Mechanism of high mobility group protein B1 enhancing migration and invasion of oral squamous cell carcinoma through macrophages

Abstract

[Objective] To explore the role of macrophage M2-type pyruvate kinase (PKM2) in high mobility group protein B1 (HMGB1)-induced migration and invasion of oral squamous cell carcinoma (OSCC), and to elucidate the molecular mechanism by which HMGB1 promotes OSCC migration and invasion through macrophages. [Methods] Human monocytic leukemia THP-1 cells were stimulated with phorbol 12-myristate 13-acetate to become M0 macrophages, and PKM2 inhibitors were used to intervene under HMGB1 stimulation. The obtained M0 macrophages were divided into control group and PKM2 inhibitor group according to whether PKM2 was inhibited or not. Human recombinant HMGB1 (rhHMGB1) was added to CAL27 tumor conditioned medium in the control group, and rhHMGB1 and PKM2 inhibitors were added to CAL27 tumor conditioned medium in the PKM2 inhibitor group. Transwell co-culture system was used to detect the migration and invasion of CAL27 cells in the two groups, flow cytometry and immunofluorescence were used to detect the positive rates of M1 macrophage marker CD86 and M2 macrophage marker CD206 in each group, and real-time quantitative PCR (qRT-PCR) was used to detect the gene expression levels of M1 macrophage markers inducible nitric oxide synthase (iNOS), tumor necrosis factor α (TNF-α) and M2 macrophage markers arginase 1 (Arg-1) and transforming growth factor β (TGF-β). [Results] Transwell results showed that the number of migrated and invaded CAL27 cells in the PKM2 inhibitor group was significantly lower than that in the control group (P < 0.001). The results of flow cytometry and immunofluorescence showed that the expression level of CD86, a specific marker of M1 macrophages, showed a significant decreasing trend in the PKM2 inhibitor-treated group compared with the control group (P < 0.01), while the expression of CD206, a specific marker of M2 macrophages, showed an up-regulation trend (P < 0.05). Experimental data from qRT-PCR showed that mRNA levels of iNOS, a marker of M1 macrophages, were significantly lower (P < 0.01) and mRNA levels of TNF-α were significantly lower (P < 0.001) in the PKM2 inhibitor group compared with the control group, while gene levels of Arg-1 and TGF-β, markers of M2 macrophages, were significantly up-regulated (P < 0.01). [Conclusion] HMGB1 induces M1-type polarization of tumor-associated macrophages through PKM2, thereby promoting the migration and invasion of OSCC cells.

Keywords

• oral squamous cell carcinoma
• migration and invasion
• tumor-associated macrophages
• pkm2
• polarization