Xenografted tumorigenicity assay in nude mice

Five-week-old male mice (BALB/c nu/nu, National Science Council Animal Center, Taipei, Taiwan) were subcutaneously injected with 3 × 10⁶ viable transfected Bm7 cells, in a total volume of 100 μL PBS, into both hind limbs. Thirty days after implantation, the mice were sacrificed, and the tumor volume (V) of the xenografts determined by measuring the length (L), width (W), and depth (D) of the tumors with a measuring caliper and using the formula: V = (L × W × D × π)/6; the average value was calculated.38^,39 The expression levels of miR-195-5p, miR-101-3p, and miR-338-5p in the excised tumor samples were detected using miRNA quantitative real-time PCR, as previously described.36^,40 All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee (IACUC) of China Medical University.