PNA-based microRNA inhibitors elicit anti-inflammatory effects in microglia cells

Abstract

Peptide nucleic acid (PNA) inhibitors of miR-221-3p (CU-PNA-221) and miR-466l-3p (CU-PNA-466) demonstrated changes in inflammatory responses. Suppression of inflammatory signalling was unexpected and further investigation led to the identification of calmodulin as a novel target of miRNA-466l-3p. These studies demonstrate that exogenous agents may suppress neuroinflammation mediated by microglial cells.

Fig. 1

PNA inhibitors and their effects on BV-2 microglia cells as analysed by quantitative real time polymerase chain reaction (qPCR). (A) The structure of a PNA miRNA inhibitor. The cell penetrating peptide (CPP, in green) used to facilitate passage across the cell plasma membranes. The PEG spacer (in red) separates the CPP from the PNA subunit (in blue). The PNA subunit presents complementary sequences for the miRNA of interest. (B) The sequences of the four PNA miRNA inhibitors used in this work. These represent the PNA sequence of the repeated subunit represented in Fig. 1A. (C) Left: The effects of CUPNA-221 upon TNF and iNOS mRNA 2 and 6 hours respectively, after a 400 ng ml⁻¹ LPS challenge. Right: The effects of CU-PNA-466 upon IL-10 and iNOS mRNA 2 and 6 hours after an LPS challenge. Both these graphs are presented on a log scale with P-values represented as follows *<0.025, **<0.010 and, ***<0.005.

Fig. 2

Changes in release of NO from BV-2 microglia cells upon miRNA modulation. The change in NO release levels from BV-2 cells under LPS stimulation (200, 400 and 800 ng ml⁻¹) with miR-221-3p or miR-466l-3p modulations. These signals are normalised to the signal from cells treated with no LPS and are all taken after a 24 hour incubation. P-values represented as follows *<0.025, **<0.010 and, ***<0.005.