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Updated: Jun 18, 2026
Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells
Published on: May 9, 2020
Stable knockdown of heparanase expression in gastric cancer cells in vitro
Li-Duan Zheng1, Guo-Song Jiang, Jia-Rui Pu
1Department of Pathology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China.
Aim:
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.
Conclusion:
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.
