The Role And Mechanism Of CCL18 In The Development Of Lung Adenocarcinoma

At present,tumor is one of the most important causes of disease and death in China,and more than 90%of tumor patients have distant metastasis of tumor cells.The microenvironment determines the development and metastasis of the tumor.In the study of anticancer therapeutic targets related to tumor-microenvironment interactions,chemokine superfamily members have also received increasing attention from scientists.CCL18,a member of the chemokine family,has been found to play a key role in the progression of malignant tumors.CCL18 was detected to be expressed in macrophages,monocytes and immature dendritic cells,which induced collagen precipitation by fibroblasts and recruited lymphocytes and immature dendritic cells.CCL18 is one of the most highly expressed factors in tumor-associated macrophages（TAMs）.Under the stimulation of stimulating factors,such as IL4,macrophages polarized into the M2phenotype,thus promoting the expression of CCL18^[1].Therefore,the increased expression of CCL18 in peripheral blood and serous cavity edema of tumor tissues associated with various malignant tumors.The high expression of CCL18 protein can inhibit the maturation and recruitment of lymphocytes and dendritic cells and destroy the immune ability of the body.It can also promote the migration and invasion of cancer cells by directly binding to cancer cells^[2-6].Early laboratory results showed that CCL18 promoted the migration of A549 cells（non-small cell lung cancer cell lines）.The paracancer and carcinoma tissues of 75 clinical patients with lung adenocarcinoma were made into tissue slices,and the tissue microarray was obtained by CCL18antibody specific staining.It was found from the data analysis that there was no significant correlation between CCL18 expression level and gender and age of lung cancer patients.Treatment group:A549 cells were treated with CCL18 for 1h,and the control group was treated with the same amount of DMSO for 1h.SILAC and lc-ms/MS were combined to quantify the dynamic changes of all proteins in the whole proteomics of A549 cells.The proteomic data identified 7,688 proteins,of which 6,352were quantified.Among the quantitative proteins,198 proteins were up-regulated and 58proteins were down-regulated compared with the control.In the acetylation results obtained by the combination of SILAC labeling and high-efficiency acetylation enrichment,we identified 1372 lysine acetylation（Kac）sites on 796 proteins in A549cells treated with CCL18 Among them,the acetylation level of 147 sites on 126 proteins was down-regulated,and the acetylation level of 57 sites on 5 proteins was up-regulated.Bioinformatic analysis further revealed that the down-regulated acetylation protein and A549 cell glycolysis,oxidative phosphorylation（OXPHOS）,tricarboxylic acid cycle（TCA）,pentose phosphate pathway（PPP）and amino acids,nucleotides and lipids Related to the synthesis.The above results indicate that CCL18 can participate in the occurrence and development of NSCLC,especially the metabolic reorganization of tumor cells,by regulating the acetylation of proteins related to cell metabolism.Through laboratory research basis and acetylation omics analysis,it was found that CCL18 had almost no effect on the expression level of Cyclin-T1 protein,but it significantly increased the acetylation level of Cyclin-T1.Cyclin-T and CDK9 are rigidly combined to form a cyclin-dependent protein kinase complex,namely positive transcription elongation factor b（positive transcription elongation factor b,P-TEFb）.The mechanism of regulating transcription is to make related negative transcription factors and RNA polymerase Phosphorylation occurs to achieve transcriptional regulation^[7-9].Studies have shown that Cyclin-T1 uses the HRD-rich domain HRD to target CTD and make it hyperphosphorylated^[10].From the results of mass spectrometry,it can be concluded that the Cyclin-T1-K492R mutation is extremely important,and this site happens to fall in the HRD domain.Combined with the previous experimental results,we conducted further research on Cyclin-T1 protein.In this study,A549 cells were time-graded with CCL18 and found that CCL18 can promote the acetylation of Cyclin-T1 but does not affect its expression;Western blot experiments confirmed that 492 acetylation mutation of Cyclin-T1 can reduce its binding to Hexim1 Cyclin-T1 K492R reduced the phosphorylation level of CDK9 at position 186 without affecting the expression of CDK9.