Nude mice of both sexes (age: 6-8 weeks, weight: 22-25 g) were purchased from HUNAN SJA LABRATORY ANIMAL CO., LTD (Hunan, China). The EC109 cells stably expressing sh-circBCAR3 or sh-nc were established by infection with corresponding lentivirus vectors. 1 x 10⁶ mL-1 (100 uL) cells were subcutaneously inoculated into the nude mice. The tumor volumes had been measured from day 5 to day 25. On day 25, the xenograft tumors were removed surgically, and the tumor weight was detected.

SPF male Sprague Dawley rats aged 8 weeks were purchased from Beijing HFK Bioscience Co., Ltd. and housed in the Experimental Animal Center of North China University of Science and Technology (licence no. SYXK(Ji)2020-007) at 22 ± 2 with 60 ± 5% humidity, 12 h light/dark cycles, and free access to food and water. The rats were raised adaptively for approximately 7 days before experimental manipulation. The animals were handled according to the National Institute of Healths Guide for the Care and Use of Laboratory Animals (1996) guidelines, and all animal experiments were approved by the Animal Care and Use Committee of North China University of Science and Technology. In addition, all efforts were made to minimize the number of animals used and their suffering.

The rats were anesthetized by intraperitoneal injection of pentobarbital sodium (40 mg/kg). Before the experiment, the rats were fasted for 8-10 h and ensured sufficient water. The left common carotid artery (CCA) and left external carotid artery (ECA) were ligated after skin incision in the middle of the neck. In the common carotid artery cut a small incision. In order to induce ischemia, a cord plug was inserted through the incision into the internal carotid artery (ICA) and then to the origin of the middle cerebral artery (MCA). After ischemia for 2 h, the rats were anesthetized and gently pulled out the cord plug. At the end of the reperfusion time, the rats were euthanized by injection of a minimal lethal dose of anesthesia.

In this study, 4-week-old specific pathogen free male nude mice were selected and assigned into 4 groups, each of which was respectively injected with U251 cells and U87 cells transfected with NC, hsa-miR-27a-3p angomir, si-TMEM161B-AS1, si-TMEM161B-AS1 + hsa-miR-27a-3p angomir. Each nude mouse was subcutaneously injected with 4 x 10⁵ cells in the right flank area for subcutaneous implantation.