miR-34a: a new player in the regulation of T cell function by modulation of NF-κB signaling

Abstract

NF-κB functions as modulator of T cell receptor-mediated signaling and transcriptional regulator of miR-34a. Our in silico analysis revealed that miR-34a impacts the NF-κB signalosome with miR-34a binding sites in 14 key members of the NF-κB signaling pathway. Functional analysis identified five target genes of miR-34a including PLCG1, CD3E, PIK3CB, TAB2, and NFΚBIA. Overexpression of miR-34a in CD4⁺ and CD8⁺ T cells led to a significant decrease of NFΚBIA as the most downstream cytoplasmic NF-κB member, a reduced cell surface abundance of TCRA and CD3E, and to a reduction of T cell killing capacity. Inhibition of miR-34a caused an increase of NFΚBIA, TCRA, and CD3E. Notably, activation of CD4⁺ and CD8⁺ T cells entrails a gradual increase of miR-34a. Our results lend further support to a model with miR-34a as a central NF-κB regulator in T cells.

Fig. 1. Schematic representation of reporter gene constructs.

The position of the predicted miR-34a-5p binding sites in the respective 3′UTR reporter constructs and additionally the sequences of the binding sites of miR-34a-5p in the different 3′UTRs as well as the mutated binding sites (underlined) are shown. a PLCG1-3′UTR reporter vector, b CD3E-3’UTR reporter vector, c PIK3CB-3′UTR reporter vector, d TAB2-3′UTR reporter vector, f NFΚBIA-3′UTR reporter vector

Fig. 2. Dual luciferase reporter assays of PLCG1, CD3E, PIK3CB, TAB2, NFΚBIA.

48 h after transfection of HEK 293 T cells with the indicated combinations of empty vectors, reporter gene constructs, empty expression plasmid pSG5 and miRNA-expression plasmids of miR-34a the cells were lysed and the luciferase activity was detected. The luciferase activity of the control vector experiments was set to 100%. The results represent the mean of four independent experiments carried out in duplicates. Three asterisks correspond to p < 0.001. Data are represented as mean ± SEM. a Results of dual luciferase assays with the PLCG1-3′UTR reporter plasmid (pMIR-RNL-TK-PLCG1-3′UTR). b Results of dual luciferase assays with the CD3E-3′UTR reporter plasmid (pMIR-RNL-TK-CD3E-3′UTR). c Results of dual luciferase assays with the PIK3CB-3′UTR reporter plasmid (pMIR-RNL-TK-PIK3CB-3′UTR). d Results of dual luciferase assays with the TAB2-3′UTR reporter plasmid (pMIR-RNL-TK-TAB2-3′UTR). e Results of dual luciferase assays with the NFΚBIA-3′UTR reporter plasmid (pMIR-RNL-TK-NFΚBIA-3′UTR)

Fig. 3. Regulation of the endogenous protein level of NFΚBIA by an altered miR-34a expression.

a Western blot analysis of NFΚBIA in miR-34a-transfected Jurkat cells. Jurkat cells were transfected either with allstars negative control (ANC) or miR-34a-5p mimic. 48 h after transfection the endogenous protein level of NFΚBIA was analyzed by western blotting using specific antibodies against NFΚBIA. GAPDH served as loading control. b Quantification of NFΚBIA levels in miR-34a-transfected Jurkat cells. The expression of NFΚBIA in three independent western blot experiments was quantified by densitometry using Image Lab Software. The expression of NFΚBIA was normalized to the corresponding GAPDH signals of the respective samples. Two asterisks correspond to p < 0.01. c, d Analysis of the impact of altered miR-34a levels on the NFΚBIA protein level in CD4⁺ cells. CD4⁺ cells were transfected either with ANC or miR-34a-5p mimic (c) and with inhibitor control (IC) or anti-miR-34a-5p (d). 48 h after transfection the endogenous protein level of NFΚBIA was analyzed by western blotting using specific antibodies against NFΚBIA. GAPDH served as loading control. e, f Quantification of endogenous NFΚBIA levels in CD4⁺ T cells with altered miR-34a expression. The NFΚBIA protein expression in miR-34a transfected CD4⁺ T cells (e) and in anti-miR-34a transfected CD4⁺ T cells (f) was quantified by densitometry using Image Lab Software. Three independent Western blot experiments were quantified each. The expression of NFΚBIA was normalized to the corresponding GAPDH signals of the respective samples. One asterisks correspond to p < 0.05 and two asterisks correspond to p < 0.01. Data are represented as mean ± SD

Fig. 4. Regulation of the endogenous protein level of NFΚBIA by an altered miR-34a expression in CD8⁺ T cells.

a, b Western blot analysis of the impact of altered miR-34a-levels on the endogenous NFΚBIA protein level in CD8⁺ cells. CD8⁺ T cells were transfected either with ANC or miR-34a-5p mimic (a) and with inhibitor control (IC) or anti-miR-34a-5p (b). 48 h after transfection, the endogenous protein level of NFΚBIA was analyzed by western blotting using specific antibodies against NFΚBIA. GAPDH served as loading control. c, d Quantification of endogenous NFΚBIA protein levels in CD8⁺ T cells with altered miR-34a expression. The NFΚBIA protein expression in miR-34a transfected CD8⁺ T cells (c) and in anti-miR-34a transfected CD4⁺ T cells (d) was quantified by densitometry using Image Lab Software. Three independent western blot experiments were quantified each. The expression of NFΚBIA was normalized to the corresponding GAPDH signals of the respective samples. One asterisks correspond to p < 0.05. Data are represented as mean ± SD

Fig. 5. Changes of miR-34a expression in CD4⁺ and CD8⁺ T cells altered the cell surface expression of CD3E and TCRA.

Primary CD4⁺ and CD8⁺ T cells were transfected either with nontargeting control (ANC allstars negative control) or miR-34a-5p mimic, respectively or with inhibitor control (IC) or anti-miR-34a-5p. a–d Mean fluorescence intensities of CD3E and TCRA expression, respectively from ANC-transfected (gray) or miR-34a-5p mimic-transfected (red and green, respectively) CD4⁺ or CD8⁺ T cells were analyzed. e, f FACS data of transfected primary CD4⁺ and CD8⁺ T cells were analyzed from three independent donor experiments performed in duplicates. One asterisk corresponds to p < 0.05 and two asterisks correspond to p < 0.01. Data are represented as mean ± SD

Fig. 6. Killing efficiency in miR-34a-5p overexpressing MART1-specific CD8⁺ T cell clones.

a, d Representative real-time killing assays in MART1-specific CD8⁺ T cell clones transfected with a miR-34a-5p mimic (red) or Allstars Negative Control (ANC) as control (black) using MART1 peptide-loaded T2 cells as target cells 30 h (a) and 50 h (d) after transfection. The effector cell to target cell ratio (E:T) was 2:1. b, e Target lysis at 60, 120, and 240 min analyzed in three independent experiments 30 h (b) and 50 h (e) after transfection (MART1-specific CD8⁺ T cell clones were independently expanded). c, f Quantification of maximal killing rates calculated from real-time killing kinetics (n = 3) 30 h (c) and 50 h (f) after transfection. qRT-PCR analysis of miRNA-34a expression in stimulated CD4⁺ and CD8⁺ T cells. g 7 h after activation of CD4⁺ and CD8⁺ cells from four different donors by CD2/CD3/CD28 beads the total RNA was isolated and miRNA-34a expression was analyzed by qRT-PCR using specific primers for miRNA-34a. The Fold change was calculated in reference to unstimulated medium controls