Study On The Effect And Mechanism Of Tumor-associated Macrophages On The Biological Behavior Of Cervical Cancer Cells

Objective:Tumor-associated macrophages（TAMs）are often shown as M2 phenotypes,involved in tumor growth,progress and treatment resistance processes,which is a potential therapeutic target for tumors.The purpose of this study is to explore the influence of TAMs from THP-1 on the biological characteristic in cervical cancer SiHa cells,in order to provide new method for improving the diagnosis,treatment and prognosis of the clinical patients.Methods:1.THP-1 cells were induced by Phorbol myristate acetate（PMA）and Interleukin-4（IL-4）as TAMs.The expression of CD204 and CD206 on their surface were determined by flow cytometry.2.The co-culture model of TAMs-SiHa cells was established.The morphological and structural changes of SiHa cell were compared.3.MTT assay was used to detect the proliferation of SiHa cells after treating with TAMs culture supernatant at different time points.4.EdU assay was used to detect the proliferation of individual SiHa cells.5.JC-1 assay was used to detect the changes of mitochondrial membrane potential of SiHa cells.6.Wound Healing and Transwell test were used to detect the abilities of migration and invasion of SiHa cells.7.Western blot analysis the expression of biological behavior-related proteins of SiHa cells.8.RT-PCR was used to detect the mRNA expression levels of transcription factor Snail of SiHa cells.9.Statistical analyses were performed using GraphPad Prism（Version 7）statistical software.The experimental results were presented as the mean±standard deviation.The Student t test was used to determine significance of changes between two groups.The comparison between two rates was analyzed by the chi-square test.Values of p<0.05 were considered to be statistically significant.Results:1.Compared with the negative control,the expression of CD204 and CD206 on the surface of cells induced by PMA and IL-4 was significantly increased（p<0.05）.The results suggest that the induction of TAMs was successful.2.SiHa cells co-cultured with TAMs showed remarkable morphological changes that their were obscure boundary,disordered arrangement,decreased intercellular fusion degree and obvious interstitial cell morphology.3.The proliferation activity of the SiHa cells in the co-culture group were remarkablely rised,and the content of the cell proliferation-related proteins CDK4 and CyclinD1 was significantly increased（p<0.05）.4.The apoptosis of SiHa cells in the co-culture group were inhibited.The content of pro-apoptotic protein Bax was significantly decreased,and the content of anti-apoptotic protein Bcl-2 was significantly increased（p<0.05）.5.Both the migratory ability and the invasive ability of SiHa cells in the co-culture group were remarkablely enhanced.The protein content of E-cadherin remarkably decreased.The proteins expression levels of N-cadherin,Vimentin and the mRNA expression levels of the transcription factor Snail increased（p<0.05）.6.The proteins content of p-AKT^ser473、p-GSK-3β^ser9 of SiHa cells in the co-culture group were significantly increased.The proteins expression levels of p-AKT^ser473、p-GSK-3β^ser9 of SiHa cells in the inhibitor group（LY294002）were significantly decreased compared with the co-culture group.The inhibition of proteins expression levels of p-AKT^ser473er473 and p-GSK-3β^ser9 by LY294002 can be partially reversed by TAMs.The proteins expression levels of total AKT and total GSK-3βin each group did not change significantly.Conclusion:1.TAMs can accelerate the proliferation and postpone the apoptosis of SiHa cells.This phenomenon may be related to the regulation of the expression of CDK4,CyclinD1,Bax and Bcl-2 proteins.2.TAMs can promote the migration and invasion of SiHa cells.This phenomenon may be caused by the activation of AKT/GSK-3β/Snail signaling pathway to accelerate the process of epithelial-mesenchymal transition of SiHa cells,thus promoting the invasion and metastasis of SiHa cells.