In vivo tumor growth assay

To evaluate the in vivo growth of NB tumors, 2-month-old female nude mice were subcutaneously inoculated in the right flank with either miR- 141-mimic- or C-miRNA-transduced IMR-132 cells (1×10⁶). The lengths and widths of the subcutaneous tumors were measured weekly, and the tumor volume (V) was calculated using the formula: V = length × width²/2. Five weeks later, the mice were sacrificed. Subcutaneous tumors were extracted and formalin-fixed and paraffin-embedded sections were prepared. Immunohistochemistry was then performed using an anti-Ki-67 antibody (Cell Signaling, USA).