Downregulation of miR-29b-3p promotes α-tubulin deacetylation by targeting the interaction of matrix metalloproteinase-9 with integrin β1 in nasal polyps

Matrix metalloproteinase (MMP)-9 is a key enzyme responsible for extracellular matrix degradation and contributes to the progressive histological changes observed in lower respiratory tract infections. Integrin beta 1 and alpha-tubulin are potential MMP-9-interacting proteins, and microRNA (miR)-29b-3p can regulate MMP-9 expression. MMP-9 is highly expressed in chronic rhinosinusitis with nasal polyps (CRSwNPs), regardless of its effects on miR-29b-3p, integrin beta 1 and alpha-tubulin expression. In the present study, samples from 100 patients with CRSwNPs were examined via reverse transcription-quantitative PCR to assess the mRNA expression of miR-29b-3p, and western blotting was performed to assess the protein expression of MMP-2, MMP-9, acetyl-alpha-tubulin, integrin beta 1 and tissue inhibitor of metalloproteinase 1 (TIMP-1). A dual-luciferase reporter assay was used to verify the direct binding of miR-29b-3p and MMP-2/MMP-9. Co-immunoprecipitation (Co-IP) and GST pull-down assays showed that integrin beta 1 and alpha-tubulin were MMP-9-interacting proteins. Cell viability, apoptosis and inflammatory cytokine levels were determined via a Cell Counting Kit-8 assay, flow cytometry and ELISA, respectively. miR-29b-3p expression was found to be positively correlated with MMP-2 and MMP-9 expression. Whereas, TIMP-1 expression was negatively correlated with MMP-2 and MMP-9 expression. The dual-luciferase assay revealed that miR-29b-3p targeted the 3 ' untranslated region of MMP-2/MMP-9. The Co-IP and GST pull-down assays showed that MMP-9 could directly bind to integrin beta 1 and indirectly bind to alpha-tubulin. Finally, the overexpression of miR-29b-3p decreased the expression of MMP-9 and increased the levels of acetyl-alpha-tubulin. By contrast, the knockdown of miR-29b-3p increased the expression of MMP-9 and decreased the levels of acetyl-alpha-tubulin. Additionally, MMP-9 expression was found to be negatively correlated with acetyl-alpha-tubulin expression. Of note, the expression of integrin beta 1 did not change following the overexpression and knockdown of MMP-9. Finally, the overexpression of miR-29b-3p not only decreased MMP-9 expression, but also alleviated lipopolysaccharide-induced inflammation in NP69 cells. The results showed that the downregulation of miR-29b-3p promoted alpha-tubulin deacetylation by increasing the number of MMP-9-integrin beta 1 complexes in CRSwNPs, thus targeting miR-29b-3p/MMP-9 may be a potential novel strategy for the clinical treatment of CRSwNPs.

Automated summary

In this study, it was found that miR-29b-3p indirectly regulated the expression of integrin beta 1 and alpha-tubulin in CRSwNPs by modulating MMP-9 expression, potentially serving as a novel target for clinical treatment of chronic rhinosinusitis with nasal polyps.