The Role Of Exosomes Derived From PM_2.5 Treated Lung Cancer Cells In Promoting The Growth Of Lung Cancer And Related Mechanisims

Background:Fine particulate matter(PM2.5)indicates atmospheric aerodynamic equivalent diameter less than or equal to 2.5 micron particles.Long-term and short-term exposure to PM2.5 directly threatens public health.PM2.5 has been related to respiratory disease and cardiovascular disease.Exposure to PM2.5 increases the risk of asthma and exacerbates established asthma.PM2.5 induces inflammation and mucus hyperproduction in the airway epithelium.PM2.5 is necessary for the migration of human bronchial smooth muscle cells,defining a novel role for PM2.5 in airway remodeling in chronic obstructive pulmonary disease.Moreover,PM2.5 is associated with the development of atherosclerosis in ApoE-/-mice.A lot of publications have demonstrated that PM2.5 is associated with cancer incidence.Suggestive evidence has shown an association between ambient air pollution and the incidence of postmenopausal breast cancer in European women.Ambient PM2.5 exposure may be a risk factor for hepatocellular carcinoma in the United States.PM2.5 is also involved in lung cancer burden.Enhanced ability of motility and proliferation were observed after PM2.5 exposure of non-small cell lung cancer cells.PM2.5 also induced epitheliamesenchymal transition of human lung cancer cells.In addition,lung cancer stem cell properties were induced by PM2.5.However,the definitive relationship between PM2.5 exposure and lung cancer has yetto be explored.Exosomes are vesicles 30-150 nm in diameter that are enriched in endosome-derived components.Exosomes have a bilayer lipid structure containing transmembrane proteins,and they enclose soluble proteins,RNA and DNA.Accumulating evidence has well recognized the important role of exosomes as couriers to mediate communication between different cells.Tumou-derived exosomes(TEXs)are closely related to tumour development.TEXs educated dendritic cells to promote tumour metastasis via the HSP72/HSP105-TLR2/TLR4 pathway.Lnc-Sox2ot of TEXs promoted EMT and stemness by acting as a ceRNA in pancreatic ductal adenocarcinoma.Tumour exosomal RNAs promoted lung pre-metastatic niche formation by activating alveolar epithelial TLR3 to recruit neutrophils.Wnt10b in cancer-associated fibroblasts has been shown to promote breast cancer cell metastasis.In addition,activation of the Wnt signalling pathway was detected in PM2.5-induced pulmonary arterial hypertension of rats.Therefore,exosomes from PM2.5-treated lung cancer cells may affect tumour progression through activation of Wnt signalling.On the one hand,studies have indicated that Wnt10b in cancer-associated fibroblasts has been shown to promote breast cancer cell metastasis.On the other hand,activation of the Wnt signalling pathway was detected in PM2.5-induced pulmonary arterial hypertension of rats.Whether exosome from lung cancer cells stimulated by PM2.5 affects the progression of lung cancer by activating Wnt signaling has not been reported before.Therefore,this study intends to use in vitro stimulation and in vivo A549 lung adenocarcinoma subcutaneous tumor model to explore the regulation of lung cancer growth by exosomes from PM2.5-stimulated A549 cell and the underlying molecular mechanisms.Methods and Results1.PM2.5 exposure increases Wnt3a protein level in EXOPM2.5.To clarify the expression of Wnt protein family in A549 lung adenocarcinoma cells treated by PM2.5,we first stimulated A549 lung adenocarcinoma cells in vitro by PM2.5.Real-time PCR and WB results showed that compared with control A549 lung adenocarcinoma cells,the levels of Wnt3a in PM2.5 stimulating A549 lung adenocarcinoma cells significantly increased,while that of Wntl,Wnt4,Wnt7a,Wnt9a,Wnt9b and Wnt10b were the same.At the same time,the gene and protein levels of Wnt3a in PM2.5 stimulating human bronchial epithelial cells HBE also significantly increased.To further clarify the levels of Wnt3a protein in exosomes derived from A549 lung adenocarcinoma cells after PM2.5 treatment,PM2.5-treated and untreated A549 cell derived exosomes were collected(EXOPM2.5 and EXOCtrl).We use electron microscopy,Nanoparticle Tracking Analysis and WB to identify and analyze these exosomes.The results showed that PM2.5 stimulation had no effect on the morphology of exosomes,while EXOPM2.5 had higher Wnt3a protein levels than EXOctri.2.EXOPM2.5 induces activation of the ?-catenin pathway.?-catenin is a key downstream effector molecule in the Wnt signaling pathway and plays an important role in the proliferation of tumor cells.PM2.5 can up-regulate Wnt3a protein level in EXOPM2.5.Therefore,we speculated that EXOPM2.5 were probably to activate the ?-catenin signaling pathway in A549 cells,thereby affecting the proliferation of lung cancer cells.To investigate whether EXOPM2.5 could activate the ?-catenin pathway in A549 lung adenocarcinoma cells,we used WB and immunofluorescence to detect the activation of ?-catenin pathway in A549 lung adenocarcinoma cells treated with and without EXOPM2.5.The results showed that the expression of ?-catenin protein in the A549 lung adenocarcinoma cells treated with EXOPM2.5 was obviously increased compared with that in the EXOctrl-treated A549 lung adenocarcinoma cells,and the nuclear ?-catenin protein was also significantly increased.The above results indicate that EXOpm2.5 can activate the ?-catenin signalling pathway in A549 lung adenocarcinoma cells.3.EXOPM2.5 does not affect A549 cell migration and invasion in vitro.The Wnt/?-catenin signaling pathway is closely related to the migration and invasion of tumor cells.To determine whether EXOPM2.5 could affect the migration and invasion ability of A549 lung adenocarcinoma cells,we performed in vitro migration and invasion experiments of A549 lung adenocarcinoma cells stimulated by EXOCtrl and EXOPM2.5.The results showed that there was no significant difference in the migration ability of A549 lung adenocarcinoma cells between these two groups,and there was no significant difference in the invasion ability of A549 lung adenocarcinoma cells in these two groups,either.The above results indicate that EXOPM2.5 does not affect the migration and invasion ability of A549 lung adenocarcinoma cells.4.EXOPM2.5 promotes A549 cell proliferation in a Wnt3a/?-catenin-dependent manner.Since ?-catenin signalling pathway promotes tumour cell proliferation,we further examined the effect of EXOPM2.5 on the proliferation ability of A549 cells.Through the CCK-8 assay,we found that compared with the EXOCtrl-stimulated A549 lung adenocarcinoma cells,the proliferation ability of the EXOPM2.5-treated A549 lung adenocarcinoma cells was significantly enhanced.In order to clarify the role of Wnt3a protein in the proliferation of A549 lung adenocarcinoma cells by EXOPM2.5,we used siRNA technology to knock down the Wnt3a protein of A549 lung adenocarcinoma cells.CCK-8 experimental results showed that exosomes derived from PM2.5-treated A549 cells transfected with negative control(NC)siRNA(EXOPM2.5/NC siRNA)were still proliferative promotion when compared with A549 lung adenocarcinoma cells treated with PBS control.However,the exosomes derived from PM2.5-treated A549 cells transfected with Wnt3a siRNA(EXOPM2.5/Wnt3a siRNA)no longer promoted the proliferation ability of A549 lung adenocarcinoma cells.These results indicate that EXOPM2.5 promotes A549 cell proliferation dependent on Wnt3a.In order to further clarify the role of Wnt3a/?-catenin signalling pathway in the proliferation of A549 lung adenocarcinoma cells by EXOPM2.5,we applied a specific inhibitor of ?-catenin signalling pathway(LF3).The results showed that,compared with A549 lung adenocarcinoma cells treated with DMSO control,the proliferative ability of A549 lung adenocarcinoma cells treated with EXOPM2.5+DMSO was still significantly increased,while the proliferative ability of A549 lung adenocarcinoma cells treated with EXOPM2.5+LF3 was not obvious changed.The above experimental results show that EXOPM2.5 enhance the proliferation of A549 lung adenocarcinoma cells is dependent on the Wnt3a/?-catenin signalling pathway.5.EXOPM2.5 promote A549 cell growth in vivo via Wnt3a.To explore whether EXOPM2.5 could promote the proliferation of A549 lung adenocarcinoma cells in vivo,we used nude mice to establish a subcutaneous tumour model of A549 lung adenocarcinoma cells,and recorded the tumour size and survival rate of tumor-bearing nude mice.The expression of Ki67(a tumour proliferation indicator protein)and ?-catenin,were detected by immunohistochemistry.The results showed that compared with the tumour-bearing nude mice in the PBS or EXOCtrl treatment group,the nude mice in the EXOPM2.5 treatment group had significantly larger tumours and a significantly shorter survival rate.Ki67 immunohistochemical staining of tumour tissues showed that EXOPM2.5 promoted the expression level of Ki67 protein in A549 lung adenocarcinoma cells,and also promoted the expression of P-catenin in tumour tissues.The above results indicate that EXOPM2.5 is likely to promote the proliferation ability of A549 lung adenocarcinoma cells by increasing the accumulation of ?-catenin.To further clarify the role of Wnt3a protein in the process of EXOPM2.5 increasing proliferation of A549 lung adenocarcinoma cells in vivo.We used nude mice to establish a subcutaneous tumour model of A549 lung adenocarcinoma cells,injected with PBS,EXOPM2.5/NC siRNA or EXOPM2.5/Wnt3a siRNA,and regularly observed the tumor size and survival rate of tumor-bearing nude mice.Tumour tissues were subjected to immunohistochemical staining of Ki67 and ?-catenin expression.The results showed that,compared with the nude mice in the PBS group,the tumours in the EXOPM2.5/NC siRNA group were significantly increased,and the survival rate was significantly shortened;the tumor size and survival rate in the EXOPM2.5/Wnt3a siRNA group were not changed.At the same time,the immunohistochemical staining of Ki67 and ?-catenin in tumour tissues also showed the same trend as above.The above results confirm that EXOPM2.5 promote the growth of A549 lung adenocarcinoma tumours in vivo dependent on the Wnt3/?-catenin signalling pathway.Conclusion:After stimulated with PM2.5,A549 lung adenocarcinoma cells secreted exosomes carrying more Wnt3a protein,and then these exosomes increase the level of?-catenin protein in A549 lung adenocarcinoma cells and promote the entry of?-catenin into the nucleus through Wnt3a,promote its regulation of target gene transcription,and ultimately accelerate the growth of lung cancer.Innovation and research significance:This study report for the first time that exosomes derived from A549 lungadenocarcinoma cells after PM2.5 treatment are rich in Wnt3a protein.It is found that Wnt3a protein in EXaPM2.5 activated the ?-catenin signalling pathway in A549 lung adenocarcinoma and promoted the growth of lung cancer.The above research results not only benefit the clarification of the relationship between environmental pollution and lung cancer,but also provide a new target for the treatment of lung cancer.