The MicroRNA-106a/20b Strongly Enhances the Antitumour Immune Responses of Dendritic Cells Pulsed with Glioma Stem Cells by Targeting STAT3

Abstract

Background: Evaluate the effect of the miRNA-106a/20b on the efficacy of DCs pulsed with GSCs in activating GSC-specific T cell responses.

Methods: We cultured GSCs and prepared GSC antigen lysates by apoptosis. Then, immature DCs were pulsed with GSC antigen lysates in vitro. STAT3 levels in DCs were assessed by Western blotting, and the expression of CD80, CD86, and MHC-II was tested by fluorescence-activated cell sorting. The production and secretion of the cytokines IL-6, IL-12, TNF-α, and IL-10 in DCs induced by GSCs were determined by enzyme-linked immunosorbent assay. Finally, the cytotoxic functions of T cells stimulated by GSC-DC fusion cells transfected with a miR-106a/20b mimic in vitro and the antitumour activity in vivo were detected.

Results: We found that the levels of miR-106a/20b were downregulated, but the expression of STAT3 was significantly upregulated. Simultaneously, the inhibition of STAT3 in the fusion cells by STAT3-specific siRNA caused significant upregulation of the expression of CD80, CD86, and MHC-II, and the secretion of the cytokines IL-6 and IL-12 was substantially increased, IL-10 was markedly decreased. These findings revealed that STAT3 is an important regulator of DC maturation. Furthermore, the interactional binding sites between the 3'-untranslated region (3'-UTR) of STAT3 mRNA and miR-106a/20b were predicted by bioinformatics and verified by a dual-luciferase assay. Moreover, the reduction in STAT3 levels in GSC-DCs enhanced the generation of CD8+ T cells and reduced the generation of Foxp3+ regulatory T cells. Meanwhile, the secretion of the T cell cytokine IFN-γ was significantly increased. Further research showed that DCs after miR-106a/20b-mimics transfection could promote the inhibition of GSC proliferation by T cells in vitro and suppress tumour growth in vivo.

Conclusions: This study indicted that the miR-106a/20b activation could be one of the important molecular mechanisms leading to enhance antitumour immune responses of GSC-mediated DCs, which downregulated the expression of STAT3 to alleviate its the inhibitory effect.

Figure 1

Characterization of GL261-induced cancer stem cells. (a) Tumour spheres expressing CD133 and DAPI. (b) After 3 days of culture in FBS-containing medium, spheres began to spread into adherent monolayers cells. (c) The results of flow cytometry of tumour spheres at the third passage. Red and blue profiles, respectively, represent test group and negative control; number indicates percentage of CD133+ cells. (d) The apoptosis rate was 54.3% + 6.4% before FACS, and 59.1% + 36.8% after FACS (Q3 and Q4).

Figure 2

Dendritic cell isolation and characterization. (a and b) The morphology of the immature DCs and mature DCs (200x magnification). (c) The expression of DC markers before and after maturation. The green lines represent the expression of LPS-induced DCs receptors; the red lines represent negative controls.

Figure 3

The expression of STAT3 and its prognostic value in glioma. (a) The expression of STAT3 was distinctly increased in GBM and LGG specimens compared with nontumour specimens based on TCGA datasets. (b) Patients with high STAT3 expression showed a shorter OS and PFS than those with low STAT3 expression in patients with GBM and LGG based on TCGA datasets.∗∗∗∗p < 0.0001.

Figure 4

The expressions of DC cell surface molecules (CD80, CD86, and MHC-II) as determined by flow cytometric analysis. (a) DCs were transfected with 500 nM nonspecific siRNA (NC-siRNA) or 100 nM, 300 nM, or 500 nM siRNA targeted against STAT3 (STAT3 siRNA). (b and c) Flow cytometry analysis of changes in the expression of costimulatory molecules and mature molecules on the cell surface of different DC groups. (d) The expression of STAT3 in each DC groups by Western blotting. (e) The cytokine secretion levels of IL-12, IL-6, TNF-α, and IL-10 in the supernatants were measured in the four groups by ELISA. The levels of IL-12 and IL-6 were significantly increased in the si-STAT3+GSC group compared with the DC+GSC group, and the level of IL-10 showed the opposite trend. Molecular weight: STAT3(88 kDa) and GAPDH(36kD). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 5

Isolation, differentiation, and cytokine secretion of T-lymphocytes. (a) The image of T cells (100x) and the expression of T cell marker CD3-FITC before and after magnetic-activated cell sorting (MACS). Numbers indicate percentages of positive cells. (b and d) CD8+ T-cell generation in the STAT3 deficiency DC group was significantly increased. (c and e). Foxp3+ CD4+ T-cell generation in the STAT3 deficiency DC group had evaluated less than other groups. (f) ELISA results showed that secretion of INF-γ by T-lymphocytes stimulated with STAT3-null DCs pulsed with GSCs was upregulated compared with that by T-lymphocytes stimulated with DCs transfected with GSCs (1.63-fold). Bars represent the mean ± SD. Comparison with the T-lymphocytes group stimulated with DCs loaded GSCs. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 6

Identification of miR-106a/20b, which regulates STAT3 expressions. (a) The predicted consequential pairing between the miRNA (miR-106a and miR-20b) and STAT3 3′-UTR; (b and c) Relative luciferase activity upon cotransfection of the empty reporter (vehicle) with pmiR-RB-STAT3-WT or pmiR-RB-STAT3-MUT and the control miRNA, miR-106a, or miR-20b mimic. ^∗∗∗p < 0.001.

Figure 7

Relative expression levels of miR-106a, miR-20b, and STAT3 mRNA in DCs after loading with GSCs. (a) miR-106a/20b expression was determined in the NC group and after 24 and 48 h of GSC stimulation by real-time PCR. (b) STAT3 mRNA expression was determined in the NC group and after 24 and 48 h of GSC stimulation by Western blotting. (c) Relative expression levels after miR-106a/20b mimic or inhibitor transfection in DCs. (d) RT-PCR of STAT3 expressions in each DC-transfected group after 12 h of GSC stimulation. (e and f) Western blotting analysis of STAT3 protein expression in each DC-transfected group. ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 8

The effect of miR-106a/20b levels on the maturation of DCs and the cytotoxicity of induced T-lymphocytes against GSCs in each group. (a) The expressions of CD80, CD86, and MHC-II in the miR-106a/20b mimic group was distinctly higher than that in the control DC group by Flow cytometry analysis. (b) LDH release assay was applied for the detection of the cytotoxicity of T cells in the miR-106a/20b mimic group; substantially higher cytotoxicity against GSCs relative to the control group at different E/T ratios was observed. (c) GSC proliferation was determined by EdU staining. ^∗∗p < 0.01.

Figure 9

miR-106a/20b mimics increased the antitumour characteristics of DCs and inhibited tumour growth in an immunocompetent syngeneic GSC model. (a) Treatment scheme for GSC-implanted mice. (b) Representative BLI images showing the final tumour volume; (c and d) Measurements of tumour volume and weight. (e) Immunohistochemistry of tumour sections stained with Ki-67 antibodies shows cellular proliferation of the tumour cells.(100x; the arrows indicate the Ki67-positive cells.). CD8+ T cell infiltration into the tumour as determined by IHC (arrows indicate the CD8-positive cells). (The red boxes indicate the details at higher magnification (200x).) ^∗∗∗p < 0.001.