Stable knockdown of heparanase expression in gastric cancer cells in vitro.
OBJECTIVE
To develop short hairpin RNA (shRNA) against heparanase, and to determine its effects on heparanase expression and the malignant characteristics of gastric cancer cells.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Zheng
Li-Duan
LD
Department of Pathology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China.
Jiang
Guo-Song
GS
Pu
Jia-Rui
JR
Mei
Hong
H
Dong
Ji-Hua
JH
Hou
Xiao-Hua
XH
Tong
Qiang-Song
QS
D002454:Cell Differentiation; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D005966:Glucuronidase / Q000096:biosynthesis*; D006801:Humans; D066298:In Vitro Techniques; D009361:Neoplasm Invasiveness; D009389:Neovascularization, Pathologic; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000201:enzymology*; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D000099175:Heparanase
D005966:Glucuronidase; D034741:RNA, Small Interfering; D013778:Tetrazolium Salts; D013844:Thiazoles; D000099175:Heparanase; C022616:thiazolyl blue