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

UNLABELLED: 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.