| Objective: To explore the effects of cigarette smoke(CS)on the function and expression profile of mices neutrophils in bone marrow,peripheral blood and tumor.Methods: The experimental mices were divided into 4 groups,tumor or without-tumor groups exposed in smoke or no-smoke condition,10/group,and the group which was neither tumor or smoke-exposed was control.MB49 cells(mice bladder cancer cell line)were inoculated on 4 to 5-week-old homologous C57BL/6J female mices.Exposure group received exposure to cigarettes smoke(CS)2 weeks before tumor inoculation,1 time / d for 4 weeks;no CS group was exposed to air as control.Simultaneously,we extracted the PMNs of blood,bone marrow and tumor of mices,and then we did the following: 1.To explore the effect of cigarette smoke on the initiation and development of bladder situ tumor.2.RNA-seq sequencing: to compare the differences of PNMs in different parts between CS exposure group and non-CS one;to analyze the clustering,function and pathway of different genes;to research the effect of smoking on the expression of PMN in tumor-bearing mice and the tumor factors.We filtrated the differentially expressed genes with significant 2-fold change,and design primes for real time RT-PCR to analyze the gene expression,and validate the PMN of blood,bone marrow and tumor of mices.After comprehensive comparison,we conducted verification preliminarily about meaningful functional genes expressed in transcription levelResult: 1.There were 3 cases of lung metastasis in cigarette smoke exposure group,1 case of lung metastasis with liver metastasis.There was just 1 case of lung metastasis in non-exposed tumor group.The tumor weight of smoke and tumor group was significantly higher than that of no-smoke and tumor group(P <0.05).The spleen of the tumor group was significantly larger than that of the non-tumor group(P <0.05).The pathology showed congestion and proliferation.The weight of no-tumor and smoke group was significantly lower than that of no-tumor and no-smoke group(P <0.05).And the weight of spleen was significantly decreased(P <0.05).The pathological showed splenic corpuscle atrophy.2.The expression of PMNs by the expression spectrum and the differential gene analysis showed that smoking and tumor factors from neutrophils began to affect the expression of neutrophils in the development of bone marrow,mainly Tumor-related signaling pathway,Glutathione metabolic pathway,Cytokine-cytokine receptor interaction signaling pathway,Chemokine signaling pathway signaling pathway and Focal adhesion signaling pathway play an important role in the immune response of cigarette-stimulated neutrophils.The expression of MMP9?Bv8?VEGF?CCL2?TRAIL?MMP8 in the pathways were significantly up-regulated or down-regulated by Real time RT-PCR tested..Conclusion: Smoke can promote the growth and progression of tumor in mice.Cigarette exposure can promote the growth and development of bone marrow neutrophils,which affect the expression of neutrophils in peripheral blood or tumor with their movement,chemotaxis,oxidative burst,adhesion as well as to promote tumor immune-related genes.Smoke not only directly affects the pathways associated with the tumor,but also together with tumor factors play a role on the tumor progression-related genes and signaling pathways.Effects of smoke and tumor on neutrophils can be in both developmental and maturation stages and functional stages.That may be an important factor in the regulation of tumor-associated neutrophils change from N1 anti-tumor phenotype to N2 pro-tumor phenotype. |
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