Mechanism Of HSP27 On The Regulation Of M2 Polarization And IL-6 Secretion Of Tumor Associated Macrophages In The Invasion And Metastasis Of Oral Squamous Cell Carcinoma

Objective:Oral squamous cell carcinoma is a destructive and lethal malignant tumor,which is prone to local recurrence and distant metastasis.Invasion and distant metastasis are the main causes of its death.However,the invasion and metastasis of oral squamous cell carcinoma has not been fully clarified.Therefore,the 5-year survival rate of patients has not changed significantly.The interaction between oral squamous cell carcinoma and tumor associated macrophages(TAMs)plays an important role in the invasion and metastasis of oral squamous cell carcinoma.As the main immune cells in the tumor microenvironment,tumor associated macrophages are mainly divided into two types: M1 and M2 phenotypes,especially M2 tumor associated macrophages can promote tumor invasion and metastasis by secreting a variety of cytokines.Studies have found that HSP27 is highly expressed in OSCC and is closely related to the poor prognosis of patients,indicating that HSP27 may play a role in the progression of oral squamous cell carcinoma.Reports have shown that HSP27 can be used as a secreted protein to regulate immune cells and promote tumor progression.Therefore,the purpose of this study is to detect the expression of HSP27 in CAL27 cells and further explore whether HSP27 can affect the epithelial mesenchyme transformation and the invasion and metastasis of CAL27 cells by regulating the polarization state and the secretion level of IL-6 of tumor associated macrophages.at the same time we explore the role of IL-6 in the occurrence of EMT in CAL27 cells and related mechanisms,so to clarify the role of tumor associated macrophages in the pathological process of oral squamous cell carcinoma,and provide a theoretical basis for using it as a target.Methods:Immortalized human normal epidermal cells Hacat,oral squamous cell carcinoma tumour CAL27 cells and mouse macrophages RAW264.7 were used as research objects.HSP27 small interfering RNA,exogenous rh HSP27,exogenous rh IL-6,TLR4 receptor inhibitor TAK-242 and IL-6 receptor inhibitor were used for intervention.Immunofluorescence was detected of rh HSP27 and TLR4colocalization;quantitative Real Time-PCR(q RT-PCR)was used to detect m RNA expression levels of HSP27,M1 and M2 type TAMs related cytokines(CD163、Arg-1、TNF-α、IL-10、i NOS、IL-12、IL-6),EMT markers(E-cadherin,N-cadherin,Vimentin);Western Blot experiment to detect protein expression levels of HSP27 and EMT markers(E-cadherin,N-cadherin);ELISA experiment to detect the secretion level of IL-6;To detect the ability of invasion and metastasis of CAL27 cells with cell scratch and Transwell assays.Results:1.Compared with human normal epidermal Hacat cells and Hacat cells intervention with TAMs supernatant,HSP27 m RNA expression was significantly increased in CAL27 cells intervention with TAMs supernatant,and compared with RAW264.7 and TAMs,HSP27 was mainly expressed by CAL27 cells.2.After HSP27 small interfering RNA knocked down the expression of HSP27 in CAL27 cells,the corresponding supernatants were used to interfere with RAW264.7 cells.It showed that the interference group TAMs down-regulated the m RNA expression of Arg-1,IL-10 and IL-6,while up-regulated the expression levels of M1 type TAMs cytokines(i NOS,IL-12,TNF-α),which implied knockdown of HSP27 inhibits the transformation of TAMs to M2 type TAMs.3.After rh HSP27 diluted with CAL27 supernatants was applied to RAW264.7,the q RT-PCR results showed that the m RNA expression levels of M2 type TAMs related cytokines(Arg-1,CD163)and IL-6 increased,while M1 type TAMs related expression of cytokines(i NOS,IL-12)were decreased.On the contrary,TAK-242 diluted with CAL27 supernatants in advance inhibited the TLR4 receptor,and then rh HSP27 was added.The results indicated that the m RNA expression of Arg-1 and IL-6 were down-regulated,but IL-12 expression level was up-regulated.ELISA results showed that after adding exogenous rh HSP27 diluted with CAL27 supernatants to interfere with RAW264.7,which promoted the secretion of IL-6,but after adding TAK-242 in advance,it obviously inhibits the secretion of IL-6.4.CAL27 supernatants diluted rh HSP27 or diluted TAK-242 and rh HSP27 simultaneously to intervene RAW264.7 cells for 12 h,the medium was discarded and the new medium was replaced and incubated for 24 h,and then collected the corresponding TAMs supernatants and acted on CAL27 cells for 24 h or 48 h.It indicated that the TAMs supernatants produced by the RAW264.7 cells with rh HSP27 intervention reduced the scratch area significantly,indicating that rh HSP27 promotes the migration ability of CAL27 cells,while after the intervention of TAK-242,inhibiting the migration ability of CAL27 cells.After inducing RAW264.7into TAMs by the above methods,Transwell experiment co-cultured CAL27 cells and TAMs,The results showed adding rh HSP27 induced TAMs significantly increased the migrated and invasive number of CAL27 cells compared to the control groups,which significantly promoted CAL27 cell migration and invasion ability,while TAMs after intervention with TAK-242 reduced the migrated and invasive number of CAL27 cells,which implied inhibited the migrated and invasive ability of CAL27 cells.5.The corresponding TAMs supernatants was obtained in the same way as result 4,and then acted on CAL27 cells.q RT-PCR results demonstrated that CAL27 cells in the HSP27 experimental group promoted the expression level of N-cadherin and Vimentin,while adding TAK-242,the expression level of Vimentin was decreased.Western Blot found N-cadherin in the HSP27 group were improved by contrasted with the control groups,while N-cadherin was reduced in the TAK-242 group.6.Before co-cultured CAL27 cells and TAMs,the upper chamber tumour cells was inhibited by IL-6 receptor inhibitor for 24 h in advance,RAW264.7 cells in the lower chamber were intervened with exogenous HSP27.The results confirmed that,by contrasted with the control groups,the migrated and invasive number of CAL27 cells in the inhibitor groups was significantly reduced,which turned out that IL-6receptor inhibitor inhibits the migrated and invasive ability of CAL27 cells.7.After knocking down the expression of HSP27 in CAL27 cells with HSP27 small interfering RNA,and then adding exogenous rh IL-6,q RT-PCR showed the expression of E-cadherin was increased compared wiith the control group,while the expression of N-cadherin was decreased;Western Blot experimental results indicated compared with the control group,the expression level of N-cadherin in the HSP27-si RNA group decreased.Conclusion:1.HSP27 is highly expressed in CAL27 cells;2.HSP27-TLR4 axis induces M2 phenotype polarization and IL-6 secretion of TAMs;3.HSP27-TLR4 axis induced TAMs promotes CAL27 cell invasion,metastasis and EMT;4.IL-6/IL-6R promotes the invasion and metastasis of CAL27 cells;5.IL-6 promotes EMT through HSP27 expressed by CAL27 cells.