Molecular Mechanism of the Antiproliferative Activity of Short Immunostimulating dsRNA

Abstract

Small double-stranded RNAs with certain sequence motifs are able to interact with pattern-recognition receptors and activate the innate immune system. Recently, we identified a set of short double-stranded 19-bp RNA molecules with 3-nucleotide 3'-overhangs that exhibited pronounced antiproliferative activity against cancer cells in vitro, and antitumor and antimetastatic activities in mouse models in vivo. The main objectives of this study were to identify the pattern recognition receptors that mediate the antiproliferative action of immunostimulating RNA (isRNA). Two cell lines, epidermoid carcinoma KB-3-1 cells and lung cancer A549 cells, were used in the study. These lines respond to the action of isRNA by a decrease in the growth rate, and in the case of A549 cells, also by a secretion of IL-6. Two sets of cell lines with selectively silenced genes encoding potential sensors and signal transducers of isRNA action were obtained on the basis of KB-3-1 and A549 cells. It was found that the selective silencing of PKR and RIG-I genes blocked the antiproliferative effect of isRNA, both in KB-3-1 and A549 cells, whereas the expression of MDA5 and IRF3 was not required for the antiproliferative action of isRNA. It was shown that, along with PKR and RIG-I genes, the expression of IRF3 also plays a role in isRNA mediated IL-6 synthesis in A549 cells. Thus, PKR and RIG-I sensors play a major role in the anti-proliferative signaling triggered by isRNA.

Keywords: IL-6; PKR; RIG-I; antiproliferative activity; gene silencing; immunostimulating dsRNA; pattern recognition receptors; shRNA.

Figure 1

The effect of isRNA/2X3-DOPE complexes (◦) or 2X3-DOPE alone (Δ) on the proliferation of parent KB-3-1 cells (A), and sublines KB-3-1-Scr (B), KB-3-1-MDA5 (C), KB-3-1-IRF3 (D), KB-3-1-RIG-I (E), and KB-3-1-PKR (F). After transfection, the relative number of living cells was measured every 4 h for 96 h. The number of living cells 4 h after transfection was set at 1. Experiments were performed in four repeats. The data represent means ± SD.

Figure 2

IL-6 levels in A549 cell sublines after transfection with isRNA/2X3-DOPE complexes (black bars) or 2X3-DOPE alone (gray bars) compared to untreated cells (white bars). The IL-6 level was determined by ELISA 24 h after transfection. Experiments were performed in duplicate. The data represent means ± SD.

Figure 3

The effect of isRNA/2X3-DOPE complexes (isRNA) or 2X3-DOPE alone (2X3-DOPE) on retardation of cell cycle progression of KB-3-1 cell sublines. The cell cycle distribution was analyzed 48 h after transfection. The data expressed as a relative number of cells from total population related to different cell cycle stages. Experiments were performed in two repeats. The data represent means ± SD.

Figure 4

RIG-I and PKR signaling pathways. Activation of RIG-I/PKR signaling by RNA ligands in cancer cells induces different mechanisms, leading to apoptosis, cell cycle arrest, type I interferon and inflammatory cytokine production.