Establishment of orthotopic osteosarcoma xenograft tumour models

The animal study was approved by the ethical committee of Zhongnan Hospital of Wuhan University.This study was executed in the Animal Laboratory Center, Zhongnan Hospital of Wuhan University. Following previous published methods [21], orthotopic osteosarcoma xenograft tumour models were established successfully. In short, 143b cells were resuspended in 100 µl PBS (the concentration of 143b cells was 10⁷/mL) and injected into the subperiosteum of the left proximal lateral tibia of 4- to 6-week old male BALB/nude mice (about 10⁶ 143b cells were injected into each nude mice). Next, the mice were divided into two groups: the miR-539 agomir (Ribobio, Guangzhou China) treatment and the agomir control group. Ten days after the cells were injected, the volume of the tumours were approximately 100 mm³. The 100 µl miR-539 agomir or the agomir control was injected intratumourly every three days. The length and width of the tumours were measured with a digital caliper every day. The tumour volume was calculated with the following equation: tumour volume (mm³) = length × width² × 0.5. Digital plain-film X-ray was used to evaluate the orthotopic tumours. After the mice were sacrificed, the tumours were excised and weighed, and the lung tissue was also removed for further analysis. The obtained tissues were fixed with paraformaldehyde, embedded in paraffin, and then stained with H&E. And we observed them by microscope for histological analysis. Embedding, fixation and H&E staining were carried out according to the previous published methods [21].