Mettl14-mediated m6A modification modulates neuron apoptosis during the repair of spinal cord injury by regulating the transformation from pri-mir-375 to miR-375

Abstract

Background: Spinal cord injury (SCI) is a disabling disorder, resulting in neurological impairments. This study investigated the mechanism of methyltransferase-like 14 (Mettl14) on apoptosis of spinal cord neurons during SCI repair by mediating pri-microRNA (miR) dependent N6-methyladenosine (m6A) methylation.

Methods: The m6A content in total RNA and Mettl14 levels in spinal cord tissues of SCI rats were detected. Mettl14 expression was intervened in SCI rats to examine motor function, neuron apoptosis, and recovery of neurites. The cell model of SCI was established and intervened with Mettl14. miR-375, related to SCI and positively related to Mettl14, was screened out. The expression of miR-375 and pri-miR-375 after Mettl14 intervention was detected. The expression of pri-miR-375 combined with DiGeorge critical region 8 (DGCR8) and that modified by m6A was detected. Furthermore, the possible downstream gene and pathway of miR-375 were analysed. SCI cell model with Mettl14 intervention was combined with Ras-related dexamethasone-induced 1 (RASD1)/miR-375 intervention to observe the apoptosis.

Results: Mettl14 level and m6A content in spinal cord tissue were significantly increased. After Mettl14 knockdown, the injured motor function was restored and neuron apoptosis was reduced. In vitro, Mettl14 silencing reduced the apoptosis of SCI cells; miR-375 was reduced and pri-miR-375 was increased; miR-375 targeted RASD1. Silencing Mettl14 inactivated the mTOR pathway. The apoptosis in cells treated with silencing Mettl14 + RASD1/miR-375 was inhibited.

Conclusions: Mettl14-mediated m6A modification inhibited RASD1 and induced the apoptosis of spinal cord neurons in SCI by promoting the transformation of pri-miR-375 to mature miR-375.

Keywords: Mettl14; Pri‐mir‐375; RASD1; Spinal cord injury; m6A modification; miR-375.

Fig. 1

Mettl14 and m6A were significantly upregulated after SCI. After the SCI model was established, the motor function of rats in each group was assessed by BBB motor function score (a) and bevel test score (b) on the 0, 5, 10, 15, 20, 25 and 30 days. The SCI at 2-cm transverse section of the spinal cord centred on the injured site was assessed by HE staining (c) on the 30th day; the neurons at the point of arrow were obviously lost; and the m6A content in total RNA of rats at the 5th day, 10th day and 30th day after SCI was assessed by EpiQuik m6A RNA Methylation quantification kit (d). The expression of Mettl14 at the 5th day, 10th day and 30th day after SCI was detected by RT-qPCR (e) and Western blot (f); and the fluorescence intensity of Mettl14 in a 2-cm transverse section of the spinal cord centred on the injured site on the 30th day was detected using immunofluorescence (g), and the arrow indicates Mettl14 positive expression. N = 8. Each experiment was repeated three times independently, and the data are expressed as mean ± standard deviation. The data in panels a, b were analysed by two-way ANOVA, and data in panels d–f were analysed by one-way ANOVA, followed by Tukey’s multiple comparisons test. Compared with the sham group, *p < 0.05, **p < 0.01

Fig. 2

ShRNA-Mettl14 can relieve SCI and promote the recovery of neurites after SCI. ShRNA-Mettl14 was injected into SCI model rats using lumbar puncture via the tail vein. The equal distance transverse section on 2-cm spinal cord centred on the injured site was made. Mettl14 levels in the spinal cord tissue of rats at the 30th day were detected by RT-qPCR (a) and Western blot analysis (b). At the 30th day after SCI, the content of m6A in the total RNA of the spinal cord was measured by EpiQuik m6A RNA methylation quantification kit (c). BBB motor function score (e) and level test score (d) were used to evaluate the motor function of the rats in each group. The rats were euthanized at 30th day, and HE staining was performed (f); the arrow shows the weakened destruction of central gray matter and peripheral white matter; Western blot analysis (g) were used to measure the expression of AcTub and MAP2. The positive expression of GFAP, NeuN and NF-200 was evaluated by immunohistochemical staining; and the arrow indicates GFAP, NeuN and NF-200 positive expression (h). N = 8. Each experiment was repeated three times independently. The data are expressed by mean ± standard deviation. The data in panels A/B/C/G were analysed by one-way ANOVA, and data in panels d–f were analysed by two-way ANOVA, followed by Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01

Fig. 3

ShRNA-Mettl14 inhibited apoptosis of spinal neurons in SCI rats. ShRNA-Mettl14 was injected into SCI model rats using lumbar puncture via the caudal vein. The equal distance transverse sections on 2-cm spinal cord centred on the injured site were made at the 30th day. The apoptosis of spinal cord cells was detected by a TUNEL and b immunofluorescence staining, and the survival number of neurons was observed by c Nissl staining; the arrows indicate TUNEL-positive cells, NeuN + caspase-3 positive cells or Nissl stained neurons. N = 8. Each experiment was repeated three times independently. The data are expressed by mean ± standard deviation. The data in panels a–c were analysed by one-way ANOVA, followed by Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01

Fig. 4

ShRNA-Mettl14 inhibited H2O2-induced apoptosis in SCI cell model. C8-DA1 and C8-B4 cells induced by H2O2 were used to establish the cell model of SCI in vitro. Mettl14 expression was detected by RT-qPCR (a). The content of m6A in the total RNA of the spinal cord was measured by EpiQuik m6A RNA methylation quantification kit (b). Then flow cytometry (c) and Hoechst staining (d) were used to evaluate the apoptosis. Each experiment was repeated three times independently. The data are expressed by mean ± standard deviation. The data in panels A/B/C were analysed by one-way ANOVA, followed by Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01

Fig. 5

Mettl14-mediated m6A modification to promote the transformation from pri-miR-375 to miR-375. RT-qPCR was used to detect miR-375 and pri-miR-375 in cells after transfection for 24 h or spinal cord tissue of rats on the 30th day of each group (a, b), and then RT-qPCR was used to detect the effect of m6A mutation in pri-miR-375 on miR-375 processing (c); RIP assay was used to evaluate the effect of overexpression of Mettl14 on pri-miR-375 and miR-375 modified by m6A (d, e). Each experiment was repeated three times independently. The data are expressed by mean ± standard deviation. The data in panel B were analysed by the t test, and the data in panels a, c–e were analysed by one-way ANOVA, followed by Tukey’s multiple comparisons test. **p < 0.01

Fig. 6

miR-375 targeted RASD1 to activate the mTOR pathway. The target binding relationship between miR-375 and RASD1 was verified by dual-luciferase assay (a). Then RT-qPCR (b) and Western blot analysis (c) were used to detect the level of RASD1 in rat spinal cord and C8-DA1/SCI cells, and Western blot analysis was used to detect the levels of mTOR-related proteins in rat spinal cord and C8-DA1/SCI cells (d). Each experiment was repeated three times independently. The data are expressed by mean ± standard deviation. The data in panels B/C were analysed by one-way ANOVA, and data in panels a, d were analysed by two-way ANOVA, followed by Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01

Fig. 7

Inhibition of miR-375 or overexpression of RASD1 partially reversed the effect of oe-Mettl14 on the apoptosis of SCI cells. Overexpression of RASD1 and oe-Mettl14, and low expression of miR-375 and oe-Mettl14 were cotransfected into C8-DA1/SCI cells. Apoptosis was evaluated by a flow cytometry and b Hoechst staining 24 h later. Each experiment was repeated three times independently. The data are expressed by mean ± standard deviation. The data in panels A/B were analysed by the t test. **p < 0.01

Fig. 8

Mechanism chart. Inhibition of Mettl14 expression can inhibit Mettl14-mediated m6A modification by regulating the transformation process from pri-miR-375 to mature pri-miR-375, thus promoting the expression of RASD1, inhibiting the apoptosis of spinal cord neurons and promoting the repair of spinal cord injury