Methylglyoxal-induced miR-223 suppresses rat vascular KATP channel activity by downregulating Kir6.1 mRNA in carbonyl stress

Abstract The vascular ATP-sensitive K+ (KATP) channel composed of Kir6.1 and SUR2B subunits regulates cellular activity by coupling intermediary metabolism to membrane excitability. Our previous studies have shown that both Kir6.1 and SUB2B are post-transcriptionally downregulated by methylglyoxal (MGO) which is a reactive carbonyl specie and can cause disruption of vascular tone regulation under diabetic conditions. We have shown that the SUB2B downregulation is mediated by the microRNA (miR) miR-9a, while the mechanism underlying Kir6.1 inhibition is still unclear. Studying the microRNA databases, we found that miR-223 has sequence similarities to the 3′ untranslated sequence (3’UTR) of Kir6.1 mRNA suggesting their potential interactions. Therefore, we explored the role of miR-233 in KATP channel regulation by up/down-regulation of miR-223 in smooth muscle cells (SMCs) and mesenteric arterials. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis showed augmentation of miR-223 expression in the cultured SMCs after 300 μM MGO exposure by 5–6 folds. miR-223 overexpression down-regulated Kir6.1 mRNA levels by ~2.6 times while miR-223 knockdown diminished the effect of 300 μM MGO by ~50% in the SMCs. Luciferase assay and mutagenesis studies showed that the effect of miR-223 was abolished when the potential interaction site in the 3’ UTR was mutated. Studies with Western blot, patch clamp, and perfused mesenteric arterial rings showed that transfection of miR-223 downregulated KATP protein expression, inhibited KATP channel activity and enhanced vasoconstriction. These results therefore suggest that miR-223 is induced by MGO exposure, which subsequently downregulates the Kir6.1 mRNA, suppresses KATP channel function, and impairs functional regulation of vascular tones. Background Methylglyoxal causes transcriptional inhibition of the vascular KATP channel. Results Exogenous miR-223 down-regulated Kir6.1. miR-223 knockdown alleviated the effect of MGO. Conclusion Vascular KATP channel is important for miR-223 targeting. Significance Regulation of the miR-223 level may be a novel strategy for clinical treatment of diabetes.