miR-540-3p partially recovers the locomotor function of spinal cord injury mice by targeting SIX4/Yap1 and inactivation of astrocytes

Spinal cord injury (SCI) lacks therapeutic reagents. miRNAs are responsible for mesenchymal stem cells (MSCs) therapy in spinal cord injury. To discover the underlying therapeutic miRNA target and its mechanism for the treatment of SCI. Two RNA sequence datasets were retrieved from the GEO Datasets database which was accessed on 30 December 2023. The targets of the top 2 ranked miRNAs (miR-540-3p and miR-433-5p) were analyzed using online databases (miRDB, miRMap, TargetScan and STRING database) and both miRNAs were screened by cell counting kit-8 (CCK-8) assay. Then, transfection and local injection of miR-540-3p were performed to examine the capacity of secretion of astrocytes and the locomotor function of SCI mice. The significantly high levels of miR-540-3p/433-5p were revealed. Transfection of miR-540-3p conferred inactivation of reactive astrocytes and weakened the capacity of secreting inflammatory cytokines of astrocytes. miR-433-5p was proven to not impact the proliferation of astrocytes. Co-culture of culture supernate from astrocytes transfected with miR-540-3p and neurons demonstrated the significantly preserved neurite length and decreased apoptotic level of neurons. Meanwhile, sine oculis homeobox (SIX4)/Yap1, as the target of miR-540-3p, is critical for abrogating inflammatory damage of neurons in vivo and in vitro, decreasing glial scar, and recovering locomotor function of spinal cord injury mice. Furthermore, SCI mice receiving a local injection of miR-540-3p showed smaller and lighter bladder volume and higher limb strength, but the period from urinary retention to autonomous urination of SCI mice showed no significance. Conclusively, miR-540 discovered from hypoxia-treated exosomes suppresses the inflammatory cytokines secreted by reactive astrocytes, partially preserves the neuronal function of spinal cord injury mice, through the SIX4/Yap1 signalling pathway.