Long noncoding RNA XIST knockdown relieves the injury of microglia cells after spinal cord injury by sponging miR-219-5p

Abstract Long noncoding RNAs have been demonstrated to play crucial roles in the pathogenesis of spinal cord injury (SCI). In this study, we aimed to explore the roles and underlying mechanisms of lncRNA X-inactive specific transcript (XIST) in SCI progression. SCI mice model was constructed and evaluated by the Basso–Beattie–Bresnahan method. The SCI cell model was constructed by treating BV2 cells with lipopolysaccharide (LPS). The levels of XIST and miR-219-5p were determined by the reverse transcription quantitative polymerase chain reaction. The concentrations of inflammatory cytokines were measured by enzyme-linked immunosorbent assay. Protein levels were measured via western blot assay. Cell viability and apoptosis were evaluated by cell counting kit-8 assay and flow cytometry analysis, respectively. The relationship between XIST and miR-219-5p was analyzed by online tool starBase, dual-luciferase reporter assay, and RNA immunoprecipitation assay. As a result, the XIST level was enhanced and the miR-219-5p level was declined in the SCI mice model. XIST was also upregulated in LPS-induced BV2 cells. LPS treatment restrained BV2 cell viability and accelerated apoptosis and inflammatory response. XIST knockdown effectively weakened LPS-induced BV2 cell injury. miR-219-5p was identified as a target of XIST. Moreover, inhibition of miR-219-5p restored the impacts of XIST knockdown on cell viability, apoptosis, and inflammation in LPS-treated BV2 cells. In addition, LPS-induced XIST promoted the activation of the nuclear factor-κB (NF-κB) pathway by sponging miR-219-5p. In conclusion, XIST silencing promoted microglial cell viability and repressed apoptosis and inflammation by sponging miR-219-5p, thus promoting the recovery of SCI.


Introduction
Spinal cord injury (SCI) is one of the most serious types of nerve injury caused by external direct or indirect factors [8,18]. The prognosis of patients with SCI is extremely dismal, causing limb movement disorders, loss of cognitive function, and even paralysis, which seriously affect people's quality of life [9,21]. Currently, although a large number of studies have explored the treatment strategies for SCI, the effects remain unsatisfactory [1,4]. Thus, it is crucial to explore the potential mechanisms of SCI development and develop novel therapeutic targets for SCI.
Long noncoding RNAs (lncRNAs) are a series of noncoding RNAs (ncRNAs) containing >200 nucleotides (nts) in length, exerting their functions mainly by sponging microRNAs (miRNAs) [19,25]. It has been demonstrated that lncRNAs regulate a variety of physiological functions, and neurological diseases, including SCI, have been demonstrated [24]. For example, Zheng et al. disclosed that the elevation of taurine upregulated gene type 1 (TUG1) repressed lipopolysaccharide (LPS)-stimulated PC-12 cell damage, as demonstrated by the promotion in cell viability and the suppression in cell apoptosis and inflammation, by decreasing miR-127 and inactivating nuclear factor-κB (NF-κB) pathway [30]. Zhou et al. claimed that metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) level was conspicuously raised in SCI mice and LPS-activated microglial cells, and MALAT1 knockdown relieved LPS-stimulated inflammatory injury by regulating miR-199b and IκB kinase β (IKKβ)/NF-κB pathway [31]. These reports indicated the vital roles of lncRNAs in SCI development. As for X-inactive specific transcript (XIST), Zhao et al. uncovered that XIST deficiency facilitated the recovery of SCI by reducing miR-27a and elevating Smurf1 [29]. Nevertheless, the molecular mechanisms of XIST in regulating SCI progression are not well understood.
miRNAs are small ncRNAs consisting of ∼22 nts and exert vital regulatory roles at the posttranscriptional level [6]. An increasing number of miRNAs have been identified to be closely linked to the progression of SCI. For instance, miR-27a-3p inhibited the inflammatory injury of SCI by interacting with toll-like receptor 4 (TLR4) [28]. miR-129-5p repressed the inflammatory response and apoptosis in LPS-stimulated BV2 cells via the modulation of high-mobility group protein B1 (HMGB1)/TLR4/ NF-κB pathway [22]. More importantly, a previous study by Zhu et al. showed that miR-219-5p was able to promote the recovery of SCI and motor function by regulating inflammation and oxidative stress [32]. However, the exact roles of miR-219-5p in SCI are largely unknown. Through online tool starBase, we found that the XIST contained potential binding sites with miR-219-5p. However, the relationship between XIST and miR-219-5p in regulating SCI progression has not been explored.
In this research, we established SCI mice and cell models and explored the effects of XIST on BV2 cell viability, apoptosis, and inflammation after SCI. Moreover, the possible mechanism and signaling pathway of XIST in SCI progression were further investigated.

Construction of SCI mice model
Adult C57bl/6J mice (female; 20-25 g) were purchased from the Vital River (Beijing, China) and divided into two groups: SCI group (n = 10) and sham group (n = 10). In SCI groups, the mice were incised along the neck after anesthesia, and then C5 lamina was excised to exhibit the dural sac. Next, adjust the hammer position of the spinal impactor on the C5 spinal cord. After that, bleeding was stopped and the incision layer by layer was sutured. In the sham group, C5 lamina was removed in the mice and C5 spinal cord was not impinged. The hindlimb locomotor activity of the mice was evaluated by Basso-Beattie-Bresnahan (BBB) Locomotor Rating Scale score. Subsequently, after 7 days of SCI, the mice were anesthetized and the spinal cord tissue specimens were collected for the following experiments. The study was allowed by the Ethics Committee of Animal Research of Yue Bei People's Hospital and conducted according to the Guidelines for Care and Use of Laboratory Animals of "National Institutes of Health."

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA in spinal cord tissues and BV2 cells was isolated utilizing TRIzol (Invitrogen). Next, the RNAs were

Enzyme-linked immunosorbent assay (ELISA)
The concentrations of inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-10) in the spinal cord tissue extracts or BV2 cell supernatants were measured by ELISA kits (ab208348; ab197742; ab100713; ab108870; Abcam, Cambridge, MA, USA) based on the guidelines of manufacturers. The absorbance was measured at 450 nm utilizing a microplate reader (Bio-Rad, Hercules, CA, USA), and the concentrations were calculated based on the standard curve.

Cell counting kit-8 (CCK-8) assay
After treatment with LPS and transfection, BV2 cells were harvested to assess cell viability through CCK-8 assay.
Briefly, BV2 cells were seeded into 96-well plates and cultivated for 24 h. Next, 10 μL CCK-8 (Beyotime, Shanghai, China) was added into each well with incubation for another 4 h at room temperature. The optical density (OD) value (at 450 nm) was recorded using a microplate reader (Bio-Rad).

Flow cytometry analysis
After treated with 100 ng/mL LPS for 24 h or transfected with indicated synthetic plasmids or oligonucleotides followed by LPS treatment for 24 h, BV2 cells were harvested and Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit (Vazyme) was utilized for the analysis of cell apoptosis. In brief, the harvested BV2 cells were rinsed with cold PBS (Sangon, Shanghai, China) and resuspended in binding buffer. BV2 cells were mixed with 5 µL Annexin V-FITC and 5 µL propidium iodide (PI) for 15 min in the dark at indoor temperature. Finally, the apoptotic cells were estimated with flow cytometry (Beckman Coulter, Atlanta, GA, USA).

RNA immunoprecipitation (RIP) assay
Magna RNA-binding protein immunoprecipitation kit (Millipore) was exploited for RIP assay. Briefly, BV2 cells were disrupted in RIP buffer, and cell extracts were cultivated with magnetic beads, which were conjugated with anti-immunoglobulin G (anti-IgG) or anti-argonaute-2 (anti-Ago2). Then, the immunoprecipitated RNAs were purified, and the enrichment of XIST and miR-219-5p was examined via RT-qPCR analysis.

Statistical analysis
All experiments were manipulated in triplicate. Data analysis was executed using the software GraphPad Prism 7, and the results were exhibited as mean ± standard deviation. The differences between two sets were estimated by Student's t-test, whereas differences among three groups were estimated by one-way analysis of variance followed by Tukey's test. It was defined as significant if P < 0.05.
Ethics approval and consent to participate: The hospital's Institutional Review Board approved the current study.

XIST was upregulated and miR-219-5p was downregulated in SCI mice model
After the mice model of SCI was established, the recovery of motor function in mice was evaluated by the BBB method. The results showed that the hindlimb locomotor activity was markedly decreased after spinal cord contusions, as indicated by the decreased BBB score in SCI groups compared with sham operation groups (Figure 1a). The results suggested that the SCI mice model was successfully established. Then, we determined the expression levels of XIST and miR-219-5p in the spinal cord tissues from SCI groups and sham groups by RT-qPCR analysis.
The results showed that the XIST level was notably elevated and miR-219-5p was conspicuously reduced in the spinal cord tissues from SCI groups compared with sham groups (Figure 1b and c). Next, we detected the levels of inflammatory cytokines (including TNF-α, IL-1β, IL-6, and IL-10) in SCI mice through ELISA. As shown in Figure 1d-g, the levels of TNF-α, IL-1β, and IL-6 were drastically elevated and the level of IL-10 was distinctly declined in SCI groups compared to sham operation groups. Herein, SCI also caused a noteworthy elevation in p-p65 protein level compared to sham groups (Figure 1h).
Collectively, XIST was abnormally increased and miR-219-5p was abnormally decreased in the SCI mice model.

LPS repressed cell viability and induced apoptosis and inflammatory response in BV2 cells
LPS-induced microglial cell is a commonly used SCI model in vitro. To establish the SCI model in vitro, microglial cells (BV2) were exposed to different concentrations of LPS (1, 10, 100, and 1,000 ng/mL) for 24 h. As illustrated by the CCK-8 assay, the viability of BV2 cells was markedly repressed by LPS in a dose-dependent manner (Figure 2a). There was no significant difference in BV cell viability between 100 ng/mL groups and 1,000 ng/mL groups; thus, 100 ng/mL LPS was utilized in the following study. Flow cytometry analysis showed that the apoptosis of BV2 cells was promoted by 100 ng/mL LPS treatment (Figure 2b). Meanwhile, we determined the levels of apoptosis-related proteins (Bcl-2, Bax, and C-caspase 3) in 100 ng/mL LPS-stimulated BV2 cells by western blot assay. The results indicated that LPS treatment led to an apparent reduction in Bcl-2 expression and an obvious elevation in Bax and C-caspase 3 expression in BV2 cells compared to control groups (Figure 2c). In addition, ELISA results showed that LPS distinctly enhanced the levels of TNF-α, IL-1β, and IL-6 and reduced the level of IL-10 in BV cells compared to control groups (Figure 2d-g). These observations suggested that LPSinduced SCI cell model was successfully constructed in vitro.

XIST knockdown abrogated LPSmediated BV2 cell viability, apoptosis, and inflammatory response
As shown in Figure 3a, the XIST level was increased in LPS-stimulated BV2 cells, indicating that XIST might be involved in the regulation of LPS-mediated microglial cell viability, apoptosis, and inflammatory cytokine production. Thus, we explored the function of XIST in LPSstimulated microglial cell progression by transfecting si-XIST or si-NC into BV2 cells and then treating the transfected cells with LPS. As demonstrated by the RT-qPCR assay, XIST silencing markedly suppressed the XIST level in LPS-induced BV2 cells (Figure 3a). CCK-8 assay showed that the inhibitory effect on cell viability mediated by LPS was restored by decreasing XIST expression in BV2 cells (Figure 3b). Flow cytometry analysis indicated that LPS-induced cell apoptosis was repressed by XIST knockdown in BV2 cells (Figure 3c). Moreover, western blot assay results showed that LPS treatment suppressed Bcl-2 expression and promoted Bax and C-caspase 3 expression in BV2 cells, while XIST knockdown effectively restored the impacts (Figure 3d). In addition, we observed that the upregulation of TNF-α, IL-1β, and IL-6 and the downregulation of IL-10 mediated by LPS were restored by reducing XIST expression in BV2 cells (Figure 3e-h). To sum up, XIST knockdown could relieve LPS-induced injury in BV2 cells.

XIST negatively regulated miR-219-5p expression by directly targeting
To explore the potential mechanism of XIST regulating BV2 cell progression, we analyzed the targets of XIST through online tool starBase (http://starbase.sysu.edu. cn/agoClipRNA.php?source=lncRNA&flag=target&clade= mammal&genome=mouse&assembly=mm10&miRNA= all&clipNum=1&deNum=0&target=Xist). The results displayed that miR-219-5p contained the complementary sequences of XIST (Figure 4a). Then dual-luciferase reporter assay and RIP assay were carried out to confirm the interaction between miR-219-5p and XIST. As suggested by the dual-luciferase reporter assay, miR-219-5p transfection markedly inhibited the luciferase activity of XIST-wt and anti-miR-219-5p transfection conspicuously elevated the luciferase activity of XIST-wt in BV2 cells, while the luciferase activity of XIST-mut was not affected by miR-219-5p or anti-miR-219-5p (Figure 4b and c). The results of the RIP assay showed that the levels of XIST and miR-219-5p were all enriched in anti-Ago2 protein complexes in BV2 cells compared to anti-IgG control groups, further confirming the interaction between XIST and miR-219-5p (Figure 4d). Thereafter, we explored the effect of XIST on miR-219-5p expression by transfecting XIST or si-XIST into BV2 cells. Our results showed that XIST transfection apparently decreased miR-219-5p level in BV2 cells, while si-XIST transfection exhibited the opposite results (Figure 4e). These observations indicated that XIST could negatively modulate miR-219-5p expression by direct interaction.
3.5 miR-219-5p inhibition reversed the effects of XIST knockdown on cell viability, apoptosis, and inflammation in LPS-stimulated BV2 cells Subsequently, we further explored whether XIST could alter LPS-induced BV2 cell injury by targeting miR-219-5p. First, anti-miR-219-5p transfection evidently reduced the level of miR-219-5p in BV2 cells compared to anti-NC and control groups, indicating that anti-miR-219-5p was successfully transfected into BV2 cells (Figure 5a). Next, BV2 cells were assigned to control, LPS, LPS + si-NC, LPS + si-XIST, LPS + si-XIST + anti-NC, and LPS + si-XIST + anti-miR-219-5p groups. The results of the CCK-8 assay and flow cytometry analysis indicated that XIST knockdown promoted cell viability and inhibited apoptosis in LPS-stimulated BV2 cells, while the impacts were partially overturned by decreasing miR-219-5p (Figure 5b and c). Western blot assay showed that the promotional role in Bcl-2 level and the suppressive roles in Bax and C-caspase 3 levels mediated by XIST silencing in LPStreated BV2 cells were ameliorated following the suppression of miR-219-5p (Figure 5d). In addition, ELISA results showed that the impacts of XIST deficiency on TNF-α, IL-1β, IL-6, and IL-10 levels were all restored by decreasing miR-219-5p expression in LPS-activated BV2 cells ( Figure  5e-h). These outcomes suggested that XIST knockdown attenuated LPS-induced BV2 cell injury by targeting miR-219-5p.

LPS-induced XIST promoted the activation of NF-κB pathway by regulating miR-219-5p
Finally, BV2 cells were divided into six groups: control, LPS, LPS + si-NC, LPS + si-XIST, LPS + si-XIST + anti-NC, and LPS + si-XIST + anti-miR-219-5p to explore the effect of XIST on LPS-mediated NF-κB signaling pathway activation in BV2 cells. As demonstrated by western blot assay, LPS treatment enhanced the protein level of p-p65 in BV2 cells, indicating the activation of the NF-κB pathway. Moreover, we found that XIST deficiency suppressed LPS-induced NF-κB pathway activation, as shown by downregulation of p-p65 protein level, while the effect was alleviated by the inhibition of miR-219-5p (Figure 6a and b). Taken together, XIST knockdown could inhibit LPS-activated NF-κB signaling pathway by modulating miR-219-5p expression in BV2 cells.

Discussion
lncRNAs have been proved as essential mediators in the development of SCI. After SCI, countless cytokines and signaling pathways have been demonstrated to mediate the apoptosis and inflammatory response [14,15]. LPSstimulated microglial cells are widely utilized to explore the pathogenesis of SCI. In this study, we successfully constructed the SCI mice model and found that XIST was drastically increased in the spinal cord tissues of SCI mice. Moreover, the SCI cell model was constructed by stimulating BV2 cells with LPS. Then, we tested cell viability, apoptosis, and the levels of inflammatory cytokines in LPS-triggered BV2 cells. We found that cell viability was repressed and cell apoptosis and inflammatory response were induced, indicating the successful construction of the SCI cell model. Thereafter, we explored the functions and mechanisms of XIST in SCI development. As a result, XIST knockdown recovered LPS-stimulated BV2 cell injury by regulating miR-219-5p and NF-κB signaling pathway.
In the past decades, the potential functions of XIST in SCI have been gradually studied. For example, Kwon et al. revealed that the XIST level was enhanced in the SCI rat model [11]. Gu et al. manifested that XIST knockdown effectively limited the apoptosis of neuronal in SCI rats by modulating miR-494/phosphatase and tensin homolog deleted on chromosome ten (PTEN)/phosphoinositide 3-kinase (PI3K)/AKT [5]. Moreover, Zhao et al. reported that XIST silencing restored the suppressive role in cell viability and the promotional role in apoptosis and inflammation mediated by LPS in microglial cells by regulating miR-27a/smad ubiquitination regulatory factor 1 (Smurf1) axis [29]. Correspondingly, our results showed that XIST was conspicuously increased in LPS-triggered BV2 cells. XIST interference enhanced cell viability and impeded apoptosis, concomitant with upregulation in Bcl-2 level and downregulation in Bax and C-caspase-3 levels in LPS-triggered BV2 cells. In addition, our results exhibited that XIST knockdown reduced TNF-α, IL-1β, and IL-6 levels and enhanced IL-10 level in LPS-activated BV2 cells, suggesting that XIST deficiency attenuated LPSinduced inflammatory response in BV2 cells. Overall, XIST knockdown could accelerate the recovery of SCI through promoting microglial cell viability and impeding apoptosis and inflammation.
It has been documented that the activation of the NF-κB pathway can trigger the production of pro-inflammatory cytokines, thereby inducing inflammatory response and apoptotic response [12,13,20]. Moreover, NF-κB pathway activation plays a positive role in the SCI development [2,10]. For example, GRB1 relieved SCI via altering miR-130b-5p/ TLR4/NF-κB pathway [23]. circ_0000962 inhibited the inflammation in the SCI cell model via activating PI3K/Akt and inactivating NF-κB by sponging miR-302b-3p [7]. Thus, we explored the impact of LPS-induced XIST in the NF-κB pathway. Our results showed that the knockdown of XIST decreased LPS-induced p-p65 level in BV2 cells, while miR-219-5p suppression restored the effect, suggesting that XIST silencing might block LPS-stimulated NF-κB pathway by targeting miR-219-5p.
In conclusion, this study uncovered that XIST was upregulated in SCI mice and LPS-activated BV2 cells. XIST knockdown ameliorated LPS-induced microglial cell apoptosis and inflammatory injury after SCI by sponging miR-219-5p and inactivating NF-κB pathway. Our study revealed the protective effect of XIST silencing in SCI, which deepened our understanding on the molecular basis in the management of SCI and might provide a novel direction for SCI therapy.

Funding information: No funds.
Conflict of interest: The authors declare that they have no conflicts of interest.
Data availability statement: The available datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.