Liu Yanyan, Sun Yingchuan
Medical College of Xuchang University, Xuchang 461000, China. *Corresponding author, E-mail:
Department of Oncology, Xuchang Central Hospital, Xuchang 461000, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2025 Aug;41(8):680-686.
Objective To investigate the regulatory effect of tumor necrosis factor-α-induced protein-8-like factor 2 (TIPE2) on the phenotype of lung cancer tumor-associated macrophages (TAM) and its influence on the stemness of lung cancer cells. Methods Mouse macrophage cell line RAW264.7 was cultured and infected with either LV-TIPE2 lentivirus or negative control LV-NC lentivirus. The TIPE2 expression in infected cells was assessed by real-time quantitative PCR (RT-qPCR) and Western blotting to verify transfection efficiency. The infected RAW264.7 cells were co-cultured with lung cancer cell line A549, and were divided into four groups: control group (RAW264.7 cells or A549 cells cultured alone), TAM group (RAW264.7 cells co-cultured with A549 cells), LV-NC group (RAW264.7 cells infected with LV-NC and co-cultured with A549 cells), LV-TIPE2 group (RAW264.7 cells infected with LV- TIPE2 and co-cultured with A549 cells). The RAW264.7 cells were collected after co-culture, and the expression of mannose receptor (CD206) protein of M2 macrophages was detected by cellular immunofluorescence staining. The proportions of M1 and M2 macrophages were detected by flow cytometry. After co-culture, A549 cells were collected, and their activity was assessed by CCK-8 assay. Self-renewal ability was evaluated using tumor cell pelleting experiment. The expression of stemness marker proteins-including cluster of differentiation 133 (CD133), transmembrane adhesion molecule (CD44), sex-determining region Y-box protein 2 (SOX2) and octamer-binding transcription factor 4 (OCT4)-was detected by Western blot. Results Compared with the control group or LV-NC group, the relative mRNA and protein expression levels of TIPE2 in RAW264.7 cells from the LV-TIPE2 group were significantly upregulated. Compared with the control group, the fluorescence intensity of M2-type macrophage marker CD206 protein in RAW264.7 cells from the TAM group was significantly increased, the proportion of M1-type macrophages was significantly decreased, and the proportion of M2-type macrophages was significantly increased. In contrast, compared with the TAM group, the fluorescence intensity of CD206 protein in RAW264.7 cells from the LV-TIPE2 group was significantly decreased, the proportion of M1-type macrophages was significantly increased, and the proportion of M2-type macrophages was significantly decreased. Compared with the control group, the proliferation activity of A549 cells in TAM group was significantly increased, the number of tumor pellet formation was significantly increased, and the relative expression levels of CD133, CD44, SOX2 and OCT4 were significantly up-regulated. However, compared with the TAM group, the proliferation activity of A549 cells from the LV-TIPE2 group was significantly decreased, the number of tumor pellet formation was significantly decreased, and the relative expression levels of CD133, CD44, SOX2 and OCT4 were significantly decreased. Conclusion TIPE2 can suppress the stemness of lung cancer cells by inhibiting the polarization of macrophages to M2-type, thereby exerting an anticancer effect.
目的 探讨肿瘤坏死因子-α诱导蛋白8样因子2（TIPE2）对肺癌肿瘤相关巨噬细胞（TAM）表型的调控作用及其对肺癌细胞干性的影响。方法 培养小鼠巨噬细胞系RAW264.7，分别用LV-TIPE2慢病毒或阴性对照LV-NC慢病毒感染。通过实时定量PCR（RT-qPCR）和蛋白质印迹法评估感染细胞中TIPE2的表达，以验证转染效率。将感染后的RAW264.7细胞与肺癌细胞系A549共培养，分为四组：对照组（单独培养的RAW264.7细胞或A549细胞）、TAM组（RAW264.7细胞与A549细胞共培养）、LV-NC组（感染LV-NC的RAW264.7细胞与A549细胞共培养）、LV-TIPE2组（感染LV-TIPE2的RAW264.7细胞与A549细胞共培养）。共培养后收集RAW264.7细胞，通过细胞免疫荧光染色检测M2巨噬细胞甘露糖受体（CD206）蛋白的表达。采用流式细胞术检测M1和M2巨噬细胞的比例。共培养后收集A549细胞，通过CCK-8法评估其活性。采用肿瘤细胞成球实验评估自我更新能力。通过蛋白质印迹法检测干性标志物蛋白（包括分化簇133（CD133）、跨膜黏附分子（CD44）、性别决定区Y框蛋白2（SOX2）和八聚体结合转录因子4（OCT4））的表达。结果 与对照组或LV-NC组相比，LV-TIPE2组RAW264.7细胞中TIPE2的相对mRNA和蛋白表达水平显著上调。与对照组相比，TAM组RAW264.7细胞中M2型巨噬细胞标志物CD206蛋白的荧光强度显著增加，M1型巨噬细胞比例显著降低，M2型巨噬细胞比例显著增加。相反，与TAM组相比，LV-TIPE2组RAW264.7细胞中CD206蛋白的荧光强度显著降低，M1型巨噬细胞比例显著增加，M2型巨噬细胞比例显著降低。与对照组相比，TAM组A549细胞的增殖活性显著增加，肿瘤球形成数量显著增加，CD133、CD44、SOX2和OCT4的相对表达水平显著上调。然而，与TAM组相比，LV-TIPE2组A549细胞的增殖活性显著降低，肿瘤球形成数量显著减少，CD133、CD44、SOX2和OCT4的相对表达水平显著降低。结论 TIPE2可通过抑制巨噬细胞向M2型极化来抑制肺癌细胞的干性，从而发挥抗癌作用。
