Role of miR-29 in mediating offspring lung phenotype in a rodent model of intrauterine growth restriction - PubMed (original) (raw)

Role of miR-29 in mediating offspring lung phenotype in a rodent model of intrauterine growth restriction

Tsai-Der Chuang et al. Am J Physiol Regul Integr Comp Physiol. 2018.

Abstract

Considerable epidemiological and experimental evidence supports the concept that the adult chronic lung disease (CLD), is due, at least in part, to aberrations in early lung development in response to an abnormal intrauterine environment; however, the underlying molecular mechanisms remain unknown. We used a well-established rat model of maternal undernutrition (MUN) during pregnancy that results in offspring intrauterine growth restriction (IUGR) and adult CLD to test the hypothesis that in response to MUN, excess maternal glucocorticoids (GCs) program offspring lung development to a CLD phenotype by altering microRNA (miR)-29 expression, which is a key miR in regulating extracellular matrix (ECM) deposition during development and injury-repair. At postnatal day 21 and 5 mo, compared with the control offspring lung, MUN offspring lung miR-29 expression was significantly decreased in conjunction with an elevated expression of multiple downstream target ECM proteins [collagen (COL)1A1, COL3A1, COL4A5, and elastin], at both mRNA and protein levels. Importantly, MUN-induced changes in miR-29 and target gene expressions were at least partially blocked in the lungs of offspring of MUN dams treated with metyrapone, a selective GC synthesis inhibitor. Furthermore, dexamethasone treatment of cultured fetal rat lung fibroblasts significantly induced miR-29 expression along with the suppression of target ECM proteins. These data, along with the previously known role of miR-29 in regulating ECM deposition in vascular tissue in the MUN offspring, suggest miR-29 to be a common mechanistic denominator for the vascular and pulmonary phenotypes in the IUGR offspring, providing a novel potential therapeutic target.

Keywords: IUGR; chronic lung disease; fetal growth restriction; fetal programming; miRNA.

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Figures

Fig. 1.

Expression levels of miRNA (miR)-29a, miR-29b, miR-29c, collagen (COL)1A1, COL3A1, COL4A5, and elastin (ELN) in maternal undernutrition (MUN) offspring lungs. The levels of miR-29a (A), miR-29b (B), and miR-29c (C) and mRNA levels of COL1A1 (D), COL3A1 (E), COL4A5 (F), and ELN (G) in pups from control and MUN group at postnatal day 1 (PND 1; n = 6), day 21 (PND 21; n = 5), and 5 mo (n = 4) by quantitative reverse transcription–polymerase chain reaction are shown. The results are presented as means ± SE with P values (*P < 0.05) indicated by corresponding lines.

Fig. 2.

Effect of metyrapone (Met) on the expression of miRNA (miR)-29a, miR-29b, miR-29c, collagen (COL)1A1, COL3A1, COL4A5, and elastin (ELN) in offspring lungs at postnatal day 21. The expression levels of miR-29a (A), miR-29b (B), and miR-29c (C) and the mRNA levels of COL1A1 (D), COL3A1 (E), COL4A5 (F), and ELN (G) in pups from control, maternal undernutrition (MUN), and MUN+Met treatment groups (all n = 6) by quantitative reverse transcription–polymerase chain reaction are shown. The results are presented as means ± SE with P values (*P < 0.05) indicated by corresponding lines.

Fig. 3.

A: representative Western blot images for the effects of metyrapone (Met) on the protein levels of collagen (COL)1A1, COL3A1, COL4A5, and elastin (ELN) in offspring lungs at postnatal day 21 of control, maternal undernutrition (MUN), and MUN+Met treatment groups (all n = 6). B: mean relative band densities presented as means ± SE and shown as a bar plot. *P < 0.05 vs. control group and #P < 0.05 vs. MUN group.

Fig. 4.

Effects of maternal undernutrition (MUN) and metyrapone (Met) treatment on the expression of collagen (COL)1A1, COL3A1, COL4A5 (red staining), and elastin (ELN) (green staining) in offspring lungs at postnatal day 21 are determined by immunohistochemistry. Representative immunohistochemistry images are shown (magnification ×20, n = 4).

Fig 5.

Complementary sequence alignments of miRNA (miR)-29 seed regions with target sites in the 3′ untranslated region (UTR) of collagen (COL)1A1, COL3A1, COL4A5, and elastin (ELN).

Fig. 6.

Relative luciferase activity in isolated embryonic day 19 fetal rat lung fibroblasts transfected with Renilla and firefly luciferase reporter carrying a 3′ untranslated region fragment of collagen (COL)1A1, COL3A1, COL4A5, and elastin (ELN). Cells were also co-transfected with pre-miR-29c (50 nM) or control oligonucleotides (NC). Ratio of firefly to Renilla was determined after 48 h and reported as relative luciferase activity compared with NC, which was independently set as 1. Values are means ± SE from three sets of independent experiments. *P < 0.05 vs. NC.

Fig. 7.

Effect of miRNA (miR)-29c overexpression or knockdown in embryonic day 19 fetal rat lung fibroblasts for 72 h on mRNA (A) and protein (B) levels of collagen (COL)1A1, COL3A1, COL4A5, and elastin (ELN) as determined by quantitative reverse transcription–polymerase chain reaction (A) and Western blotting (B), with relative band densities shown (C). Results are presented as means ± SE of at least three independent experiments with P values (*P < 0.05) indicated by corresponding lines. Multiple gels were used to resolve the proteins; each assay included GAPDH as a loading control; one representative blot is shown here. aNC, anti-miR negative control; NC, miR negative control

Fig. 8.

Effects of dexamethasone (DEX; 10 nM) and mifepristone (Mif; 1μM) on the expression of miRNA (miR)-29a, miR-29b, and miR-29c (A) and protein expression of collagen (COL)1A1, COL3A1, COL4A5, and elastin (ELN) (B) in embryonic day 19 fetal rat lung fibroblasts for 7 days as determined by quantitative reverse transcription–polymerase chain reaction (A) and Western blotting (B), with relative band densities (C) shown. Results are presented as means ± SE of at least three independent experiments. *P < 0.05 vs. DMSO group and #P < 0.05 vs. DEX group. Multiple gels were used to resolve the proteins; each assay included GAPDH as a loading control; one representative blot is shown here.

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Role of miR-29 in mediating offspring lung phenotype in a rodent model of intrauterine growth restriction - PubMed (original) (raw)