microRNA-98 mediated microvascular hyperpermeability during burn shock phase via inhibiting FIH-1

Abstract

Background: microRNA is a small non-coding RNA molecule and functions in RNA silencing and post-transcriptional regulation of gene expression. This study was designed to evaluate the role of miR-98 in the development of microvascular permeability and its molecular pathogenesis.

Methods: Forty-eight healthy adult Wistar rats were divided into the control group (n = 8) and burn group (n = 40) that inflicted with 30% total body surface area third-degree burn. Groups were processed at 2, 4, 8, 12, and 24 h post-burn. Plasma for vascular endothelial cell culture was collected from control and 12 h post-burn rats. Organic microvascular permeability and serum miR-98 level were measured. In vitro, rat aorta endothelial cells were stimulated with burn serum. Level of miR-98 and protein of hypoxia-inducible factor-1 (HIF-1), factor inhibiting HIF-1α (FIH-1), and tight junction-associated proteins were determined.

Results: Organic microvascular permeability began to rise at 2 h post-burn and maintained the same character throughout the experiment except in lung tissue that was still rising at 12 h; the serum level of miR-98 was elevated (P < 0.05). In vitro, burn serum stimulation increased rat aorta endothelial monolayer cell permeability as well as upregulated miR-98 expression (P < 0.05). As shown in the result of transfection experiment, miR-98 negatively regulated FIH-1 and tight junction-associated protein expression (P < 0.05).

Conclusions: The findings of the present study suggest severe microvascular permeability due to burns; and the underlying mechanism bases on the promotion of miR-98 level to the extent that it activated HIF-1 gene expression, resulting in junction-associated protein deficiency.

Figure 1

Burns induced increase of vascular permeability. Mice burn model was established, and then on different time points after surgery, mice were euthanatized for the following detection. (A) Vascular permeability was evaluated using Evans blue contents in tissues. (B) Levels of miR-98 in serum was determined using quantitative real-time PCR. The data were represented with mean ± SD. ^* P < 0.05 compared with corresponding control.

Figure 2

Burn serum stimulation increased permeability of rat aorta endothelial cell monolayers. Rat aorta endothelial cells were incubated with burn serum for 12 h. (A) Level of miR-98 expression was determined using quantitative real-time PCR. (B) Profiles of tight junction-associated proteins expression were evaluated using Western blot. (C) Transendothelial electrical resistance and (D) permeability coefficient of albumin were performed with monolayer endothelial cells. The data were represented with mean ± SD. ^* P < 0.05 compared with control plasma.

Figure 3

Regulated role of miR-98 in FIH-1 expression in rat aorta endothelial cell. Knock down of miR-98 by miR-inhibitor (A) increased FIH 3ʹUTR activity, (B) suppressed HIF-1 expression, and (C) inactivated HIF-1 transcription. Overexpression of miR-98 by miR-98 mimic transfection (D) inhibited FIH 3ʹUTR activity, (E) promoted HIF-1 expression, and (F) increased HIF-1 transcription activity. The data were represented with mean ± SD. ^* P < 0.05 compared with negative control.

Figure 4

Silenced miR-98 abrogated burn serum-increased permeability in rat aorta endothelial cells. After transfected with miR-98 inhibitor for 48 h, cells were incubated with burn serum or normal serum (as control) followed. (A) Profiles of protein expression were determined using Western blot. (B) Transendothelial electrical resistance and (C) permeability coefficient of albumin were examined with monolayer endothelial cells. The data were represented with mean ± SD. ^* P < 0.05 compared with cell incubated with control plasma; ^# P < 0.05 compared with cell incubated with burn plasma.

Figure 5

Co-silenced FIH-1 abrogated silenced miR-98-reduced permeability in rat aorta endothelial cells. Cells were pretreated with siRNA-FIH-1 followed by transfection with miR-98 inhibitor. After 48 h, the cells were incubated with burn serum or control serum. (A) Profiles of protein were determined using Western blot. (B) Transendothelial electrical resistance and (C) permeability coefficient of albumin were performed with monolayer endothelial cells. The data were represented as mean ± SD. ^* P < 0.05 compared with control plasma; ^# P < 0.05 compared with burn serum; ^*# P < 0.05 compared with burn serum + miR-98 inhibitor.

Figure 6

Signaling mechanism by which miR-98 mediated burn-induced increase of microvascular permeability.