The Functional Mechanisms of miR-30b-5p in Acute Lung Injury in Children

Abstract

BACKGROUND Acute lung injury in children is a complicated disease linked to the inflammation response. MicroRNA (miRNA) plays a vital role in acute lung injury. However, the role of miR-30b-5p in the pathogenesis of acute lung injury is not clear. The purpose of our study was to investigate the alteration of miR-30b-5p, suppressor of cytokine signaling 3 (SOCS3), in children with acute lung injury, and also in a mouse model of acute lung injury induced by the endotoxin lipopolysaccharide (LPS). MATERIAL AND METHODS The levels of miR-30b-5p, SOCS3, FKN (fractalkine), tumor necrosis factor (TNF)-α, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B), interleukin-6 (IL-6), and IL-8 were detected by ELISA (enzyme-linked immunosorbent assay), western blot, and qRT-PCR (quantitative reverse transcription polymerase chain reaction) assay. The alveolar permeability index and the ratio of wet weight/dry weight (W/D) were measured. Then, we examined the inflammation and apoptosis using hematoxylin and eosin (H&E) staining and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay. Additionally, SOCS3 was investigated as a direct target of miR-30a-5p in RAW264.7 cells by dual-luciferase reporter assays. RESULTS Our study indicated that the level of miR-30b-5p was decreased and the levels of SOCS3, FKN, TNF-α, NF-κB, IL-6, and IL-8 were increased in lung tissue, serum, and bronchoalveolar lavage fluid of mice with acute lung injury induced by LPS. In addition, LPS increased alveolar permeability index and the ratio of W/D and induced inflammatory responses, including the activation of the NF-kB pathway in a mouse model. Furthermore, SOCS3 was confirmed to be a target of miR-30a-5p in RAW264.7 cells. CONCLUSIONS Our data demonstrated an important role for miR-30b-5p in acute lung injury inflammation and suggested that miR-30b-5p might be an important therapy target in children with acute lung injury.

Figure 1

The levels of miR-30b-5p, SOCS3, FKN, TNF-α, NF-κB, IL-6, and IL-8 in children with pneumonia. (A) miR-30b-5p expression was downregulated in the experimental group compared with the control group. (B–G) The mRNA levels of SOCS3, FKN, TNF-α, NF-κB, IL-6, and IL-8 were upregulated in patients in the experimental group comparing to the control group. (** P<0.01, *** P<0.001, experiment versus control.) SOCS3 – suppressor of cytokine signaling 3; FKN – fractalkine; TNF-α – tumor necrosis factor-α; NF-κB – nuclear factor kappa-light-chain-enhancer of activated B; IL-6 – interleukin-6; IL-8 – interlukein-8.

Figure 2

The alveolar permeability index, the ratio of W/D, lung tissue injury, apoptosis, and the levels of inflammatory factors in mice with ALI. (A) The alveolar permeability index was significantly increased in mice with ALI compared to the control group. (B) The ratio of W/D was significantly increased in ALI group compared with the control group. (* P<0.05, ** P<0.01, ALI versus control.) (C) Representative images of lung injury by hematoxylin and eosin staining. (D) Representative images of apoptosis by TUNEL. (E) The expression of SOCS3, FKN, TNF-α, and NF-κB were remarkably upregulated in lung tissue of mice with ALI by immunocytochemistry analysis compared with the control group. As the picture shows, the number of brownish yellow particles in the ALI group was significantly higher than that of the control group. ALI – acute lung injury; W/D – wet weight/dry weight; TUNEL – terminal deoxynucleotidyl transferase dUTP nick end labeling; SOCS3 – suppressor of cytokine signaling 3; FKN – fractalkine; TNF-α – tumor necrosis factor-α; NF-κB – nuclear factor kappa-light-chain-enhancer of activated B.

Figure 3

Expression of SOCS3, FKN, IL-6, and IL-8 in the serum and BALF of mice with ALI. The expression of SOCS3, FKN, IL-6, and IL-8 were significantly upregulated in serum (A) and BALF (B) of mice with ALI by ELISA assay. (** P<0.01, *** P<0.001, ALI versus control.) ALI – acute lung injury; SOCS3 – suppressor of cytokine signaling 3; FKN – fractalkine; IL-6 – interleukin-6; IL-8 – interlukein-8; BALF – bronchoalveolar lavage fluid; ELISA – enzyme-linked immunosorbent assay.

Figure 4

The mRNA levels of miR-30b-5p, SOCS3, FKN, TNF-α, NF-κB, IL-6, and IL-8 in lung tissue of mice with ALI. The qRT-PCR assay suggested that the mRNA levels of miR-30b-5p was decreased (A) and the mRNA levels of SOCS3, FKN, TNF-α, NF-κB, IL-6, and IL-8 (B–G) were increased in the lung tissue of mice with ALI compared with the control group. (** P<0.01, *** P<0.001, ALI versus control.) ALI – acute lung injury; SOCS3 – suppressor of cytokine signaling 3; FKN – fractalkine; TNF-α – tumor necrosis factor-α; NF-κB – nuclear factor kappa-light-chain-enhancer of activated B; IL-6 – interleukin-6; IL-8 – interlukein-8; qRT-PCR – quantitative reverse transcription polymerase chain reaction.

Figure 5

The protein levels of SOCS3, FKN, TNF-α, NF-κB, IL-6, and IL-8 in lung tissue, serum, and BALF of mice with ALI. Western blotting assay also suggested that the protein levels of SOCS3, FKN, TNF-α, NF-κB, IL-6, and IL-8 were increased in lung tissue (A), serum (B), and BALF (C) of mice with ALI compared with the control group. (** P<0.01, *** P<0.001, ALI versus control.) ALI – acute lung injury; SOCS3 – suppressor of cytokine signaling 3; FKN – fractalkine; TNF-α – tumor necrosis factor-α; NF-κB – nuclear factor kappa-light-chain-enhancer of activated B; IL-6 – interleukin-6; IL-8 – interlukein-8; BALF – bronchoalveolar lavage fluid.

Figure 6

miR-30b-5p directly targeted SOCS3 and miR-30b-5p mimics suppressed SOCS3 expression. (A) The putative binding of miR-30b-5p on SOCS3 gene 3′UTR are shown. RAW264.7 cells were co-transfected with luciferase plasmids with SOCS3 3′UTR-WT or with SOCS3 3′UTR-MUT. The relative luciferase activities were measured by a dual-luciferase assay in different groups (* P<0.05). (B) The mRNA expression of SOCS3 in RAW264.7 cells, and SOCS3 is downregulated in miR-30b-5p mimics group (** P<0.01, *** P<0.001 versus miR-30b-5p mimics control and NC group.) SOCS3 – suppressor of cytokine signaling 3.