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

Background: Spinal cord injury (SCI) lacks therapeutic reagents. miRNAs are responsible for mesenchymal stem cells (MSCs) therapy in spinal cord injury.

Purpose: To discover the underlying therapeutic miRNA target and its mechanism for the treatment of SCI.

Method: 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.

Results: 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.

Conclusions: 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.