The Study Of N6-methyladenosine(m6A) Modification In Degeneration Of Lumbar Intervertebral Disc In Mouse

Objective: The model of lumbar disc degeneration was established by inducing the mice to stand on their feet and increase the vertical stress of spine.Method: Twenty C57 BL / 6-week-old male mouse were randomly divided into control group and experimental group,with 10 in each group.The mouse in the experimental group were placed in a 500 ml beaker filled with a small amount of water,and forced to stand for 8 hours a day by the water-escape habit of mice.The treatment of the mouse in the control group was the same as that in the experimental group except that there was no water in the beaker.The mouse were sacrificed at 6 weeks and 10 weeks of standing,and the lumbar specimens of the mouse were taken out for micro-CT scanning to observe and compare the disc height index of two groups.After scanning,the lumbar specimens of mice were stained with pathology and immunohistochemistry to evaluate the lumbar intervertebral disc degeneration.Results: micro-CT results showed that: at 6 weeks,the disc height index of the control group was 5.2%±0.5%,and the experimental group was 4.9%±0.4%.There was no statistically significant difference between the two groups(P﹥0.05).At 10 weeks,the disc height index of the control group was 5.1%±0.5%,and the experimental group was 4.5%±0.3%.The disc height decreased significantly,and the difference between the two groups was statistically significant(P﹤0.05),and the disc height index gradually decreases with the extension of standing time.HE staining and safranin green staining showed that the nucleus pulposus cells and extracellular matrix of the experimental group were reduced compared with the control group,the arrangement of annulus fibrosus gradually became disordered and ruptured,and the height of the cartilage endplate gradually decreased over time.Immunohistochemical staining showed that:Compared with the control group,the expression of COL2A1 in the intervertebral disc of the experimental group gradually decreased with the extension of standing time,while the expression of MMP-13 increased.Conclusion: Bipedal standing mouse model can induce degeneration of lumbar intervertebral discs.Objective: To explore the m6 A modification mode of nucleus pulposus tissue after intervertebral disc degeneration.Methods: Ten C57 BL/6-week-old male mouse were randomly divided into control group and experimental group,with 5 in each group.The mouse were first induced to stand to establish intervertebral disc degeneration model.Then the lumbar nucleus pulposus tissues of the experimental group and the control group were taken out at 10 weeks.Using Me RIP-seq to analyze the m6 A methylation transcription profile of nucleus pulposus tissue,q RT-PCR to detect the expression of methylationrelated enzymes,gene ontology and Kyoto Encyclopedia of Genes and Genomes to annotate gene functions and analyze related pathways of differentially expressed genes.Results: Using Me RIP-Seq,we identified a total of 985 differentially expressed genes,of which 363 genes were significantly up-regulated,and 622 genes were significantly down-regulated.In addition,among the 9648 genes counted,1319 m6 A peaks with significant differential methylation were identified,of which 933 were significantly up-regulated and 386 were significantly down regulated,and the related functions and possible pathways of differentially expressed genes were identified.The q RT-PCR results showed that: compared with the control group,the expression of KIAA1429,METTL14,METTL3,METL4,WTAP,DGCR8,EIF3 A and YTHDC1 in the experimental group was relatively increased,while the expression of ELAVL1,HNRNPC1,SRSF and FTO were relatively decreased.And methylase(RBM15 and RBM15B),demethylase(ALKBH5)and methylation recognition enzyme(EIF3B,YTHDC2,YTHDF1,YTHDF2 and YTHDF3)have no significant differences between the two groups.Besides,the joint analysis of Me RIP-seq and m RNA-seq identified 30 genes with significant changes between m6 A methylation levels and m RNA expression.Conclusion: m6 A modification may play a role in degeneration of lumbar intervertebral disc in mice by affecting the metabolism of cells in the intervertebral disc.