TAMs (Tumor Associated Macrophages) Promote The Progression Of Lung Cancer Through NF-?B/PP2Ac/COL6A1 Pathway

Part ?: The role of TAMs in the progression of lung cancer Objective:To verify whether TAMs could promote the progression of lung cancerMethods:The expression of CD163 and Ki67 in 339 lung cancer patients was detected with immunohistochemistry;The correlation between the expression of CD163 and Ki67 was assessed;Chronic pneumonia model and orthotopic xenograft nude mouse model were performed in vivo to investigate the influence of TAMs on lung cancer growth;MTT was used in vitro to investigate the influence of TAMs on lung cancer growth;Wound healing assay was applied in vitro to investigate the influence of TAMs on lung cancer migration;Immunofluorescence was performed in vitro to investigate the influence of TAMs on the process of EMT in lung cancer.Results:The positive correlation between the expression of CD163 and Ki67 was detected;TAMs colud promote the growth,migration and the procession of EMT of lung cancer.Conclusion:TAMs could promote the progression of lung cancer.Part ?: The prediction of the potential key genes involved in the procession of TAMs promoting lung cancer progressionObjective:To verify the key genes involved in the procession of TAMs promoting lung cancer progression.Methods:GEO database was used to analyze the genes involved in the procession of TAMs promoting lung cancer progression;R language was applied to draw the heat maps of differentially expressed genes;GO analysis was performed to annotate and visualize differentially expressed genes;KEGG pathway was exploited to analyze the pathways which the differentially expressed genes was enriched in;STRING was used to establish protein-protein interaction(PPI)network of differentially expressed genes.Results:With GEO database,176 differential expressed genes were found between MCM-treated group and control group;With GO term analysis,these differential expressed genes were rich in biological functions including extracellular matrix,angiogenesis,protein integration,etc;With KEGG pathway database,these differential expressed genes were involved in many signaling pathways,including ECM-receptor interaction,P13K-Akt,TNF,TGF-beta,MAPK,NF-?B,p53;Protein–protein interactions(PPI)made by cytoscape software predicted that PP2Ac?,POLR2 F,POLR2D and RBBP4 were obviously down-regulated,wheras PTGS2,PDGFB were obviously up-regulated.Conclusion:TAMs promoting lung cancer progression might be dependent on the down-regulation of the expression of PP2 Ac.Part ?:The investigation of the potential key genes involved in the procession of TAMs promoting lung cancer progression Objective:To verify whether PP2 Ac was the key gene of TAMs promoting the progression of lung cancerMethods:Immunohistochemistry was performed to investigate the expression of PP2 Ac in lung cancer patients and nude mice;The relationship between the expression of CD163 and PP2 Ac was analyzed;Western blot was applied to investigate the expression of PP2Ac;The overexpression PP2Ac? cell line was established through Tet-ON regulation system.;The influence of the expression of PP2 Ac on the growth of lung cancer was investigated with subcutaneous xenograft nude mice;Wound healing assay was used to investigate the influence of the expression of PP2 Ac on lung cancer migration;Immunofluorescence was applied to investigate the influence of the expression of PP2 Ac on the process of EMT in lung cancer.Results:The negative correlation between the expression of PP2 Ac and CD163 was detected;The expression of PP2 Ac in MCM-treated group was lower than that in the control group;The tumours in the overexpression PP2Ac? group were significantly smaller than those in the control group;The overexpression of PP2 Ac could impair the ability of TAMs promoting the migration and the procession of EMT of lung cancer cells.Conclusion:TAMs promoting the lung cancer progression was dependent on the down-regulation of the expression of PP2 Ac.Part ?: The key pathway invovled in the procession of TAMs promoting lung cancer progressionObjective:To investigate the key pathway of TAMs promoting the progression of lung cancer through down-regulation of PP2 AcMethods:Immunohistochemistry was applied to investigate the expression of CD163 and p-IKK in lung cancer;The association between CD163 and p-IKK was assesed;Western blot was performed to investigate the expression of p-IKK and IKK;DN-IKK?(S176/180A)and DN-I?B?(S32/36A)cell lines were established through Tet-ON regulation system.Results:The positive correlation between the expression of CD163 and p-IKK was found;The level of p-IKK and IKK in MCM-treated group was higher;The expression of PP2 Ac was lower in the DN-IKK?(S176/180A)?DN-I?B?(S32/36A)groups.Conclusion:TAMs could activate NF-?B pathway and down-regulate the expression of PP2 Ac in an NF-?B pathway dependent manner.Part ?: The key downstream genes affected by TAMs activating NF-?B/PP2 Ac pathwayObjective:To investigate the downstream target genes which could be affected after activation of NF-?B/PP2 Ac pathwayMethods:Bioinformatics were used to predict the potential downstream target genes of TAMs activating NF-?B/PP2 Ac pathway;Real Time-PCR was performed to investigate these genes;Kaplan-Meier plotter was applied to evaluate the relationship between these genes and the overall survival(OS)in lung cancer patients,respectively.Results:CXCL1 and COL6A1 were the potential target genes of TAMs activating NF-?B / PP2 Ac pathway;Down-regulating the expression of PP2 Ac could up-regulate the expression of CXCL1 through ERK,NF-?B,and SRC pathways,wheras up-regulate the expression of COL6A1 through NF-?B pathway;The expression of COL6A1 was negatively correlated with the OS in lung cancer patients.Conclusion:TAMs could activate the NF-?B/PP2 Ac pathway to up-regulate the expression of downstream target gene COL6A1,which is associated with poor prognosis of lung cancer.