Spinal cord injury (SCI) is a devastating neurological disorder that affects thousands of individuals each year. As about 150–180 hundred new cases increasing every year, the amount of the SCI patient population was estimated to be over 3 million cases around the world (David et al., 2019). However, not only the clinical treatment but also the functional rehabilitation is still the highly problematic issue in clinic, which greatly affects the physiological, psychological, and social activity of the SCI patients (Silva et al., 2014).

Methylprednisolone (MP), a kind of glucocorticoid, is used to be one of the most important medicine in SCI treatment at acute phase. Although several randomized controlled trial studies demonstrated the effectiveness of MP in SCI treatment (Bracken et al., 1984, 1990, 1997), more and more controversies arose recently because more complications but less effectiveness of MP were observed in patients with SCI (Evaniew et al., 2016). Previously, our study in non-human primates with SCI demonstrated that MP resulted in the dysfunction of neural stem cells (NSCs), which may help to explain the controversy of MP in SCI (Ye et al., 2018). NSCs are a kind of adult pluripotent stem cells located in nervous tissue in vivo (Ortiz-Alvarez et al., 2019). NSCs can differentiate into multiple cell lineages including neuron, astrocyte, and oligodendrocyte, which greatly contribute to nervous tissue repairment and regeneration (Tang et al., 2017). However, the molecular mechanism that MP affects the functions of NSCs is still unclear.

Long non-coding RNA (LncRNA), a novel member of non-coding RNA, is characterized by the length larger than 200 nt and without protein-coding potential (Ransohoff et al., 2018). Researches have demonstrated that LncRNA played an important role in regulation of cell function, which was greatly involved in physiological and pathological processes in vivo (Gil and Ulitsky, 2020). Moreover, it has been reported that LncRNA expression was up- or downregulated after glucocorticoid stimulation (Bunch, 2018). Whether LncRNAs contribute in the MP mediating NSC dysfunction needs to be addressed.

In this study, we investigate the expression profiles of LncRNA and mRNA of NSCs treated with MP using a microarray, followed by bioinformatics analysis. These results provide greater insight into the precise mechanisms of MP mediating NSC dysfunction in SCI.

NSCs, a kind of adult multipotential stem cell, are one of the critical cells in nervous tissue repairment after SCI. Once the spine cord injury occurs, endogenous NSCs are firstly activated from quiescent status, followed by migration to the injured sites, and then exhibit their healing function through differentiation into neuron or secreting cytokines (Stenudd et al., 2015). Except for the endogenous NSCs, more and more studies have demonstrated that exogenous NSC transplantation improved the recovery of nervous tissue in SCI models, emphasizing the important role of NSCs in SCI treatment (Yousefifard et al., 2016). However, these reparative processes are widely and usually affected by kinds of factors in vivo and in vitro.

MP is a widely used glucocorticoid for many diseases in clinic, in which its effectiveness is nevertheless a controversial focus of SCI treatment (Ahuja et al., 2017). Recently, our research found that MP inhibited the proliferation of NSCs in a non-human primate with SCI. Besides, MP affected the differentiation potentials of NSCs in a rat model with SCI (Wang et al., 2014). Moreover, high dose MP after SCI significantly reduces the proliferation rate of NSCs in vitro (Schroter et al., 2009). These results remind us that the ineffectiveness of MP in SCI may partially come from its effect on NSCs, but the detailed mechanism is largely unknown.

NSC functions were under the control of kinds of intracellular regulatory mechanisms at gene transcription and protein translation levels, among which LncRNAs were one of the most important mechanisms (Kim, 2016; Ramos et al., 2016). LncRNAs widely exist in cells and regulate the cells’ function through trans/cis-acting or endogenous competitive RNA (ceRNA) mechanisms (Guil and Esteller, 2015). Previously, many studies have shown the large involvement of LncRNA in regulating functions of NSCs. A LncRNA named Pnky was determined to be a neural-specific LncRNA to inhibit the neuronal differentiation of NSCs (Ramos et al., 2015). Moreover, another LncRNA, LncRNA-158, was proven to promote the NSC differentiation into oligodendrocytes through the NFκB signaling pathway (Li et al., 2018). Nevertheless, the LncRNA expression profiles are easily affected by external stimulation, which in turn results in function alternation of NSCs (Lu et al., 2018). In this study, in order to figure out the role of LncRNA in MP mediating NSC dysfunction, a LncRNA–mRNA microarray was performed to scan their expression in NSCs after MP stimulation. There were 63 differentially expressed LncRNAs and 174 differentially expressed mRNAs identified in MP treated NSCs compared to those control NSCs without stimulation. These results indicated that not only the mRNA expression profile but also the LncRNA expression profile was altered by MP treatment. Moreover, the possibility that MP affected the NSC function through regulation of their LncRNA expression existed.

A cell’s mRNA expression profile reflects its status and function. To investigate the MP affected functions of NSCs, GO was performed to enrich the differentially expressed mRNA. In the biological process domain of GO terms, functions about cell migration, secretion, and stimulation reaction were enriched, suggesting that MP stimulation at a concentration of 10 μg/ml for 24 h mainly affects these functions of NSCs. It is worth noting that NSCs migrating to the sites of injury and secreting large amount of neurotrophic factors are two key steps in neural restoration except for differentiation directly (Stenudd et al., 2015). Therefore, we suggest that MP may also affect the NSC migration and secretion potential in addition to the previously reported ability in proliferation and differentiation. To explore the enriched signaling pathway, we then performed the KEGG analysis based on the differentially expressed mRNA profile. Although 22 signaling pathways were enriched, the PI3K–Akt signaling pathway was determined to be the largest significant difference pathway with the highest gene ratio. It has been widely proven that the PI3K–Akt signaling pathway contributed greatly to the functional regulation of NSCs (Lee et al., 2016; Zhang et al., 2017; Wang et al., 2018). To our knowledge, for the first time, we demonstrated that MP may affect the NSCs by modulating the PI3K–Akt signaling pathway. Song et al. (2017) found that LncRNA named IGF2AS could inactivate the Akt signaling pathway and then protected the NSC derived neurons from apoptosis. We speculated that differentially expressed LncRNA may activate or inactivate the PI3K–Akt signaling pathway, which ultimately caused the dysfunction of NSCs. Functional experiments in vivo and in vitro should be performed to confirm this hypothesis.

LncRNAs always show a positive or negative correlation in expression level to their target gene (Chen, 2016). To further clarify the relationship and regulatory mechanism of differentially expressed mRNA and LncRNA, we identified the predicted target mRNA and their related LncRNA using bioinformatics methods based on the CNC network of those differentially expressed mRNA and LncRNA in this study. Among the five identified pair, NONMMUT067949-MMP12 was the most remarkable combination. Previous study by Shan et al. demonstrated that MMP12 was a critical molecular in the activation and development of NSCs (Shan et al., 2018). As our results, NONMMUT067949 may be positively related to MMP12 expression. After MP stimulation, NONMMUT067949 expression decreased, which downregulated the expression of MMP12 through cis-acting mechanism, and finally resulted in the dysfunction of NSCs. These relationships and the regulatory mechanisms are also need to be addressed in the future study.

In summary, we investigated the LncRNA and mRNA expression profiles of NSCs treated with MP. This study may help to understand the mechanism of MP to regulate NSCs’ function and the role of LncRNA in this process. However, limitations still exist in this study. The detailed regulatory mechanisms of these identified LncRNA are still unknown in cells and mice models. Besides, the LncRNA expression profiles of NSCs treated by MP at different concentrations or stimulation times have not been clarified. Further studies should be addressed to elucidate these questions.

The data of microarrays can be obtained from the NCBI SRA database at this link: https://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA727403&o=acc_s%3Aa.

JY, YT, and ZX designed research studies, conducted the experiments, analyzed the data, and wrote the manuscript. MM and YL conducted the experiments, analyzed data, and wrote the manuscript. KD conducted the experiments. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.