Transwell invasion assay

The same sterile Transwell membrane insert was used to perform the invasion assay. Before commencing the experiment, the BD matrigel (#356234, BD Biosciences, Franklin Lakes, NJ, USA) was placed in a −4 °C refrigerator for 24 h. The BD matrigel would turn into liquid. It was diluted to 1:9 ratios with RPMI 1640. The bottom of the Transwell upper chamber was filled with 50 ul of the diluent. The Transwell insert was incubated for 3 h until the matrigel clotted. The similar steps were performed as the migration assay: 1 × 10⁵ DU145 cells were loaded into the upper chamber of a 24-well matrigel invasion assay plate, the lower chamber was filled with 0.75 ml of RPMI 1640 medium containing 10 % FBS. After 24 h of incubation, cells that had migrated to the lower surface of the filter were fixed with 4 % paraformaldehyde and stained with 0.1 % crystal violet solution. Cell count was performed in nine random fields per insert.

Fifteen 6-week-old male BALB/c nude mice were supplied by the Experimental Animal Center of Guangdong province, China. All experimental animal protocols were approved by the Animal Care and Use Ethics Committee. The animals were maintained in a special pathogen free facility. All the mice were randomly divided into three groups of five mice each. DU145 cells were collected, washed, and resuspended in serum-free medium at a concentration of 5 × 10⁷ cells/ml. Aliquots of 100 ul cells from each group were subcutaneously injected into the right side of axillary region of each animal. The animals were followed for 4 weeks, the mice were sacrificed, and the tumors were dissected and put them into liquid nitrogen container for Quantitative RT-PCR and Western blot.