Decreased Dicer expression elicits DNA damage and up-regulation of MICA and MICB

Abstract

RNA interference (RNAi) acts constitutively to silence the innate immune response, and innate immunity genes are misregulated in Dicer-deficient Caenorhabditis elegans. Here, we show that inhibition of Dicer expression by RNAi in human cells up-regulates major histocompatibility complex class I-related molecules A and B (MICA and MICB). MICA and MICB are innate immune system ligands for the NKG2D receptor expressed by natural killer cells and activated CD8(+)T cells. We reveal that knockdown of Dicer elicits DNA damage. Up-regulation of MICA and MICB by Dicer knockdown is prevented by pharmacologic or genetic inhibition of DNA damage pathway components, including ataxia telangiectasia mutated (ATM) kinase, ATM- and Rad3-related kinase, or checkpoint kinase 1. Therefore we conclude that up-regulation of MICA and MICB is the result of DNA damage response activation caused by Dicer knockdown. Our results suggest that RNAi is indirectly linked to the human innate immune system via the DNA damage pathway.

Figure 1.

DNA damage accumulation in Dicer knockdown cells. (A) DNA damage assayed by immunostaining for γ-H2AX and RPA70. Mean ± SD indicates the fraction of γ-H2AX– or RPA-positive cells in mock-transfected (M), control siRNA-transfected (C), Dicer siRNA1-transfected (D1), and Dicer siRNA2-transfected (D2) cells. Bar, 10 μM. (B) Immunoblot analysis revealed an increase of Chk1 phosphorylation on S345 in Dicer knockdown cells. (C, top) Representative comet assay showing formation of DNA strand breaks (formation of a “comet tail”). (bottom) Mean ± SD indicates the fraction of cells containing a comet tail. (D) Levels of DNA damage–induced transcripts determined by real-time RT-PCR. (E) Transcription of DNA damage–induced genes assayed by RNAPol-ChIP. Data in D and E represent means ± SD from three independent experiments.

Figure 2.

Up-regulation of MICA and MICB in Dicer knockdown cells. (A) Up-regulation of MICA and MICB mRNAs determined by real-time RT-PCR. Cells treated with 4 μM aphidicolin for 16 h were used as positive control. (B) Transcription of MICA and MICB was monitored by RNAPol-ChIP. Data in A and B represent means ± SD from three independent experiments. (C) Cell surface expression of MICA and MICB measured by FACS. Dicer siRNA1-transfected (red) and Dicer siRNA2-transfected (blue) cells were compared with mock-transfected (black) and control siRNA-transfected (green) cells. Filled histograms indicate isotype control antibody staining. The x axis depicts staining intensity and the y axis depicts the relative number of cells. (D) Knockdown of Dicer sensitizes HepG2 and HEK293T cells to lysis by NKL cells. NKL cells were incubated with Dicer knockdown (squares) or control siRNA-transfected (circles) cells in the absence (closed) or presence (open) of anti-NKG2D antibody. The effector to target ratio (E:T) is indicated. Data represent means ± SD from three independent experiments.

Figure 3.

Inhibition of DNA damage response prevents MICA and MICB up-regulation in Dicer knockdown cells. Dicer knockdown-induced MICA and MICB up-regulation was prevented by incubation with caffeine (C) or staurosporine (S) and by cotransfection with ATR siRNA (R), ATM siRNA (M), or Chk1 siRNA (K). MICA and MICB mRNA levels were determined by real time RT-PCR, and were normalized to these in mock-transfected cells, which are defined as 1. Data represent means ± SD from three independent experiments.

Figure 4.

DNA damage progresses in parallel with the kinetics of up-regulation of MICA and MICB in Dicer knockdown cells. Cells were transfected with Dicer siRNAs. (A) γ-H2AX (▪) and RPA70 (•) staining was performed at the indicated time points. (B) Levels of MICA (▪), MICB (•), and Dicer (broken lines) transcripts were measured by real time RT-PCR. Means ± SD of three independent experiments are shown.