The etiological factors for peripheral nerve injury (PNI), a frequent clinical disorder, are accidents, natural disasters, compression, or surgical procedures. PNI often results in disability and is an economic burden for patients(Noble et al., 1998; Williams et al., 2020; Zhang et al., 2018). In contrast to the neurons of the central nervous system (CNS) that have a poor regenerative capacity, the neurons of the peripheral nervous system (PNS) can undergo spontaneous regeneration after damage (Abe and Cavalli, 2008; Afshari et al., 2009). However, clinical nerve regeneration is not optimal owing to the low axonal growth rate of the damaged neurons (Nocera and Jacob, 2020). Axon regeneration is dependent on neuronal survival, axonal growth rate, and specificity for reinnervation of peripheral target organs (Liuzzi and Tedeschi, 1991). The dorsal root ganglion (DRG) and cortical neurons are excellent models for studying the regeneration of peripheral nerves (Aparicio et al., 2023; Dubový et al., 2019; Jones et al., 2018; Nascimento et al., 2018; Qian and Zhou, 2020; Varadarajan et al., 2022). Transcription factors and microRNAs (miRNAs) are required for the transition of neurons from a signal transmission site to a regeneration site after injury (Seijffers et al., 2007; Yang et al., 2022; Zhao et al., 2021). Hence, the molecular mechanisms underlying axon regeneration must be elucidated to improve the functional outcomes of nerve repair.

Extracellular vesicles (EVs), which participate in diverse physiological and pathological processes,which are involved in various physiological and pathological processes, have low immunogenicity and facilitate the transfer of nucleic acids, proteins, and lipids between cells (Wang et al., 2022). EVs are mainly synthesized in the cytoplasm, RNAs are incorporated into the EVs during processing and are protected against degradation by the phospholipid bilayer membrane. EVs comprise various RNAs, including messenger RNA (mRNA), miRNA, Piwi-interacting RNA, small nucleolar RNA, small nuclear RNA, ribosomal RNA and transfer RNA. In addition to providing useful information after liquid biopsy, exosomal RNAs serve as effector molecules of EVs to regulate cellular functions(Buzas, 2023). For example, EVs from various sources can mediate nerve injury repair by promoting neurovascular regeneration(Dong et al., 2019; Zhang et al., 2020a). Previously, our research group had demonstrated that skin precursor cell-induced Schwann cell-secreted EVs (SKP-SC-EVs) and sciatic nerve-derived fibroblast-secreted EVs (Fb-EVs) significantly enhance the growth of neuronal axons(Wu et al., 2020). Genomic studies have reported that 98% of the entire genome is transcribed into non-coding RNAs (Wilusz et al., 2009). >60% of human genes are estimated to have putative mRNA targets regulated by miRNAs. These findings indicate that miRNAs play a role in the development of .various diseases (Friedman et al., 2009). Previous research has shown that miR-222 (Zhou et al., 2012), miR-29a (Zou et al., 2015b), and miR-29c (Zou et al., 2015a) enhance the growth of neurites by targeting PTEN. miRNAs, such as miR-26a can also target signaling mediators. The miR-26a-GSK3β-Smad1 signaling pathway is responsible for regulating axonal regeneration in mammals (Jiang et al., 2015). Downregulating miR-192-5p promotes the expression of XIAP in injured sciatic nerves, suppressing neuronal apoptosis and promoting regeneration (Liu et al., 2020). Additionally, miRNAs, including miR-9 (Jiang et al., 2017; Zhou et al., 2014) and miR-29a-3p (Shen et al., 2022), are reported to promote peripheral nerve regeneration. Therefore, the elucidation of the roles of individual miRNAs can improve our understanding of the processes involved in PNI and regeneration and aid in the identification of novel therapeutic targets.

In this study, 12 overlapping miRNAs in two types of EVs that promote nerve regeneration were screened. miR-25-3p facilitated the process of axon regeneration in both DRG and cortical neurons at various stages of development. Tgif1 was discovered to be the miR-25-3p downstream target gene using transcriptome sequencing and biosignature analyses. Furthermore, Tgif1 knockdown in DRG and retinal ganglion cell (RGC) neurons promoted axon regeneration. Our study provided novel insights into the regulatory role and the molecular mechanism of miR-25-3p in axon regeneration. Additionally, it indicated that miR-23-3p has the potential to be targeted therapeutically for repairing nerve injuries.