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. 2016 Oct 7:7:389.
doi: 10.3389/fimmu.2016.00389. eCollection 2016.

MicroRNA-7 Deficiency Ameliorates the Pathologies of Acute Lung Injury through Elevating KLF4

Juanjuan Zhao  1 , Chao Chen  1 , Mengmeng Guo  1 , Yijin Tao  1 , PanPan Cui  1 , Ya Zhou  2 , Nalin Qin  1 , Jing Zheng  1 , Jidong Zhang  1 , Lin Xu  1
Affiliations

MicroRNA-7 Deficiency Ameliorates the Pathologies of Acute Lung Injury through Elevating KLF4

Juanjuan Zhao et al. Front Immunol. .

Abstract

Recent evidence showed that microRNA-7 (miR-7) played an important role in the pathologies of lung-related diseases. However, the potential role of miR-7 in acute lung injury (ALI) still remains poorly understood. Here, we assessed the effect of miR-7 deficiency on the pathology of ALI. We, first, found that the expression of miR-7 was upregulated in lung tissue in murine LPS-induced ALI model. Notably, we generated miR-7 knock down mice by using miRNA-Sponge technique and found that miR-7 deficiency could ameliorate the pathologies of lung as evidenced by accelerated body weight recovery, reduced level of bronchoalveolar lavage (BAL) proinflammatory cytokines and decreased number of BAL cells in ALI mice. Moreover, the proportion and number of various immune cells in BAL, including innate immune cell F4/80+ macrophages, γδT cells, NK1.1+ T cells, and CD11c+DCs, as well as adaptive immune cell CD4+ T cells and CD8+ T cells, also significantly changed, respectively. Mechanistic evidence showed that KLF4, a target molecule of miR-7, was upregulated in lung tissues in ALI model, accompanied by altered transduction of NF-κB, AKT, and ERK pathway. These data provided a previously unknown role of miR-7 in pathology of ALI, which could ultimately aid the understanding of development of ALI and the development of new therapeutic strategies against clinical inflammatory lung diseases.

Keywords: KLF4; acute lung injury; cytokines; immune cells; miR-7KD.

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Figures

Figure 1
Figure 1
The generation of miR-7 deficiency mice. The sketch map of construction of eukaryotic expression vector encoding miR-7-Sponge (termed as p-miR-7-Sp) (A) and generation of miR-7 KD mice (B). (C) The identification of DNA. DNA was derived from wild-type (WT) or miR-7KD mice. And the expected product (505 bp size) was amplificated by PCR, respectively. (D) The relative expression of miR-7 in lung derived from WT mice and miR-7KD mice (n = 6) were detected by Real-time PCR assay. (E) The expression of miR-7 in lung tissue derived from WT mice and miR-7KD was analyzed by In situ hybridization. Representative data of three independent experiments were shown. **p < 0.01.
Figure 2
Figure 2
miR-7 deficiency ameliorated the pathology of ALI. FVB/N6 mice (WT, n = 6) and miR-7KD mice (n = 6) were administered with i.p. 10 mg/kg LPS, respectively. (A) Schematic representation of the animal study. (B) The change on the body weight of WT and miR-7KD mice at indicated time point was observed. (C) FVB/N6 mice (WT, n = 6) and miR-7KD mice (n = 6) were administered with i.p. 10 mg/kg LPS, respectively. After 48 h, the morphology, (D) the body weight, and (E) the weight index (tissue weight/body weight), as well as (F) the pathology, of lung tissues were analyzed, respectively. (G) The relative expression of BAD, BAX, BCL-XL, and p53 in lung tissue also were analyzed by Real-time PCR assay. (H–M) FVB/N6 mice (WT, n = 6) and miR-7KD mice (n = 6) were administered with i.p. 10 mg/kg LPS, respectively. After 48 h, BAL was collected and the level of IFN-γ, IL-1β, TNF-α, IL-4, and IL-10, as well as TGF-β, was determined with ELISA, respectively. Representative data of three independent experiments were shown. *p < 0.05, **p < 0.01.
Figure 2
Figure 2
miR-7 deficiency ameliorated the pathology of ALI. FVB/N6 mice (WT, n = 6) and miR-7KD mice (n = 6) were administered with i.p. 10 mg/kg LPS, respectively. (A) Schematic representation of the animal study. (B) The change on the body weight of WT and miR-7KD mice at indicated time point was observed. (C) FVB/N6 mice (WT, n = 6) and miR-7KD mice (n = 6) were administered with i.p. 10 mg/kg LPS, respectively. After 48 h, the morphology, (D) the body weight, and (E) the weight index (tissue weight/body weight), as well as (F) the pathology, of lung tissues were analyzed, respectively. (G) The relative expression of BAD, BAX, BCL-XL, and p53 in lung tissue also were analyzed by Real-time PCR assay. (H–M) FVB/N6 mice (WT, n = 6) and miR-7KD mice (n = 6) were administered with i.p. 10 mg/kg LPS, respectively. After 48 h, BAL was collected and the level of IFN-γ, IL-1β, TNF-α, IL-4, and IL-10, as well as TGF-β, was determined with ELISA, respectively. Representative data of three independent experiments were shown. *p < 0.05, **p < 0.01.
Figure 3
Figure 3
miR-7 deficiency altered the immune cell composition in BAL of mice from ALI. FVB/N6 mice (WT, n = 6) and miR-7KD mice (n = 6) were administered with i.p. 10 mg/kg LPS, respectively. After 48 h, (A) the total numbers of BAL cells were calculated. (B) The proportion of F4/80+ Mφ and γδ+ T cells were analyzed by FCM. The percentage and the absolute number of cells were calculated, respectively (C). (D) The expression of CD86 and MHC-II on F4/80+ Mφ were analyzed by FCM. The percentage and the absolute number of cells were calculated, respectively (E). (F) The proportion of CD11c+ dendritic cells and NK1.1+ T cells were analyzed by FCM. The percentage and the absolute number of cells were calculated, respectively (G). (H) The proportion of CD4+ T cells and CD8+ T cells were analyzed by FCM. The percentage and the absolute number of cells were calculated, respectively (I). (J) The expression of CD62L and CD69 on CD4+ T cells were analyzed by FCM. And, the percentage and the absolute number of cells were also calculated, respectively. (K) The expression of CD62L and CD69 on CD8+ T cells were analyzed by FCM. And, the percentage and the absolute number of cells were also calculated, respectively. Representative data of three independent experiments were shown. *p < 0.05, **p < 0.01.
Figure 4
Figure 4
KLF4 was upregulated in lung tissues in LPS-treated miR-7KD mice. (A) FVB/N6 mice (WT, n = 6) and miR-7KD mice (n = 6) were administered with i.p. 10 mg/kg LPS, respectively. After 48 h, the relative expression of the potential target genes of miR-7 in the lung tissue were analyzed by Real-time PCR assay. (B) Putative miR-7-binding sites in the 3′UTR of KLF4. (C) FVB/N6 mice (WT, n = 6) were administered with i.p. 10 mg/kg LPS. The relative expression of miR-7 and KLF4 were detected by Real-time PCR at indicated time points. (D) FVB/N6 mice (WT, n = 6) and miR-7KD mice (n = 6) were administered with i.p. 10 mg/kg LPS, respectively. After 48 h, KLF4 expression in lung tissue was analyzed by Western Blot and (E) immunohistochemical staining, respectively (original magnification 200×, 400×). Representative data of three independent experiments were shown. *p < 0.05, **p < 0.01.
Figure 5
Figure 5
The signaling pathway change of miR-7 deficiency in ALI. FVB/N6 mice (WT, n = 6) and miR-7KD mice (n = 6) were administered with i.p. 10 mg/kg LPS, respectively. After 48 h, the expression level of ERK, p-ERK, AKT, p-AKT, and p-NF-kB in lung tissue were determined with Western Blot (A) and calculated (B). (C) The expression of NF-κB was also detected by the immunohistochemical staining. Representative data of three independent experiments were shown. *p < 0.05, **p < 0.01. (D) Schematic representation of the underlying mechanism of miR-7 deficiency on ALI. miR-7 deficiency leads to upregulation of KLF4, which could successively alter the transduction of NF-κB, ERK, and AKT signaling pathway and contribute to the ameliorated pathologies of ALI.
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