Abstract:

Objective To investigate the effect of targeting protein for targeting protein for xenopus kinesin-like protein 2 （TPX2） on immune escape in the non-small cell lung cancer A549 cells， and to clarify its mechanism. Methods The TPX2 small interfering RNA plasmid （si-TPX2） and its negative control plasmid （si-NC） were transfected into the A549 cells using the liposome transfection method； the cells were divided into blank control group， si-TPX2 group， and si-NC group. The conditioned medium （CM） from A549 cells was collected and used to treat the CD8⁺ T lymphocytes； the cells were divided into CD8⁺ T group， CD8⁺ T+CM/blank group， CD8⁺ T+CM/si-NC group， and CD8⁺ T+CM/si-TPX2 group. The transfected A549 cells were pretreated with the wingless-type MMTV integration site family（Wnt）/β-catenin signaling pathway activator CHIR-99021； the cells were divided into si-NC group， si-TPX2 group， si-NC+CHIR-99021 group， and si-TPX2+CHIR-99021 group. A co-culture system of A549 cells and CD8⁺ T lymphocytes was established using Transwell chambers； the cells were divided into A549 group， A549si-NC group， A549si-TPX2 group， A549/T group， A549si-NC/T group， A549si-TPX2/T group， A549si-NC+CHIR-99021/T group， and A549si-TPX2+CHIR-99021/T group. Cell counting kit-8（CCK-8） method was used to detect the survival rates of the cells in various groups； flow cytometry was used to detect the levels of human leukocyte antigen DR （HLA-DR）， and programmed death-ligand 1 （PD-L1） and apoptotic rate in the cells in various groups； enzyme-linked immunosorbent assay（ELISA） method was used to detect the levels of interferon-γ （IFN-γ）， tumor necrosis factor-α （TNF-α）， interleukin-2 （IL-2）， transforming growth factor-β （TGF-β）， and IL-10 in supernatant of the cells in various groups； lactate dehydrogenase （LDH） release assay was used to detect the killing effect of CD8⁺ T lymphocytes on A549 cells； real-time fluorescence quantitative PCR（PT-qPCR） method was used to detect the expression levels of TPX2 mRNA in the cells in various groups； Western blotting method was used to detect the expression level of β-catenin protein in the cells in various groups. Results Compared with CD8⁺ T+CM/blank group， the survival rate of CD8⁺ T cells， the level of HLA-DR on cell surface， and the levels of IFN-γ， TNF-α， and IL-2 in the supernatant in CD8⁺ T+CM/si-TPX2 group were significantly increased （P<0.05）. Compared with A549si-TPX2 group， the survival rate of A549 cells in A549si-TPX2/T group was significantly decreased （P<0.05）. Compared with A549/T group， the survival rate of A549 cells in A549si-TPX2/T group was further decreased （P<0.05）， while the apoptotic rate and mortality rate of A549 cells were significantly increased （P<0.05）. Compared with blank group， the expression levels of total and nuclear β-catenin protein， the level of PD-L1 on cell surface， and the levels of TGF-β and IL-10 in the supernatant of A549 cells in si-TPX2 group were significantly decreased （P<0.05）. Compared with si-TPX2 group， the level of PD-L1 on A549 cell surface and the levels of TGF-β and IL-10 in the supernatant in si-TPX2+CHIR-99021 group were significantly increased （P<0.05）. Compared with A549si-TPX2/T group， the survival rate of A549 cells in A549si-TPX2+CHIR-99021/T group was significantly increased （P<0.05）， while the apoptotic rate and mortality rate of A549 cells on A549 cells were significantly decreased （P<0.05）. Conclusion TPX2 gene silencing inhibits immune escape in non-small cell lung cancer A549 cells， and its mechanism is related to blocking the activation of the Wnt/β-catenin signaling pathway.

Key words: Lung neoplasms, Carcinoma， non-small-cell lung, Immune escape, Targeting protein for Xklp2, Wnt/β-catenin signaling pathway, Killing effect