Characterization of a Murine Model System to Study MicroRNA-147 During Inflammatory Organ Injury

Abstract

Inflammatory organ injury and sepsis have profound impacts on the morbidity and mortality of surgical and critical care patients. MicroRNAs are small RNAs composed of 20-25 nucleotides that have a significant contribution to gene regulation. MicroRNA-147 (miR-147), in particular, has been shown to have an emerging role in different physiological functions such as cell cycle regulation and inflammatory responses. However, animal model systems to study tissue-specific functions of miR-147 during inflammatory conditions in vivo are lacking. In the present study, we characterize miR-147 expression in different organs and cell types. Next, we generated a transgenic mouse line with a floxed miR-147 gene. Subsequently, we used this mouse line to generate mice with whole-body deletion of miR-147 (miR-147 ^-/-) by crossing "floxed" miR-147 mice with transgenic mice expressing Cre recombinase in all tissues (CMVcre mice). Systematic analysis of miR-147 ^-/- mice demonstrates normal growth, development, and off-spring. In addition, deletion of the target gene in different organs was successful at baseline or during inflammation, including the heart, intestine, stomach, liver, spleen, bone marrow, lungs, kidneys, or stomach. Moreover, miR-147 ^-/- mice have identical baseline inflammatory gene expression compared to C57BL/6 mice, except elevated IL-6 expression in the spleen (7.5 fold, p < 0.05). Taken together, our data show the successful development of a transgenic animal model for tissue and cell-specific deletion of miR-147 that can be used to study the functional roles of miR-147 during inflammatory organ injury.

Keywords: Inflammation; MicroRNA; MicroRNA-147; Organ injury; Transgenic mouse model.

Fig. 1

MiR-147 expression across different cell lines, tissue, and cells. a Transcript level of miR-147 in various human cell lines (kidney-derived cell lines including HEK293T and HK2; endothelial cell line, HMEC1; colon-derived cell lines including T84, Caco2, and HL60; lung-derived cell lines including A549 and Calu3; peripheral blood–derived cell line, THP1). Expression level was normalized to U6 snoRNA. b Transcript level of miR-147 in human primary cells including human cardiomyocytes (HCM), human pulmonary alveolar epithelial cells (HPAEpiC), and human monocyte-derived macrophages (MDM). Expression level was normalized to U6 snoRNA. c Screening of miR-147 expression in different organs collected from C57BL/6J mice. Expression level was normalized to U6 snoRNA. d Transcript level of miR-147 in murine primary epithelial cells derived from airway, colon, and renal tubules. Expression level was normalized to U6 snoRNA. e Transcript level of miR-147 in immune cells such as lymphocytes, monocytes, and neutrophils isolated from the peripheral blood of C57BL/6J mice. Expression level was normalized to U6 snoRNA. f Transcript level of miR-147 in Th0, Th1, Th2, Th17, and Treg cells differentiated from naïve CD4 T cells of C57BL/6J mice. Data was normalized to Th0. All graphs represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA with Bonferroni’s multiple comparisons.

Fig. 2

Organ inflammation and miR-147 levels after CLP. CLP was conducted on 8- to 12-week-old C57BL/6J mice, and the indicating organs were collected in 24 h. a, b Induction of inflammatory genes including Cxcl1 and Il6 in CLP group. c Relative transcript level of miR-147 in CLP group compared to sham group. All graphs are presented as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001 relative to sham, by Mann-Whitney test.

Fig. 3

Strategy for the generation of miR-147 floxed mice and miR-147^−/− mice. a Schematic illustration for the generation of miR-147 conditional knockout mice. b Southern blotting with Neo and 3′ probes to confirm targeted ES cells. I-IC7 (lane 1, 2) was selected for chimera generation. c Southern blot data of DNA extracted from the litter of chimera and flipase mice. d PCR conditions for genotyping of miR-147 ^−/− mice. e Primer sequences are complementary to miR-147 allele. f Gel image from PCR-based genotyping to detect deletion of miR-147 allele.

Fig. 4

Confirmation of miR-147 KO in various tissues and breeding characteristics and growth curve of miR-147^−/− (KO) mice. a Basal transcript level of miR-147 in the brain, heart, intestine, liver, lung, kidney, spleen, and stomach from C57BL/6J (WT) and miR-147^−/− mice (KO) (n = 6/group). b Transcript level of miR-147 in the lung, stomach, and kidney from WT and miR-147^−/− mice following CLP procedure (n = 6 for WT; n = 5 for KO mice). c Comparison of gender ratio and average litter size from WT and KO mice that were used for the present study. P value from Fisher’s exact test and unpaired t test, respectively. d Growth curve based on body weight of all animals from WT and KO mice over 42 days. e Growth curve based on body weight of females from WT and KO mice. f Growth curve based on body weight of males from WT and KO mice. Graphs for mRNA expression represent mean ± SEM and growth curves represent mean ± SD. *P < 0.05, **P < 0.01 relative to WT, by Mann-Whitney test.

Fig. 5

Characterization of miR-147^−/− mice. a, b Baseline mRNA expression level of inflammatory genes including Cxcl1 and Il6 in various organs isolated from WT and miR-147^−/− mice (KO). c Representative images of H&E staining from mouse tissues including the brain, heart, intestine, liver, lung, kidney, spleen, and stomach to compare histology of WT and miR-147^−/− mice (scale bar: 200 μm). All graphs represent mean ± SEM. *P < 0.05 (n = 6 mice/group) relative to WT, by two-way ANOVA with Bonferroni’s multiple comparisons.