，内皮细胞管腔形成实验检测细胞体外血管生成能力。
特异性 shRNA 转染可显著降低 SGC-7901 细胞乙酰肝素酶 mRNA 和蛋白的表达，而对照 shRNA 及空载体转染对乙酰肝素酶的表达无明显影响。乙酰肝素酶表达下调并不影响 SGC-7901 细胞的增殖能力，但可显著降低细胞的体外侵袭和转移能力，同时下调乙酰肝素酶的表达还可降低 SGC-7901 细胞的体外血管生成能力。
稳定下调乙酰肝素酶的表达可显著抑制人胃癌细胞的侵袭、转移和血管生成能力，而对细胞的增殖无明显影响。
