Role And Mechanism Of Regulation Of HMGB1 On Tumor-associated Macrophages In Invasion And Metastasis Of Oral Squamous Cell Carcinoma

Objective:Oral squamous cell carcinoma(OSCC)is one of the most invasive and most common malignant tumors with the highest incidence in the head and neck.The five-year survival rate of patients is less than 50%.OSCC has a high local infiltration rate,strong metastatic ability,and is prone to relapse at a later stage.The characteristics of invasion and metastasis are the main reasons for the high mortality of OSCC.The pathogenesis of OSCC invasion and metastasis has not been fully elucidated.In the tumor microenvironment(TME),tumor-associated macrophages(TAMs)can regulate tumor invasion and metastasis by producing various factors and metalloproteases.TAMs can also affect tumor invasion and metastasis by affecting the M2 type polarization state.TAMs play an important role in the regulation of TME.High mobility group protein B1(HMGB1)is a member of the high mobility group protein family,mainly derived from tumor cells,and plays a key role in the invasion,metastasis and prognosis of various cancers.Clinical studies have shown that HMGB1 is highly expressed in OSCC tissues,suggesting that HMGB1 may play an important role in the occurrence and development of OSCC.Studies have shown that HMGB1 can regulate the cytokines secreted by TAMs,and then interfere with tumor progression.This topic mainly explores the role of HMGB1 produced by OSCC tumor cells on the regulation of cytokines secreted by TAMs in OSCC invasion and metastasis,and further clarifies the interaction between tumor cells and TAMs in the OSCC tumor microenvironment and its possible mechanism,and provide new experimental basis and potential therapeutic strategies for intervention strategy of oral tumor.Methods:CAL27 cells and Raw264.7 cells were used as experimental subjects in this study.HMGB1 siRNA and human recombinant HMGB1 protein were used for intervention.Real-time PCR and Western blot experiments were used to detect the expression of HMGB1 gene and protein in OSCC tumor cells.The cell scratch test and Transwell test were used to detect the ability of OSCC tumor cell invasion and metastasis.Real-time PCR and ELISA experiments were used to detect gene and protein expression of cytokines secreted by TAMs.Results:1.At the gene level and protein level,compared with Hacat normal epidermal cells,HMGB1 showed high expression in Cal27 cells.After transfection of HMGB1 siRNA into Cal27 cells for 36 h,compared with the negative control group,the level of gene expression of HMGB1 in Cal27 cells decreased by about 50%.After transfection of HMGB1 siRNA into Cal27 cells for 48 h,compared with the negative control group,at the protein level,the expression of HMGB1 in Cal27 cells decreased by about 40%.2.After transfection of HMGB1 siRNA into Cal27 cells for 36 h,and the supernatants of Cal27 cells were collected and acted on Raw264.7 cells for 12 h,then the fresh medium was used to act on Raw264.7 cells for 12 h,and the supernatants of Raw264.7 cells were collected and acted on newly incubated Cal27 cells for 24 h or 48 h.The results of scratch experiment showed that compared with the negative control group,the ability of Cal27 cells to transfer was inhibited.The results of Transwell experiment showed that compared with the negative control group,the number of metastasis and invasion cells of Cal27 cells decreased.3.The supernatants of Cal27 cells and exogenous rhHMGB1 protein were added to Raw264.7 cells for 12 h,then the fresh medium was used to act on Raw264.7 cells for 12 h,and the supernatants of Raw264.7 cells were collected and acted on newly incubated Cal27 cells for 24 h or 48 h.The results of scratch experiment showed that compared with the control group,the transfer ability of Cal27 cells was enhanced.The results of Transwell experiment showed that compared with the control group,the number of metastasis and invasion cells of Cal27 cells significantly increased.4.After transfection of HMGB1 siRNA into Cal27 cells for 24 h,the supernatants of Cal27 cells were collected and acted on Raw264.7 cells for 12 h,then the fresh medium was used to act on Raw264.7 cells for 12 h.ELISA results showed that compared with the negative control group,the protein expression level of IL-6 secreted by TAMs decreased significantly,and the protein expression level of IL-10 secreted by TAMs decreased.Real-time PCR results showed that compared with the negative control group,at the protein gene levels,cytokines of IL-6 and IL-10 secreted by TAMs decreased.5.After the supernatants of Cal27 cells and exogenous rhHMGB1 protein were added to Raw264.7 cells for 12 h,then the fresh medium was used to act on Raw264.7 cells for 12 h.ELISA results showed that compared with the negative control group,at the protein expression levels,cytokines of IL-6 and IL-10 secreted by TAMs increased.Real-time PCR results showed that compared with the control group,at the protein gene levels,cytokines of IL-6 and IL-10 secreted by TAMs increased.Conclusion:1.HMGB1 is highly expressed in OSCC tumor cells.2.HMGB1 derived from OSCC tumor cells up-regulates the expression of genes and proteins of cytokines IL-6 and IL-10 secreted by TAMs.3.HMGB1 derived from OSCC tumor cells promote the invasion and metastasis of OSCC tumor cells by up-regulating the expression levels of IL-6 and IL-10 secreted by TAMs.