| Objective:To determine the differential expressions of NR3C1(nuclear receptor subfamily 3group C member 1)among different types of macrophages derived from THP-1(Human monocytic leukemic cell line)and its influence on the polarization direction of macrophages.To explore the gastric cancer cells effect of proliferation and migration with NR3C1 expression in macrophages,and provide new perspectives and theoretical support for the diagnosis and treatment of gastric cancer.Methods:1.NR3C1 was identified as the study objection through literature review.THP-1differentiation into different types of macrophages induced by Phorbol 12-Myristate13-Acetate(PMA),Lipopolysaccharide(LPS),Interferon-γ(IFN-γ),Interleukin 4(IL-4).Quantitative real time polymerase chain reaction(q RT-PCR)and Western Blot techniques were used to detect the differences in m RNA and protein expression of NR3C1 in different macrophages.2.The construction of knockdown and overexpression models in THP-1-derived macrophages were used by small interfering RNA(si RNA)and overexpressed plasmids in vitro,and q RT-PCR and Western Blot techniques were used to detect the effect of NR3C1 on the polarization of macrophages.3.The construction of co-culture models between macrophages and gastric cancer cells in vitro.The effect of proliferation of gastric cancer cell were detected by Cell Counting Kit-8 assay(CCK8),cell clone formation assay and cell cycle assay with inhibition and overexpression of NR3C1 in THP-1-derived macrophages.Scratch healing assay and Transwell assay were detected the migration ability of gastric cancer cells.The effect of NR3C1 expression in macrophages on gastric cancer related proliferation and epithelial-mesenchymal transition related protein expression were detected by Western Blot.Results:1.q RT-PCR results indicated that the inducer successfully induced different differentiated types of macrophages from THP-1.q RT-PCR and Western Blot results showed that the relative expression level of NR3C1 in M1 macrophages was lower,while that in M2 macrophages was higher.2.The results of q RT-PCR and Western Blot experiments revealed that NR3C1 could promote the M2 type polarization of THP-1-derived macrophages and inhibit the M1 type polarization.3.The results of CCK8,cell clone formation assay and cell cycle assay,scratch healing experiment and Transwell experiment displayed that the inhibition of NR3C1 in macrophages could reduce the proliferation and migration activity of gastric cancer cells,and promote cell apoptosis.Western Blot experiment showed that the expression of PCNA and Bcl-2 protein were decreased and the expression of BAX protein was increased through the co-culture of macrophages with NR3C1 inhibition.Epithelial markers E-cadherin protein expression was increased,and mesenchymal markers N-cadherin and Vimentin protein expression were decreased,suggesting that macrophages NR3C1 Knockdown could weaken the proliferation and migration of gastric cancer cells.4.Functional experiment results showed that macrophages with overexpressing NR3C1 could strengthen the proliferation and migration activity of gastric cancer cells,and enhance the role of M2 type macrophages in promoting the proliferation and migration of gastric cancer cells.Western Blot experiments indicated that macrophages with NR3C1 overexpression could up-regulate proliferation and expression of mesenchyme-related proteins,reducing apoptosis and expression of epithelial-related proteins.Conclusion:NR3C1 was relatively higher in M2 macrophages.It promoted the polarization of THP-1-derived macrophages towards M2 macrophages,thus enhanced the proliferation and migration of gastric cancer cells,inhibiting cell apoptosis,and further strengthened the role of M2 macrophages to gastric cancer cells on tumor promotion effect. |
PDF Full Text Request