Research Paper Volume 13, Issue 16 pp 20258—20276

DNMT3B decreases extracellular matrix degradation and alleviates intervertebral disc degeneration through TRPA1 methylation to inhibit the COX2/YAP axis

DNMT3B inhibited the expression of TRPA1 through methylation. (A) RT-qPCR showing the expression of TRPA1 in the intervertebral disc tissue of IVDD rats (n = 10) and sham-operated rats (n = 10). (B) Western blot showing the expression of TRPA1 in the intervertebral disc tissue of IVDD rats). (C) mRNA expression of DNMT3B and TRPA1 in NP cells after 24 hours of transfection with oe-DNMT3B or oe-NC was detected by RT-qPCR. (D) mRNA expression of DNMT3B and TRPA1 in NP cells after 48 hours of transfection with oe-DNMT3B or oe-NC was detected by Western blot. (E) A C-phosphate-G (CpG) island in the TRPA1 promoter region was identified in UCSC website. (F) The methylation of the TRPA1 promoter was measured through MSP in NP cells after 24 hours of transfection with oe-DNMT3B. (G) The luciferase reporter assay of luciferase activity in promoter of the TRPA1-WT and TRPA1-MUT in NP cells after 24 hours of transfection with oe-DNMT3B. (H) RT-qPCR showing the mRNA expression of DNMT3B and TRPA1 in NP cells after 24 hours of transfection with aza or oe-DNMT3B. (I) Western blot showing the protein expression of DNMT3B and TRPA1 in NP cells after 48 h treatment with aza or oe-DNMT3B. Measurement data are expressed as the mean ± standard deviation (n = 3) and analyzed using independent sample t-tests between two groups or using one-way ANOVA between multiple groups. **, p

Figure 3. DNMT3B inhibited the expression of TRPA1 through methylation. (A) RT-qPCR showing the expression of TRPA1 in the intervertebral disc tissue of IVDD rats (n = 10) and sham-operated rats (n = 10). (B) Western blot showing the expression of TRPA1 in the intervertebral disc tissue of IVDD rats). (C) mRNA expression of DNMT3B and TRPA1 in NP cells after 24 hours of transfection with oe-DNMT3B or oe-NC was detected by RT-qPCR. (D) mRNA expression of DNMT3B and TRPA1 in NP cells after 48 hours of transfection with oe-DNMT3B or oe-NC was detected by Western blot. (E) A C-phosphate-G (CpG) island in the TRPA1 promoter region was identified in UCSC website. (F) The methylation of the TRPA1 promoter was measured through MSP in NP cells after 24 hours of transfection with oe-DNMT3B. (G) The luciferase reporter assay of luciferase activity in promoter of the TRPA1-WT and TRPA1-MUT in NP cells after 24 hours of transfection with oe-DNMT3B. (H) RT-qPCR showing the mRNA expression of DNMT3B and TRPA1 in NP cells after 24 hours of transfection with aza or oe-DNMT3B. (I) Western blot showing the protein expression of DNMT3B and TRPA1 in NP cells after 48 h treatment with aza or oe-DNMT3B. Measurement data are expressed as the mean ± standard deviation (n = 3) and analyzed using independent sample t-tests between two groups or using one-way ANOVA between multiple groups. **, p < 0.01; ns, not significant.