MicroRNA-143-5p Regulates AMPK Pathway In Cervical Disc Degeneration And Its Mechanism

Part 1 Pathological changes of intervertebral disc degeneration and miRNA gene expression screeningObjective: Screen and explore the differentially expressed miRNAs in the cartilage endplate tissue of the cervical intervertebral disc degeneration group and the normal group through miRNAs gene chip sequencing.Methods: 21 patients with cervical disc degeneration who needed surgical treatment were enrolled.13 patients without cervical disc degeneration were included in the normal group.The gene chip was used for sequencing.The cervical disc cartilage endplate tissues of the degeneration group and the normal group passed total RNA Extraction,respectively perform RNA quality evaluation,c DNA library establishment,sequencing quality evaluation,miRNAs gene expression changes and differential expression profile analysis;use miRNAs sequencing to screen differentially expressed miRNAs and use GO function analysis and KEGG pathway analysis to predict miRNAs targets.Detailed analysis of genes.Five miRNAs changes that meet the candidate criteria were verified by qT-PCR.Results: Both total RNA extraction and RNA quality analysis showed that the sequencing results met the quality requirements.The differentially expressed miRNAs between the degenerative group and the normal group were screened by miRNAs gene chip sequencing;a total of 35 miRNA expressions were screened for significant differences,22 were up-regulated and 13 were down-regulated.Using GO and KEGG enrichment analysis showed that the differentially expressed miRNAs target genes of the degenerative group are mainly involved in biological processes such as DNA-templating and transcription regulation,and involved in signal pathways such as AMPK,MAPK,Wnt,and Hippo.A total of hsa-miR-143-5p,hsa-miR-222-5p,hsa-miR-140-5p-F,hsa-miR-199a-5p and hsa-miR-132 are expressed in the normal group and degenerative group Significant differences.The verification by q RT-PCR showed that the hsa-miR-143-5p of the degeneration group had significant differences,hsa-miR-222-5p,hsa-miR-140-5p-F,hsa-miR-199a-5p and hsa-miR-132.There was no significant difference in miR-132 between the normal group and the degenerative group.Conclusion: Screening by gene chip sequencing and qT-PCR verification showed that miR-143-5p is significantly up-regulated in cervical disc degeneration.miR-143-5p can be used as a new therapeutic target in intervertebral disc degeneration.Part Ⅱ: miRNA-143-5p mediates cervical disc degeneration through AMPKObjective: To explore whether miRNA-143-5p mediates cervical disc degeneration through AMPK and its possible regulatory mechanism;Methods: 21 patients with cervical disc degeneration who needed surgical treatment were enrolled.13 patients without cervical disc degeneration were classified as normal group,and 13 patients were traumatic and required cervical spine surgery to remove the cartilage tissue of the intervertebral disc.The HE method was used to explore the pathological changes of the intervertebral disc cartilage endplates in the degeneration group and the normal group;Western Blotting and immune tissue were used to explore the changes in the protein expression of p-AMPK,AMPK,and EEF2 in the degenerative cartilage endplates.Results: Compared with the normal group,the pathological structure of patients with cervical intervertebral disc degeneration was disordered with obvious changes;bioinformatics showed that e EF-2 is a theoretical target gene.WB showed a significant increase in p-AMPK and a significant decrease in EEF-2 in the degenerative group,and AMPK changes were not restricted.Conclusion: miR-143-5p is significantly up-regulated in cervical disc degeneration,and its mechanism may be related to AMPK pathway.miR-143-5p can be used as a new therapeutic target in intervertebral disc degeneration.