Experiments in vivo

Six-week-old nude mice were purchased from Shanghai Laboratory Animal Center, Laboratory Animal Company (Shanghai, China). For the tumor xenograft experiment, 8 nude mice were randomly divided into two groups. Each nude mouse was subcutaneously injected in the right armpit with 5×10⁶ A549 cells which stably expressed LV-let-7b-3p-inhibitor or LV-NC-inhibitor. Tumor formation was monitored every 4 days up to 24 days post inoculation. The volumes of tumors were estimated using the following equation: V = (length × width²)/2. The mice were euthanized and the tumors were harvested at the end of the study (day 24). Then, 6 other nude mice were randomly divided into two groups to establish the lung metastasis experimental model. Each nude mouse received a tail vein injection with 2×10⁶ A549 cells that expressed LV-let-7b-3p-inhibitor or LV-NC-inhibitor. The lungs of the mice were harvested after 2 months, and their metastatic nodes were counted. All the specimens were further subjected to HE staining and IHC. The animal experiments were performed under a project license (SCXK Lu 20090001) granted by the Shandong University Animal Research Ethics Committee, in compliance with institutional guidelines for the care and use of animals.