The Mechanism Of Lactic Acid Regulation On Polarization And GPNMB Expression Of Macrophages In Facilitating OSCC Cell Migration And Invasion

Objective: Oral squamous cell carcinoma(OSCC)is the most common pathological type of oral and maxillofacial malignancies,with frequent regional lymph node metastasis and distant metastasis in advanced stages.Since the molecular mechanism of OSCC invasion and metastasis has not been clarified yet,the clinical prognosis of OSCC is poor.Tumorassociated macrophages(TAMs)represent the main immune cells in the tumor microenvironment.Lactic acid is the major product of aerobic glycolysis and mediates the interaction between OSCC cells and macrophages.However,the molecular mechanism by which lactic acid regulates macrophages and thus affects tumor cell migration and invasion have not been elucidated.In the present study,we investigated the regulation of lactic acid on the polarization state and GPNMB expression of macrophages,and further explored the effect and molecular mechanism of GPNMB in facilitating OSCC cell migration and invasion.Methods: 1.Tumor-conditioned medium(TCM)of CAL27 cells derived from human tongue squamous cell carcinoma was collected to induce tumor-associated macrophages(TAMs)in vitro.2.TCM,lactic acid and monocarboxylic acid transporter inhibitor α-CHC were used to intervene THP-1-derived macrophages to verify the polarization state and GPNMB expression levels.3.CCK8 assay was used to detect the cell viability of THP-1cells treated with different concentrations of lactic acid and α-CHC,respectively.4.RTq PCR,Western blot and immunofluorescence were used to detect the expression levels of M1/M2 markers and GPNMB in macrophages.5.ELISA assay was used to detect GPNMB secretion levels.6.Wound-healing assay,Transwell assay were used to investigate the effects of GPNMB on tumor cell migration and invasion ability.7.Western blot were used to investigate the effect of TAMs-CM supplemented with rh GPNMB on the epithelialmesenchymal transition(EMT)of CAL27 cells.8.Bioinformatics analysis was applied to screen GPNMB potential interacting proteins.9.The co-localization of GPNMB and CD44 in tumor cells was detected by immunofluorescence.10.The interaction between GPNMB and CD44 in tumor cells was detected by immunoprecipitation.11.The expression of CD44 in CAL27 cells was knocked down by RNA interference technique,and the transfection efficiency was verified by RT-q PCR and Western blot.12.The phosphorylation levels of PI3K/AKT/m TOR signaling pathway protein after rh GPNMB intervention in tumor cells was detected by Western blot.Results:1.CCK8 assay showed that no more than 10 m M lactic acid and 4 m M α-CHC were safe concentrations to interfere with THP-1 cells.RT-q PCR results showed that the m RNA expression levels of M2 markers CD206 and ARG1 were up-regulated but M1 marker i NOS was down-regulated in lactic acid-induced macrophages and it showed a concentration dependent effect(P < 0.05),and there were no significant differences in the m RNA expression levels of M1-marker CD86 at this time point(P > 0.05).Western blot showed that 10 m M lactic acid promoted ARG1 protein expression levels(P < 0.05).Immunofluorescence showed higher CD206 fluorescence intensity in lactic acid-induced macrophages.2.GPNMB m RNA levels were higher in THP-1-derived macrophages by comparison with CAL27 cells(P < 0.05).Compared with control group,GPNMB m RNA levels was further upregulated in TCM-induced macrophage(P < 0.05).GPNMB m RNA levels in IL-4-induced M2-type macrophages were significantly higher than M0-stage macrophages(P <0.05).RT-q PCR showed that 10 m M lactic acid in TCM increased GPNMB m RNA and protein levels in macrophages(P < 0.05),and α-CHC supplementation in TCM inhibited GPNMB m RNA level in macrophages(P < 0.05).Western blot and immunofluorescence verified that lactic acid promoted the protein expression of GPNMB in macrophages.ELISA results showed that lactic acid up-regulated GPNMB protein secretion levels,whileα-CHC down-regulated GPNMB protein secretion levels(P < 0.05).3.rh GPNMB significantly facilitated the migration and invasion ability of CAL27 cells and promoted the expression of mesenchymal marker N-cadherin in CAL27 cells,while inhibited the expression of epithelial marker E-cadherin(P < 0.05).4.The STRING database screened out 10 potential interacting proteins of GPNMB,among which CD44 is closely related to cell proliferation,migration,and invasion.OSCC cell lines CAL27 and SCC9 cells had higher m RNA expression levels than Ha Ca T cells(P <0.05).TAMs-CM promoted the m RNA and protein levels of CAL27 cells.Immunofluorescence results showed that GPNMB and CD44 were co-localized on CAL27 cells.The results of co-immunoprecipitation showed that GPNMB interacted with CD44 in CAL27 cells and SCC9 cells.5.The knockdown of CD44 expression significantly inhibited the migration and invasion ability of CAL27 cells,and the effect of rh GPNMB was significantly attenuated under this condition(P < 0.05).Western blot showed that there were no significant changes in EMTrelated molecular markers after CD44 receptor inhibition.6.After the stimulation of rh GPNMB at different time points,the total protein expression of PI3 K,AKT and m TOR in CAL27 cells did not change significantly,but the phosphorylation levels of PI3 K,AKT and m TOR protein showed a time-dependent increase.rh GPNMB stimulation after transfection of CD44-si RNA had no significant effect on the phosphorylation levels of AKT.Conclusions: 1.Lactic acid induces polarization of macrophages towards M2-type.2.Lactic acid promotes the expression and secretion of GPNMB in macrophages.3.The GPNMB/CD44 axis facilitates OSCC cell migration,invasion,and EMT through activation of the AKT signaling pathway.