METTL14 promotes apoptosis of spinal cord neurons by inducing EEF1A2 m6A methylation in spinal cord injury

Abstract

Spinal cord injury (SCI) is a devastating traumatic condition. METTL14-mediated m6A modification is associated with SCI. This study was intended to investigate the functional mechanism of RNA methyltransferase METTL14 in spinal cord neuron apoptosis during SCI. The SCI rat model was established, followed by evaluation of pathological conditions, apoptosis, and viability of spinal cord neurons. The neuronal function of primary cultured spinal motoneurons of rats was assessed after hypoxia/reoxygenation treatment. Expressions of EEF1A2, Akt/mTOR pathway-related proteins, inflammatory cytokines, and apoptosis-related proteins were detected. EEF1A2 was weakly expressed and Akt/mTOR pathway was inhibited in SCI rat models. Hypoxia/Reoxygenation decreased the viability of spinal cord neurons, promoted LDH release and neuronal apoptosis. EEF1A2 overexpression promoted the viability of spinal cord neurons, inhibited neuronal apoptosis, and decreased inflammatory cytokine levels. Silencing METTL14 inhibited m6A modification of EEF1A2 and increased EEF1A2 expression while METTL14 overexpression showed reverse results. EEF1A2 overexpression promoted viability and inhibited apoptosis of spinal cord neurons and inflammation by activating the Akt/mTOR pathway. In conclusion, silencing METTL14 repressed apoptosis of spinal cord neurons and attenuated SCI by inhibiting m6A modification of EEF1A2 and activating the Akt/mTOR pathway.

Fig. 1. EEF1A2 was poorly expressed and Akt/mTOR pathway was inhibited in SCI.

A differential expression gene map of SCI chip GSE45006, the abscissa represents –log (p value), the ordinate represents log2FC, red dots indicate highly-expressed genes and green dots indicate lowly-expressed genes; (B) Venn map of the intersection of differentially low-expression genes and GeneCards database; (C) gene co-expression network by Coexpedia; (D) bar graph of the correlation of gene-disease, the abscissa represents correlation score; (E) expression heatmap of candidate genes, the color scale represents gene expression from low (blue) to high (red); (F) enriched terms visualized in barplot, each row represents an enriched function, and the length of the bar represents the enrich ratio, which is calculated as “input gene number”/“background gene number”. The color of the bar is the same as the color in the circular network in above, which represents different clusters. For each cluster, if there are more than five terms, top 5 with the highest enrich ratio will be displayed; (G) significant low expression of EEF1A2 in chip GSE45006; (H) locomotor ability of rats detected by BBB locomotor rating scale; (I) neuronal function detected by inclined plane test; (J) pathological conditions of spinal cord assessed by HE staining; (K) apoptosis of spinal cord tissues detected by TUNEL staining; (L) expressions of EEF1A2 and Akt/mTOR pathway-related proteins detected by Western blot. Data were expressed as mean ± SD. Pairwise comparisons were analyzed using independent t test, N = 5. * vs sham group, p < 0.05.

Fig. 2. EEF1A2 overexpression inhibited SCI progression in rats.

A expression of EEF1A2 in spinal cord tissues detected by Western blot; (B) locomotor ability detected by BBB locomotor rating scale; (C) neuronal function detected by inclined plane test; (D) pathological conditions of spinal cord assessed by HE staining; (E) apoptosis of spinal cord tissues detected by TUNEL staining; (F) expressions of TNF-α, IL-1β and IL-6 detected by ELISA. Data were expressed as mean ± SD and analyzed using independent t test, N = 5. * vs SCI + oe-NC group, p < 0.05.

Fig. 3. EEF1A2 overexpression promoted viability and inhibited apoptosis of H/R-treated spinal cord neurons.

A neuronal viability detected by CCK-8; (B) cell toxicity detected by LDH release assay; (C) neuronal apoptosis detected by flow cytometry; (D) neuronal apoptosis detected by TUNEL staining; (E) expressions of Bax, cleaved caspase3, and bcl-2 detected by Western blot; (F) expression of EEF1A2 detected by Western blot. Data were expressed as mean ± SD and analyzed using independent t test. The experiment was repeated three times. * vs control group, p < 0.05; # vs OGD + oe-NC group, p < 0.05.

Fig. 4. Mettl14 downregulated EEF1A2 in SCI by mediating m6A methylation of EEF1A2.

A possible m6A methylation sites of EEF1A2; (B) m6A level in spinal cord tissues and H/R neurons detected by Dot blot assay; (C) expression of Mettl14 in rat spinal cord tissues detected by Western blot (N = 5); (D) expression of Mettl14 in spinal cord tissues detected by immunofluorescent staining (N = 5); (E) expression of Mettl14 in H/R neurons detected by Western blot; (F): expression of Mettl14 detected by Western blot; (G) total m6A level detected by Dot blot assay; (H) mRNA expression of EEF1A2 detected by RT-qPCR; (I) EEF1A2 protein expression detected by Western blot; (J) m6A modification level of EEF1A2 detected by Me-RIP assay. Data were expressed as mean ± SD and analyzed using independent t test. The experiment was repeated three times. * vs sham group, control group, sh-NC group, p < 0.05; # vs oe-NC group, p < 0.05.

Fig. 5. Mettl14 modulated the function of H/R-treated spinal cord neurons by mediating EEF1A2.

A expressions of Mettl14 and EEF1A2 in neurons detected by Western blot; (B) total m6A level detected by Dot blot assay; (C) neuronal viability detected by CCK-8; (D) cell toxicity assessed by LDH release assay; (E) neuronal apoptosis detected by flow cytometry; (F) neuronal apoptosis detected by TUNEL staining; (G) expressions of Bax, cleaved caspase3 and bcl-2 detected by Western blot. Data were expressed as mean ± SD and analyzed using independent t test. The experiment was repeated three times. * vs OGD + sh-NC group, p < 0.05; # vs OGD + sh-Mettl14 group, p < 0.05.

Fig. 6. Mettl14 modulated SCI by mediating EEF1A2 expression.

A expressions of Mettl14, EEF1A2 proteins in spinal cord tissues detected by Western blot; (B) total m6A level detected by Dot blot assay; (C) locomotor ability detected by BBB locomotor rating scale; (D) neuronal function detected by inclined plane test; (E) pathological conditions of spinal cord assessed by HE staining; (F) apoptosis of spinal cord tissues detected by TUNEL staining; (G) expressions of TNF-α, IL-1β and IL-6 detected by ELISA. Data were expressed as mean ± SD and analyzed using independent t test, N = 5. * vs SCI + sh-NC group, p < 0.05; # vs SCI + sh-Mettl14 + sh-EEF1A2 group, p < 0.05.

Fig. 7. EEF1A2 promoted viability and inhibited apoptosis of spinal cord neurons by activating Akt/mTOR pathway.

A expressions of EEF1A2 and Akt/mTOR pathway-related proteins detected by Western blot; (B) neuronal viability detected by CCK-8; (C) cell toxicity assessed by LDH release assay; (D) neuronal apoptosis detected by flow cytometry; (E) neuronal apoptosis detected by TUNEL staining; (F) expressions of Bax, cleaved caspase3 and bcl-2 detected by Western blot. Data were expressed as mean ± SD and analyzed using independent t test. The experiment was repeated three times. * vs OGD + oe-EEF1A2 + H2O group, p < 0.05.

Fig. 8. EEF1A2 affected SCI by activating the Akt/mTOR pathway.

A expressions of EEF1A2 and Akt/mTOR pathway-related proteins in spinal cord tissues detected by Western blot; (B) locomotor ability detected by BBB locomotor rating scale; (C) neuronal function detected by inclined plane test; (D) pathological conditions of spinal cord assessed by HE staining; (E) apoptosis of spinal cord tissues detected by TUNEL staining; (F) expressions of TNF-α, IL-1β and IL-6 detected by ELISA. Data were expressed as mean ± SD and analyzed using independent t test, N = 5. * vs SCI + oe-EEF1A2 + H2O group, p < 0.05.