Mechanism of Fat Mass and Obesity-Related Gene-Mediated Heme Oxygenase-1 m6A Modification in the Recovery of Neurological Function in Mice with Spinal Cord Injury

Abstract

Objectives: This study examined the mechanism of fat mass and obesity-related gene (FTO)-mediated heme oxygenase-1 (HO-1) m6A modification facilitating neurological recovery in spinal cord injury (SCI) mice. FTO/HO-1 was identified as a key regulator of SCI as well as a potential target for treatment of SCI.

Methods: An SCI mouse was treated with pcDNA3.1-FTO/pcDNA3.1-NC/Dac51. An oxygen/glucose deprivation (OGD) cell model simulated SCI, with cells treated with pcDNA3.1-FTO/si-HO-1/Dac51. Motor function and neurobehavioral evaluation were assessed using the Basso, Beattie, and Bresnahan (BBB) scale and modified neurological severity score (mNSS). Spinal cord pathology and neuronal apoptosis were assessed. Further, FTO/HO-1 mRNA and protein levels, HO-1 mRNA stability, the interaction of YTHDF2 with HO-1 mRNA, neuronal viability/apoptosis, and HO-1 m6A modification were evaluated.

Results: Spinal cord injury mice exhibited reduced BBB, elevated mNSS scores, disorganized spinal cord cells, scattered nuclei, and severe nucleus pyknosis. pcDNA3.1-FTO elevated FTO mRNA, protein expression, and BBB score; reduced the mNSS score of SCI mice; decreased neuronal apoptosis; improved the cell arrangement; and improved nucleus pyknosis in spinal cord tissues. OGD decreased FTO expression. FTO upregulation ameliorated OGD-induced neuronal apoptosis. pcDNA3.1-FTO reduced HO-1 mRNA and protein and HO-1 m6A modification, while increasing HO-1 mRNA stability and FTO in OGD-treated cells. FTO upregulated HO-1 by modulating m6A modification. HO-1 downregulation attenuated the effect of FTO. pcDNA3.1-FTO/Dac51 increased the HO-1 m6A level in mouse spinal cord tissue homogenate, reduced BBB, boosted mNSS scores of SCI mice, aggravated nucleus pyknosis, and increased neuronal apoptosis in spinal cord tissues, confirming that FTO mediated HO-1 m6A modification facilitated neurological recovery in SCI mice.

Conclusion: The fat mass and obesity-related gene modulates HO-1 mRNA stability by regulating m6A modification levels, thereby influencing HO-1 expression and promoting neurological recovery in SCI mice.

Keywords: Fat mass and obesity‐related genes; HO‐1; Neuron; Spinal cord injury; m6A.

FIGURE 1

Fat mass and obesity‐related genes (FTO) alleviated neuronal apoptosis and promoted neurological function recovery in SCI mice. (A) In vivo experimental scheme in mice. (B) Basso, Beattie, and Bresnahan (BBB) motor function scores, N = 12; (C) modified neurological severity score (mNSS) tests, N = 12; (D) hematoxylin–eosin (HE) staining to assess the pathological condition of spinal cord tissues, N = 6; (E) TdT‐mediated dUTP‐biotin nick end‐labeling (TUNEL) staining to assess neuronal apoptosis in spinal cord tissues, N = 6; (F) reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) to determine FTO mRNA expression, N = 6; (G) western Blot to assess FTO protein expression, N = 6. Data were expressed as mean ± standard deviation. One‐way analysis of variance (ANOVA) was utilized for data comparisons among multiple groups. Tukey's multiple comparisons test was applied for post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2

Oxygen/glucose deprivation (OGD) reduced fat mass and obesity‐related genes (FTO) expression, and upregulation of FTO alleviated OGD‐induced neuronal apoptosis. (A) Cell experiment scheme; (B) 2, 5‐diphenyltetrazolium bromide (MTT) assay to assess neuronal viability; (C) flow cytometry to assess neuronal apoptosis; (D) reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) to measure FTO mRNA level; (E) and western blot to determine the FTO protein level. The cellular experiments were repeated three times, and the data were expressed as mean ± standard deviation. One‐way analysis of variance (ANOVA) was used to compare the data among multiple groups, and Tukey's multiple comparisons test was conducted for post hoc tests. *p < 0.05, ***p < 0.001.

FIGURE 3

Fat mass and obesity‐related genes (FTO) upregulated heme oxygenase‐1 (HO‐1) expression by modulating m6A modification. (A) Reverse transcription quantitative polymerase chain reaction (RT‐qPCR) to measure HO‐1 mRNA level; (B) western blot to determine HO‐1 protein expression; (C) SRAMP database to predict m6A methylation modification sites on the HO‐1 mRNA sequences; (D) methylated RNA immunoprecipitation (Me‐RIP) assay to assess HO‐1 m6A modification level; (E) actinomycin D to detect the stability of HO‐1 mRNA in cells; (F) RIP to determine the interaction of YTHDF2 with HO‐1. Cell experiments were repeated three times. Data were expressed as mean ± standard deviation, and one‐way analysis of variance (ANOVA) was implemented to compare data among multiple groups. Tukey's multiple comparisons test was used for post hoc tests. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

Heme oxygenase‐1 (HO‐1) knockdown partially reversed the suppressive effect of fat mass and obesity‐related genes (FTO) on oxygen/glucose deprivation (OGD)‐induced neuronal apoptosis. (A) Reverse transcription quantitative polymerase chain reaction (RT‐qPCR) to assess HO‐1 mRNA level; (B) western Blot to measure HO‐1 protein level; (C) 2, 5‐diphenyltetrazolium bromide (MTT) to evaluate neuronal viability; (D) flow cytometry to assess neuronal apoptosis. The cellular experiments were repeated three times. Data were expressed as mean ± standard deviation, and one‐way analysis of variance (ANOVA) was used to compare the data among multiple groups. Tukey's multiple comparisons test was used for post hoc test. *p < 0.05, ***p < 0.001.

Figure 5

Fat mass and obesity‐related genes (FTO)‐mediated heme oxygenase‐1 (HO‐1) m6A modification facilitated neurological function recovery in spinal cord injury (SCI) mice. (A) Methylated RNA immunoprecipitation (Me‐RIP) to test HO‐1 m6A modification level in mouse spinal cord tissue homogenate, N = 6; (B) Basso, Beattie, and Bresnahan (BBB) motor function score, N = 12; (C) modified neurological severity score (mNSS) tests, N = 12; (D) hematoxylin–eosin staining to assess the pathological condition of spinal cord tissue, N = 6; (E) TdT‐mediated dUTP‐biotin nick end‐labeling (TUNEL) staining to evaluate apoptosis in spinal cord tissues, N = 6. Data were expressed as mean ± standard deviation. One‐way analysis of variance (ANOVA) was applied for comparisons of data among multiple groups, and Tukey's multiple comparisons test was used for post hoc tests. *p < 0.05, **p < 0.01, ***p < 0.001.