CXCL7-Mediated Stimulation of Lymphangiogenic Factors VEGF-C, VEGF-D in Human Breast Cancer Cells

Figure 3

CXCL7 siRNA decreased heparanase activity and invasion of CXCL7-stably transfected cells. The CXCL7 MISSION siRNA was used to effectively knockdown heparanase activity and the invasion of CXCL7-stably transfected cells while the nontargeting siRNA was used as a control. CXCL7-stable transfected MCF10AT cells ($2\times {10}^5$) were transfected with 100 pmol of each siRNA by using the Lipofectamine 2000 transfection reagent (Invitrogen) after 48 hours in culture. (a) Quantifying heparanase activity. The CM was collected 24 hours later and analyzed by heparanase-degrading enzyme assay kit. Indicated amounts of cell lysate were incubated with biotinylated heparan sulfate at ${37}^{°}\text{C}$ for 45 minutes, and enzyme activity was determined using an ELISA-type assay. Color was developed using the substrate supplied in the kit, and plates were read at 450 nm using a microplate reader. Decreased heparanase activity in CXCL7 siRNA. (b) Invasion of Matrigel. Using a BD BioCoat Matrigel invasion assay (6-well plates), $1.25\times {10}^5$ cells/ml were inoculated onto the membrane in the upper chamber in serum-free medium. The lower chamber contained complete medium, and control membrane did not have Matrigel to measure migration of cells. Triplicate assays per group were completed. Results reported per cell line as % invasion = (mean number of cells invading Matrigel membrane/mean number of cells migrating through control membrane) $\times 100$. CXCL7 siRNA significantly inhibited the invasion of CXCL7 stable transfected MCF10AT cell, while the control siRNA did not. Results compared by one-way ANOVA ($P<.01$).

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