| Objective: Lung cancer is the most common malignant tumor with the highest mortality.Tumor metastasis is a key obstacle for the effective treatment of lung cancer,so it is urgent to explore the mechanism of lung cancer metastasis and develop targeted treatment strategies.Tumor microenvironment has attracted widespread attention in recent years because it involves in the occurrence and development of tumor from many aspects.Cancer-associated fibroblasts(CAFs)are one of the majorcell components in the tumor microenvironment and can promote tumor progression through direct contact with tumor cells or paracrine cytokines,so they have become potential therapeutic targets in many solid tumors.Autophagy is a highly evolutionarily conservative cellular process,which enables cells to survive through degradation of cell contents in the face of stress such as lack of nutrients.It has been found that autophagy plays an important role in tumorigenesis,tumor development and tumor treatment.However,the role of autophagy level of CAFs in tumor growth and metastasis is not clear.In the present study,we investigated the role of autophagy of CAFs in lung cancer cell proliferation,invasion and metastasis,and further explored the underling mechanisms.This study can improve the understanding of the mechanism of lung cancer metastasis and provide novel strategies for the treatment of lung cancer.Methods: Part 1: In vitro study 1.The isolation and autophagy level of CAFs: CAFs and normal fibroblasts(NFs)were isolated,cultured and identified from lung cancer specimens.Western blot,GFP-LC3 puncta assay and acridine orange staining were performed to detect the level of autophagy in NFs and CAFs.2.The effect of inhibition of CAF autophagy on lung cancer cell proliferation,invasion and migration: CAFs were treated with autophagy specific inhibitors 3-MA,CQ or knockdowned the expression of autophagy-related gene ATG5 by RNAi method.CAF-conditioned medium(CAF-CM)was collected and the lung cancer cells were cultured with this CAF-CM.CCK-8,wound healing and transwell assays were used to investigate the effect of CAFs on lung cancer cell A549 and H661 proliferation,invasion and migration after inhibition of CAF autophagy.Quantitative real-time PCR and Western blot were performed to detect the expression of metastasis-related genes(MMP2,Twist,MMP9)and EMT-related markers(E-cadherin,N-cadherin,Vimentin)in lung cancer cells.3.The effect of activation of CAF autophagy on lung cancer cell proliferation,invasion and migration: CAFs were treated with a specific autophagy activator RAPA.CAF-CM was collected and the lung cancer cells were cultured with this CAF-CM.CCK-8,wound healing,transwell assays and Western blot were performed to detect the effect of CAFs on lung cancer cell proliferation,invasion,migration and the expression of metastasis-and EMT-related genes in lung cancer cells after upregulation of CAF autophagy.4.The effect of autophagy level of CAFs on secretion of high mobility group protein B1(HMGB1): the expression of HMGB1 in NFs and CAFs was detected by Western blot,and the secretion of HMGB1 in lung cancer cells,NFs and CAFs was detected by ELISA.CAFs were treated with 3-MA,CQ,RAPA or knockdowned the ATG5 expression by RNAi method.ELISA was performed to detect the effect of regulating the autophagy level of CAFs on the secretion of HMGB1 by CAFs.5.The effect of HMGB1 on CAF autophagy: CAFs was treated with recombinant HMGB1 and GFP-LC3 puncta assay and Western blot were performed to detect the change of CAF autophagy level after intervention with HMGB1.6.The effect of HMGB1 secreted by CAFs on proliferation,invasion and metastasis of lung cancer cells: lung cancer cells were cultured with CAF-CM supplemented with HMGB1 neutralization antibody or HMGB1 inhibitor,or normal medium supplemented with recombinant HMGB1.CCK-8,wound healing,transwell assays and Western blot were used to evaluate the effect of HMGB1 secreted by CAFs on the proliferation,invasion and metastasis of lung cancer cells.7.The effect of HMGB1 secreted by CAFs on TLR4/NF-κB signaling pathway in lung cancer cells: lung cancer cells were cultured with CAF-CM which was collected from CAFs treated with 3-MA,CQ,RAPA or si ATG5.Western blot was used to detect the effect of CAFs on the expression of key molecules TLR4,p-IκBα,IκBα and p-p65 in TLR4/NF-κB signaling pathway of lung cancer cells after regulating the autophagy level of CAFs.Combined with HMGB1 neutralizing antibody and recombinant HMGB1,the role of HMGB1 secreted by CAFs in TLR4/NF-κB signaling pathway of lung cancer cells was evaluated.Part 2: In vivo study In vivo experiment in nude mice: the subcutaneous tumor model in nude mice was established by subcutaneously injecting the mixture of CQ-treated CAFs and A549 into the back of nude mice,and untreated CAFs were used as control.After 5 weeks of feeding,the tumor tissue was stripped off,and the volume and weight of tumor tissue were measured.The expression levels of key molecules in the TLR4/NF-κB signaling pathway,metastasis-related genes and EMT-related markers in tumor tissues were detected by Western blot.Results: 1.CAFs have a heightened level of basal autophagy compared to NFs in lung cancer.2.Inhibition of CAF autophagy could weaken the effect of CAFs on promoting lung cancer cell proliferation,invasion and metastasis,and up-regulating the expression of metastasis-related genes MMP2,Twist,MMP9 and mesenchymal markers N-cadherin and Vimentin,and down-regulating the expression of epithelial marker E-cadherin in lung cancer cells.3.Up-regulation of CAF autophagy could enhance the effect of CAFs on promoting lung cancer cell proliferation,invasion and metastasis,and up-regulating the expression of metastasis-related genes and mesenchymal markers,and down-regulating the expression of epithelial marker in lung cancer cells.4.CAFs could secrete more HMGB1 than NFs and lung cancer cells.Regulation the autophagy level of CAFs could affect the secretion of HMGB1 by CAFs,and HMGB1 could further up-regulate the autophagy level of CAFs,indicating that there is a positive feedback regulation between CAFs and HMGB1.5.HMGB1 secreted by CAFs activated the TLR4/NF-κB signaling pathway in lung cancer cells,and then promoted the proliferation,invasion and metastasis of lung cancer cells.6.In vivo studies showed that CAFs promoted the growth of lung cancer cells in vivo.Inhibiting the autophagy level of CAFs could effectively weaken the effect of CAFs on activating EMT and TLR4/NF-κB signaling pathway in lung cancer cells,and then weaken the effect of CAFs on promoting the growth of lung cancer cells in vivo.Conclusions: 1.CAFs have a high level of basal autophagy in lung cancer.2.The high autophagy level of CAFs mediates the role of CAFs in promoting the proliferation,invasion and metastasis of lung cancer cells.3.There is a positive feedback regulation relationship between the high autophagy level of CAFs and the HMGB1 secreted by CAFs.4.CAFs activates the TLR4/NF-κB signaling pathway in lung cancer cells by secreting HMGB1,which promotes the proliferation,invasion and metastasis of lung cancer cells.5.Inhibiting the autophagy level of CAFs could effectively weaken the effect of CAFs on promoting the growth of lung cancer cells in vivo. |
