Experimental animals and middle cerebral artery occlusion (MCAO) model

All animal experiments were performed with the approval and in accordance with standard principles approved by the Institutional Animal Care and Use Committee of China Resources & WISCO General Hospital (approval no. 2020-005; Hubei, China). The Experimental Animal Center of the Chinese Academy of Medical Sciences (Beijing, China) provided 90 male C57BL/6J mice (age, 8–10 weeks; body weight, 22–30 g) for the current experiments. The mice were maintained at 25°C in 40–60% humidity on a 12 h light/dark cycle with free access to food and water. All mice were divided into seven groups: i) Sham + Adeno-associated virus (AAV)-short hairpin RNA (sh)-negative control (NC) group (n=20); ii) I/R+ AAV-sh-NC group (n=20); iii) I/R + AAV-sh-XIST group (n=10); iv) I/R + AAV-sh-FOXO3 group (n=10); v) sham + AAV-NC mimic group (n=10); vi) I/R + AAV-NC mimic group (n=10); and vii) I/R + AAV-miR-27a-3p mimic group (n=10).

In all I/R groups, mice were subjected to MCAO. After anesthesia with pentobarbital sodium (45 mg/kg), a monofilament nylon suture was inserted into the internal carotid artery through the external carotid artery, followed by occluding the origin of the MCA. The occlusion continued for 2 h, and the suture was slowly removed to allow blood flow and induce reperfusion. The mice in the sham group were treated in the same way, but without occlusion of the MCA. Mice that died or failed to show an 80% reduction in cerebral blood flow after occlusion were excluded from the following experiments. A total of 24 h after reperfusion, measurements, including Longa's scores, cerebral blood flow and infarct volume, were conducted. The experiments were performed for ~26 h, with 2 h for occlusion and 24 h for reperfusion. The health and behavior of rats were monitored every 6 h during reperfusion. Animals (n=90) were immediately euthanized when they showed signs of weight loss >20%, dehydration or were non-ambulate, using intraperitoneal injection of 45 mg/kg pentobarbital sodium followed by cervical dislocation. Mouse death (n=90) was confirmed by bilateral thoracotomy and the subsequent absence of pupillary response, respiratory movement and heartbeat. When the I/R model was successfully established, the brain tissues were instantly collected and used for detection of the RNA expression.