Roles Of C-raf Kinase And Its Binding Protein IRS4 In Environment Related Lung Cancer Development

As the first killer of human health,the incidence of lung cancer is as twice as the national average.Non-small cell lung cancer becomes the most difficult problem because of its characteristics of chemotherapy non-sensitivity,tumor metastasis and tumor recurrence.This makes the exploration of new early diagnosis of non-small cell lung cancer target and personalized treatment target imminent.It is generally believed that the occurrence of lung cancer is associated with genetic factors and environmental degradation.Studies have shown that the pollution in the Xuanwei,Yunnan is more severe than that in other regions,and the expression of EGFR and Ras genes is different from that of other regions.Whether genetic or environmental factors,the intracellular signal transduction must changed ultimately then lead to cell and tissue carcinogenesis.RAF family as a downstream signal molecule of EGFR and Ras,are key proteins in cell signaling.the RAF family play an indispensable role in tumorigenesis.The signals received from the upstream oncogene RAS and then RAF transmited the signal to MEK and ERK.The phosphorylated ERK controls a wide range of biological functions:cell survival,proliferation,migration,differentiation and immune response,etc.The RAF family has three members:A-Raf,B-Raf and C-Raf(Rafl).As the first discovered member of the Raf family,C-Raf often appear high expression and no mutation in the tumor tissue.C-Raf inhibitors are pan-Raf inhibitors that blocking the MAPK signal pathway's abnormal activation.So the side effects caused by these inhibitors can not be ignored.Therefore,a comprehensive analysis of C-Raf function is particularly important.We can not fully understand the function of C-Raf protein because the previous works are focused on the biological function of C-Raf itself,while ignoring many C-Raf interacting and ignoring the gene expression that C-Raf controls.Therefore,this study used immunoprecipitation and interaction proteomics to find new C-Raf interacting protein,and compared the different gene expression profile between before and after C-Raf knockout by transcriptomics.Increased the perspective to the system level to find C-Raf and its biological changes,provided a reliable basis for C-Raf function in-depth understanding.The analysis of the r interaction proteomics results suggests that our C-Raf can bind 198 proteins in a steady state,16 of which were previously reported.These proteins were 85%positive in binding verification by western blot.Functional enrichment analysis and categorization of these proteins indicated that the function of the C-Raf binding protein covered the ten hallmarks of cancer,and were also involved in some bacterial and viral infections.And these are previously not found or summarized in the C-Raf function researches.These provide new targets for the diagnosis and treatment of lung cancerThe data of the transcriptome revealed that C-Raf protein controlled the expression of 3353 genes under resting conditions or activation conditions.Among them,1993 genes were over expressed after CRAF knockout and 1420 genes were inhibited.The intersection of the changed genes under activation condition and rest condition are the gene changed only dependent on C-Raf(1007 up-regulated,660 down-regulated).Some of the up-regulated genes are involved in opioid addiction,cardiomyopathy and systemic lupus erythematosus.The predicted relationship between these diseases and C-Raf has not been reported in previous studies.We also demonstrated the expression changes of nine genes associated with morphine addiction in independent repetitive experiments(qPCR).The down-regulated genes are enriched in HTLV-I and Epstein-Barr virus infections,and amino acid metabolism,and the correlation between the first two functions has been already reported.This huge amount of informations suggests that C-Raf's functions are far more than we have known.For some of C-Raf interacting or regulating proteins/genes are not functional clearly in the development of lung cancer,also directly or indirectly indicate some of the unknown functions of C-Raf.We selected IRS4 as a specific research object and explored its role in the development of lung cancer.The results showed that the growth of lung cancer cells,the cell migration and transformation ability were positively correlated with the expression level of IRS4.After the decrease of IRS4 expression,the sensitivity of cells to cisplatin was decreased,and the ability of angiogenesis was also decreased.We determine that IRS4 is a factor that promote the occurrence of lung cancer,but the low expression of IRS4 was promoted by lung cancer cells to tolerance to cisplatin.These results are being validated at the animal level.In conclusion,we not only explored the function of C-Raf interacting proteins and the gene regulation from the macroscopic level of the interaction proteome and differential transcriptome.We successfully identified 182 new C-Raf binding proteins and 3353 genes regulated by C-Raf.We also explored the role of one of the C-Raf interacting proteins from the microscopic level in the development of tumors,and identified of enhancement role of IRS4 in the occurrence of lung cancer.Even we traced back to its expression level in human tumor samples which shown the strong individual difference of IRS4 expression in lung cancer tissues.These will provide a theoretical basis for the exploration of C-Raf's new functions and mechanism of non-small cell lung cancer;and will provide new targets for the clinical treatment researches of non-small cell lung cancer.