Fig. 2
The miR-23a/27a/24 − 2 cluster inhibits T cell mediated immune response in NSCLC. (A) The concentration of IFN-γ in the culture medium after co-culturing CD8+ T cells and NSCLC cells in a 10:1 ratio for 12 h. (B) CD8+ T cell-induced NSCLC cells death was measured by flow cytometry. Indicated NSCLC cells were transfected with vector or miR-23a/27a/24 − 2 cluster, and then co-cultured with activated CD8+ T cells at 1:20 ratio for 6 h. (C) Relative migration of human CD8+ T cells incubated with culture medium supernatant from H1299 or H1650 cells that transfected with vectors or miR-23a/27a/24 − 2 cluster expressing constructs. (D) The growth and weight of subcutaneous tumors in C57BL/6J mouse xenograft model constructed using LLC cells transfected with vectors or miR-23a/27a/24 − 2 cluster expressing constructs. (E) Representative immunofluorescence (IF) image of CD8+ T cells, and the number of infiltrating CD8+ T cells in C57BL/6J xenograft tumors (100 × 100 μm square area). Tumor tissues from (D) (scale bar: 50 μm) and green is CD8+ T cells. (F) IFN-γ concentration in tumor tissues of C57BL/6J xenograft model. Tumor tissues from (D). (G) The growth and weight of subcutaneous tumors in C57BL/6J mouse xenograft model that constructed using LLC cells transfected with scramble or miR-23a/27a/24 − 2 cluster shRNA expressing constructs. (H) Representative IF images of CD8+ T cells and the number of infiltrating CD8+ T cells (100 × 100 μm square area) in C57BL/6J xenograft tumor tissues. Tumor tissues from (G) and green is CD8+ T cells (scale bar: 50 μm). (I) IFN-γ concentration in C57BL/6J xenograft tumor tissues. Tumor tissues from (G). (J) The growth and weight of subcutaneous tumors in SCID mouse xenograft models that treated with PBMCs. Xenograft models were constructed in SCID mouse using H1299 cells transfected with scramble or miR-23a/27a/24 − 2 cluster shRNA expressing constructs. (K) Representative IF images of CD8+ T cells and the number of infiltrating CD8+ T cells (100 × 100 μm square area) in SCID xenograft tumor tissues. Tumor tissues from (J) (scale bar: 50 μm) and red is CD8+ T cells. (L) The growth and weight of subcutaneous tumors in SCID mouse xenograft models without PBMCs treatment. Xenograft models were constructed in SCID mouse using H1299 cells transfected with scramble or miR-23a/27a/24 − 2 cluster shRNA expressing constructs. (M) Tumor inhibition rate of miR-23a/27a/24 − 2 cluster silencing on SCID xenograft models with or without PBMCs treatment. Tumor growth data from (J) and (L). In animal experiments, each group consists of 10 mice. In vitro experiments were conducted in three independent experiments. Data shown are means ± SD. p value was calculated by t test. *, p < 0.05 compared to vector or scramble control; **, p < 0.01 compared to vector or scramble control; ***, p < 0.001 compared to vector or scramble control. LLC, Lewis lung cancer; IFN-γ, interferon-γ; miR-cluster, miR-23a/27a/24 − 2 cluster; sh miR-cluster, shRNA of miR-23a/27a/24 − 2 cluster; PBMCs, peripheral blood mononuclear cells