| Objective: ①The study is focus on the relationship between KIT,EGFR and non small cell lung cancer and predict the survival of these patients. ② We further explore the effect of mast cells derived exosomes on bio-function of non small cell lung cancer cells, such as proliferation and cell cycle. ③ Finally, we discuss the mechanism of mast cells derived exosomes in non small cell lung cancer proliferation.Methods: ①Tumors specimens from 146 non-small cell lung cancer patients who underwent surgical resection. The paraffin sections were stained with KIT expression by immunohistochemistry, and the expression of EGFR was evaluated by fluorescence in situ hybridization. The relationships of the distribution of KIT and EGFR with clinicopathologic parameters and survival of patients were analyzed. ② We used uptake experiment, Brd U cell proliferation and PI staining to explore the effect of mast cells derived exosomes on biological function of non-small cell lung cancer cells including proliferation and cell cycle in vitro. ③To investigate the mechanism of mast cells derived exosomes on proliferation of non-small cell lung cancer cells, we detected the KIT protein transfer from the mast cells to the lung cancer cells by PCR and western blot, and then discussed KIT-SCF and the downstream PI3K/Akt signaling pathway.Results: ①KIT was a significant negative prognostic factor in both univariate and multivariate analyses. The expression of KIT was correlated with poorly differentiation, pleura involvement and smoking(P=0.043, 0.007 and 0.032 respectively), while the difference of KIT-positive survival was statistically significant(P=0.048). The median follow-up time was 19 months after surgery. EGFR polysomy tended to be negatively related to poorly differentiated and smoking history(P=0.023 and 0.044 respectively). However, there was no statistical significant difference in the influence of EGFR expression in the patients. The co-expression of KIT and EGFR tended to add to the degree of mortality in non-small cell lung cancer patients. ② Exosome can be isolated from the supernatant of cultured HMC-1 cells and the protein fraction isolated from the pellet after ultracentrifugation was positive for the traditional exosome markers CD81 and TSG101. Flow cytometry showed an increased fluorescence intensity of the lung cancer cells after the addition of mast cell-derived exosomes, suggesting cellular uptake. The fluorescence of A549 cells was obvious already after two hours, and increased over time, indicating initiation of uptake of the exosomes by A549 cells. Exosomes from HMC-1 cells could influence cell proliferation in A549 cell at 4hours. Cell cycle phases were determined with PI stain by flow cytometry, indicating that in the presence of HMC-1 exosomes the number of cells in the S-G2/M phase significantly increased(from 27.2 to 35.6%), and the number of cells in G1 phase decreased from approximately 73.3 to 64.3%. ③When we explored the mechanism of proliferation in non small cell lung cancer cells, our identification of KIT protein but not c-kit m RNA in the HMC-1 exosomes, and the transfer of the protein to lung adenocarcinoma cells, suggests the transfer of protein rather than m RNA transcript. PI3 Kinase and its downstream targets, AKT, and GSK3β showed both increased phosphorylation compared to media or control exosomes treated A549 cells by western blot, but not phosphorylation of Cyclin D1.However, the total cyclin D1 protein is increased.Conclusion: KIT and EGFR is a strong and independent negative prognostic factor in NSCLC.The purified exosomes from mast cells take up by lung cancer cells. Our results also show that exosomes express and transfer KIT protein to A549 cells and subsequently activate PI3K/Akt signal transduction, which increase cyclin D1 expression, enhance the cell proliferation, and accelerate the cell cycle in the human lung adenocarcinoma cells. Our study first explored the mast cells derived exosomes transfer the KIT to the lung cancer cells at the protein level and effect on the lung cancer cells, cell-signaling pathway as well. Therefore,we have a new opinion on the KIT-containing exosomes derived from mast cells in the development and progression in the lung cancer. |
