N6-Methyladenosine-induced miR-143-3p promotes intervertebral disc degeneration by regulating SOX5.

Abstract

Intervertebral disc degeneration is the basic cause of lumbocrural pain, which not only causes pain and but also serious economic burdens on patients. Increasingly more evidence has shown that tumor necrosis factor-α (TNF-α) is involved in the pathological process of intervertebral disc degeneration, but the specific molecular mechanism is still unclear. This study investigated the potential mechanism and function of methyltransferase-like 3 (METTL3)/miR-143-3p/SOX5 regulatory axis in nucleus pulposus cells under the action of TNF-α. Human nucleus pulposus cells were treated with TNF-α to construct an in vitro model of intervertebral disc degeneration. Flow cytometry, quantitative reverse-transcription PCR (RT-qPCR), Western blot (WB) and luciferase assays were used to identify the mechanism of action of miR-143-3p in the course of intervertebral disc degeneration in vitro and the downstream targeted regulatory molecules. The role of miR-143-3p in intervertebral disc degeneration was also validated by in vivo. RT-qPCR, WB, coimmunoprecipitation (Co-IP) and flow cytometry were used to verify the regulatory effect of METTL3 on miR-143-3p maturation. RT-qPCR and WB were adopted to detect differences in METTL3, miR-143-3p and SOX5 expression in human nucleus pulposus tissue. miR-143-3p in nucleus pulposus cells was involved in the regulation of extracellular matrix metabolism and apoptosis after TNF-α stimulation, and intervertebral disc degeneration was relieved by effectively regulating miR-143-3p expression. Subsequent experiments showed that the downstream direct target gene of miR-143-3p was SOX5 and that miR-143-3p negatively regulated the expression of SOX5. In addition, METTL3 promoted miR-143-3p maturation, and METTL3 and miR-143-3p were significantly upregulated in degenerative nucleus pulposus, an effect that was significantly negatively correlated with low SOX5 expression. In conclusion, TNF-α upregulates METTL3, METTL3 promotes miR-143-3p maturation, and miR-143-3p inhibits the transcriptional activity of SOX5 through targeted binding, thereby inducing intervertebral disc degeneration. The inhibition of METTL3 or miR-143-3p expression may be an effective way to treat intervertebral disc degeneration.