【摘要】 目的:探究肺癌细胞外泌体miR-24-3p调控CD8~+T细胞抗肿瘤的功能及机制。方法:将16HBE exo、A549 exo、H522 exo、H460 exo、miR-NC exo、miR-24-3p exo与CD8~+T细胞共孵育(16HBE exo组、A549 exo组、H522 exo组、H460 exo组、miR-NC exo组、miR-24-3p exo组),与PBS共孵育组(PBS组)作为对照，CD8~+T与miR-24-3p exo共孵育后转染FGF11质粒(miR-24-3p exo+FGF11组)。CCK8法检测CD8~+T细胞的增殖能力。双荧光素酶报告基因实验验证miR-24-3p和FGF11的靶向关系。将各组CD8~+T细胞与A549细胞以20∶1的比例共孵育4 h(16HBE exo/CD8~+T组、A549 exo/CD8~+T组、H522 exo/CD8~+T组、H460 exo/CD8~+T组、miR-NC exo/CD8~+T组、miR-24-3p exo/CD8~+T组、PBS/CD8~+T组、miR-24-3p exo+FGF11/CD8~+T组),Elisa检测共孵育后细胞上清中INF-γ和IL-2的浓度，LDH法检测CD8~+T细胞对A549细胞的杀伤能力。结果:相比于PBS组，A549 exo组、H522 exo组、H460 exo组CD8~+T细胞增殖能力显著降低(P<0.05)。A549 exo/CD8~+T组、H522 exo/CD8~+T组、H460 exo/CD8~+T组细胞上清中INF-γ和IL-2的浓度显著下降(P<0.001),CD8~+T细胞杀伤能力亦显著下降(P<0.001)。双荧光素报告基因检测结果显示FGF11为miR-24-3p的靶基因。miR-24-3p exo组CD8~+T细胞增殖能力显著低于miR-NC exo组，miR-24-3p exo+FGF11组CD8~+T细胞增殖能力显著高于miR-24-3p exo组(P<0.05)。miR-24-3p exo/CD8~+T组细胞上清中INF-γ和IL-2浓度及CD8~+T细胞杀伤能力均显著低于miR-NC exo/CD8~+T组(P<0.001), miR-24-3p exo+FGF11/CD8~+T组细胞上清中INF-γ和IL-2浓度及CD8~+T细胞杀伤能力显著高于miR-24-3p exo/CD8~+T组(P<0.001)。结论:肺癌细胞外泌体miR-24-3p可通过靶向抑制FGF11而抑制CD8~+T细胞的抗肿瘤功能。更多还原

【Abstract】 Objective:To explore the function and mechanism of lung cancer cells exosomal miR-24-3p on the anti-tumor function of CD8~+T cells.Methods:16HBE exo, A549 exo, H522 exo, H460 exo, miR-NC exo, miR-24-3p exo and PBS were co-incubated with CD8~+T cells(16HBE exo group, A549 exo group, H522 exo group, H460 exo group, miR-NC exo group, miR-24-3p exo group, PBS group),CD8~+T was co-incubated with miR-24-3p exo and transfected into FGF11 plasmid(miR-24-3p exo+FGF11 group).The proliferation ability of CD8~+T cells was detected by CCK8 method.The dual-luciferase reporter gene experiment verified the targeting relationship between miR-24-3p and FGF11.The above groups of CD8~+T cells and A549 cells were co-incubated for 4 h at a ratio of 20∶1(16HBE exo/CD8~+T group, A549 exo/CD8~+T group, H522 exo/CD8~+T group, H460 exo/CD8~+T group, miR-NC exo/CD8~+T group, miR-24-3p exo/CD8~+T group, PBS/CD8~+T group, miR-24-3p exo+FGF11/CD8~+T group).Elisa was used to detect the concentrations of INF-γ and IL-2 in the supernatant, and the killing ability of CD8~+T cells in each group was detected by LDH method.Results:Compared with the PBS group, the proliferation ability of CD8~+T cells in the A549 exo group, H522 exo group and H460 exo group was significantly decreased(P<0.05).The concentrations of INF-γ and IL-2 in the supernatant of A549 exo/CD8~+T group, H522 exo/CD8~+T group and H460 exo/CD8~+T group were significantly decreased(P<0.001),and the killing ability of CD8~+T cell was also significantly decreased(P<0.001).The results of dual fluorescein reporter gene assay showed that FGF11 was the target gene of miR-24-3p.The proliferation ability of CD8~+T cells in miR-24-3p exo group was significantly lower than that in miR-NC exo group, and the proliferation ability of CD8~+T cells in miR-24-3p exo+FGF11 group was significantly higher than that in miR-24-3p exo group(P<0.05).The concentrations of INF-γ and IL-2 in the supernatant and CD8~+T cell killing ability of the miR-24-3p exo/CD8~+T group were significantly lower than those of the miR-NC exo/CD8~+T group(P<0.001),and the concentrations of INF-γ and IL-2 in the supernatant and the CD8~+T cell killing ability of the miR-24-3p exo+FGF11/CD8~+T group were significantly higher than those in miR-24-3p exo/CD8~+T group(P<0.001).Conclusion:Exosomal miR-24-3p in lung cancer cells can inhibit the anti-tumor function of CD8~+T cells by targeting FGF11.更多还原