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. 2017 Aug;14(8):712-720.
doi: 10.1038/cmi.2015.113. Epub 2016 Mar 21.

miR-146a negatively regulates NK cell functions via STAT1 signaling

Dongqing Xu  1 , Qiuju Han  1 , Zhaohua Hou  1 , Cai Zhang  1 , Jian Zhang  1
Affiliations

miR-146a negatively regulates NK cell functions via STAT1 signaling

Dongqing Xu et al. Cell Mol Immunol. 2017 Aug.

Abstract

It is known that natural killer (NK) cell function is downregulated in chronic hepatitis B (CHB)-infected patients and in hepatic carcinoma (HCC) patients, but the mechanisms underlying this functional downregulation are largely unclear. In this study, microRNA (miR)-146a expression increased in NK cells from CHB and HCC patients compared with NK cells from healthy donors, and miR-146a levels were negatively correlated to NK cell functions. Overexpression of miR-146a reduced NK cell-mediated cytotoxicity and the production of interferon (IFN)-γ and tumor necrosis factor-α, which were reversed upon inhibition of miR-146a. In NK cells, miR-146a expression was induced by interleukin (IL)-10 and transforming growth factor-β, but reduced after treatment with interleukin-12, IFN-α and IFN-β. We further revealed that miR-146a regulated NK cell functions by targeting STAT1. Taken together, upregulated miR-146a expression, at least partially, attributes to NK cell dysfunction in CHB and HCC patients. Therefore, miR-146a may become a therapeutic target with great potential to ameliorate NK cell functions in liver disease.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
NK cell cytotoxicity is negatively associated with miR-146a expression levels. NK cells were isolated from CHB and HCC patients using a MACS NK cell isolation kit. (a) The cytotoxicity of NK cells to HepG2 cells was detected using an LDH cytotoxicity assay. HD (n=12), CHB (n=9) and HCC (n=9). (b) miR-146a expression levels were quantified by qPCR. The results were normalized to an endogenous control, RNU6B. HD (n=12), CHB (n=9) and HCC (n=13). (c) The correlation between the cytotoxicity of NK cell and the expression level of miR-146a (n=34). (d) Primary NK cells were treated with serum from CHB and HCC patients for 4 h, and then the expression of miR-146a was quantified by qPCR. Statistical significance was determined as *P<0.05; **P<0.01 compared with the control. CHB, chronic hepatitis B; HCC, hepatic carcinoma; HD, healthy donors; LDH, lactate dehydrogenase; miR, microRNA; NK, natural killer; qPCR, quantitative PCR.
Figure 2
Figure 2
miR-146a negatively regulates NK cell function. The cytolytic activity of NK92-146a-over cells (left) and NK92-146a-inh cells (right) against HepG2 cells was analyzed using a CFSE/7AAD assay. (b, c) The protein levels of molecules associated with NK cell cytolysis were analyzed by flow cytometry. (d) Primary NK cells from healthy donors were transduced with miR-146a-overexpressing or miR-146a-inhibiting lentivirus; 24 h later, the cytotoxicity of these NK cells against HepG2 cells was analyzed using an LDH cytotoxicity assay. (e) NK cells from CHB (n=6) and HCC (n=5) patients were transduced with miR-146a inhibitor lentivirus, and the cytotoxicity was detected using an LDH cytotoxicity assay at E:T=10:1. Data are representative of three independent experiments, statistical significance was determined as *P<0.05 and **P<0.01 compared with the control. CHB, chronic hepatitis B; HCC, hepatic carcinoma; LDH, lactate dehydrogenase; miR, microRNA; NK, natural killer.
Figure 3
Figure 3
miR-146a expression is regulated by cytokines associated with NK cell activation. (a) Primary NK cells were treated with IL-10 (30 ng/ml), TGF-β (30 ng/ml) and IL-6 (30 ng/ml). After 4 h, miR-146a expression was quantified by qPCR. (b) Primary NK cells were stimulated with poly(I:C) (500 IU/ml), IL-12 (20 ng/ml), IL-15 (20 ng/ml) and IFN-α (1,000 IU/ml)., , After 12 h, miR-146a expression was quantified by qPCR. Data are representative of three independent experiments, statistical significance was determined as *P<0.05 and **P<0.01 compared with the control. Ctrl, control; IL, interleukin; INF, interferon; miR, microRNA; NS, nonsignificant; NK, natural killer; TGF, transforming growth factor; qPCR, quantitative PCR.
Figure 4
Figure 4
miR-146a targets STAT1 in NK cells. The protein expression of IRAK1, TRAF6 and STAT1 in (a) miR-146a-overexpressing NK92 cells and (b) miR-146a-inhibited NK-92 cells was measured by western blot. (c) Primary NK cells were transduced with miR-146a overexpression lentivirus; 24 h later, the expression of STAT1 was detected by western blot. (d) STAT1 expression of the mixed NK cells from CHB patients (n=8) or HCC patients (n=8) was detected. Data are representative of three independent experiments, statistical significance was determined as *P<0.05 and **P<0.01 compared with the control. CHB, chronic hepatitis B; HCC, hepatic carcinoma; miR, microRNA; NS, nonsignificant; NK, natural killer.
Figure 5
Figure 5
STAT1 signaling contributes to NK cell cytotoxic activity. NK-92 cells (a) or primary NK cells (c) were treated with IFN-α (1000 IU/ml), fludarabine (50 μM; Flu) or both for 24 h; then, the cytotoxicity of these NK cells against HepG2 cells was analyzed. NK92-146a-over cells (b) or primary NK cells transduced with miR-146a overexpression lentivirus for 24 h (d) were treated with or without fludarabine, and then the cytotoxicity was detected. (e) NK cells from patients of CHB (n=10) and HCC (n=8) were transduced with has-STAT1-ORF lentivirus; 24 h later, the cytotoxicity was detected. Data are representative of three independent experiments, statistical significance was determined as *P<0.05 and ***P<0.001. CHB, chronic hepatitis B; HCC, hepatic carcinoma; INF, interferon; miR, microRNA; NK, natural killer.
Figure 6
Figure 6
miR-146a expression in NK cells from HCC or CHB patients can be reversed by cytokine-based therapy in vitro. Primary NK cells isolated from CHB and HCC patients were treated with IL-12 (20 ng/ml), IFN-α (1,000 IU/ml) or IFN-β (1,000 IU/ml) for 12 h. (a) Changes in miR-146a expression levels were detected by qPCR; CHB (n=5) and HCC (n=5). (b) CD107a expression was detected by flow cytometry; CHB (n=9) and HCC (n=7). Statistical significance was determined as *P<0.05 and **P<0.01 compared with the control. CHB, chronic hepatitis B; HCC, hepatic carcinoma; IL, interleukin; INF, interferon; miR, microRNA; NK, natural killer; qPCR, quantitative PCR.
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References

    1. Moretta A, Bottino C, Vitale M, Pende D, Cantoni C, Mingari MC et al. Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis. Annu Rev Immunol 2001; 19: 197–223. - PubMed
    1. Alli RS, Khar A. Interleukin-12 secreted by mature dendritic cells mediates activation of NK cell function. FEBS Lett 2004; 559: 71–76. - PubMed
    1. Biron CA. Interferons alpha and beta as immune regulators–a new look. Immunity 2001; 14: 661–664. - PubMed
    1. Sun C, Fu B, Gao Y, Liao X, Sun R, Tian Z, Wei H. TGF-beta1 down-regulation of NKG2D/DAP10 and 2B4/SAP expression on human NK cells contributes to HBV persistence. PLoS Pathog 2012; 8: e1002594. - PMC - PubMed
    1. Fehniger TA, Cai SF, Cao X, Bredemeyer AJ, Presti RM, Ley TJ. Acquisition of murine NK cell cytotoxicity requires the translation of a pre-existing pool of granzyme B and perforin mRNAs. Immunity 2007; 26: 798–811. - PubMed