Attenuation of miR-17∼92 Cluster in Bronchopulmonary Dysplasia

Abstract

Rationale: Bronchopulmonary dysplasia remains a significant cause of neonatal morbidity; however, the identification of novel targets to predict or prevent the development of bronchopulmonary dysplasia remains elusive. Proper microRNA (miR)-17∼92 cluster is necessary for normal lung development, and alterations in expression are reported in other pulmonary diseases. The overall hypothesis for our work is that altered miR-17∼92 cluster expression contributes to the molecular pathogenesis of bronchopulmonary dysplasia.

Objectives: The current studies tested the hypothesis that alterations in miR-17∼92 cluster and DNA methyltransferase expression are present in bronchopulmonary dysplasia.

Methods: miR-17∼92 cluster expression, promoter methylation, and DNA methyltransferase expression were determined in autopsy lung samples obtained from premature infants who died with bronchopulmonary dysplasia, or from term/near-term infants who died from nonrespiratory causes. Expression of miR-17∼92 cluster members miR-17 and -19b was measured in plasma samples collected in the first week of life from a separate cohort of preterm infants at a second institution in whom bronchopulmonary dysplasia was diagnosed subsequently.

Measurements and main results: Autopsy tissue data indicated that miR-17∼92 expression is significantly lower in bronchopulmonary dysplasia lungs and is inversely correlated with promoter methylation and DNA methyltransferase expression when compared with that of control subjects without bronchopulmonary dysplasia. Plasma sample analyses indicated that miR-17 and -19b expression was decreased in infants who subsequently developed bronchopulmonary dysplasia.

Conclusions: Our data are the first to demonstrate altered expression of the miR-17∼92 cluster in bronchopulmonary dysplasia. The consistency between our autopsy and plasma findings further support our working hypothesis that the miR-17∼92 cluster contributes to the molecular pathogenesis of bronchopulmonary dysplasia.

Keywords: bronchopulmonary dysplasia; miR-17∼92 cluster; microRNA.

Figure 1.

Decreased miR-17∼92 cluster expression in bronchopulmonary dysplasia (BPD). Expression was determined by quantitative real-time polymerase chain reaction from control (n = 7) and BPD (n = 8) autopsy lung samples. Data, normalized to RNU38B, are expressed as fold expression ± SD vs. control tissue (*P < 0.05).

Figure 2.

Altered localization of miR-19b and -20a in bronchopulmonary dysplasia (BPD). In situ hybridization was performed on autopsy tissues from four BPD and three term/near-term control patients using locked nucleic acid–modified DNA probes for miR-let-7c (positive control), -19b, and -20a. Scrambled probes were used as a negative control. Representative images are shown and each contains a terminal respiratory bronchiole (1,000×). Positively stained cells for miR-19b or -20a (blue, indicated by arrows) in control lungs are mostly epithelial, whereas positively stained cells in BPD lungs are predominantly stromal and not epithelial.

Figure 3.

Increased miR-17∼92 promoter methylation in bronchopulmonary dysplasia (BPD). Data are presented as average percent of unmethylated and methylated DNA in control (n = 7) and BPD (n = 8) tissues.

Figure 4.

Increased DNA methyltransferase (DNMT) expression in bronchopulmonary dysplasia (BPD). Because of sample limitations, expression of (A) DNMT-1, (B) -3a, and (C) -3b was determined by quantitative real-time polymerase chain reaction in a subset of control (n = 4) and BPD (n = 6) tissues. Using adenylate cyclase-associated protein (Cap1) as an endogenous control, the average relative copy number (RCN) ± SD was calculated (*P < 0.01).

Figure 5.

For plasma analyses, no differences were detected between control and bronchopulmonary dysplasia (BPD) groups in (A) birth weight, (B) gestational age at birth, and (C) Fi_o2 at the time of sample acquisition.

Figure 6.

Decreased plasma miR-17 and -19b expression in the first week of life in infants who subsequently developed bronchopulmonary dysplasia (BPD). (A and B) Expression (mean ± SD) of miR-17 and - 19b in control (n = 10) and BPD (n = 11) plasma samples was determined using lipoplex nanoparticles containing molecular beacons. (C and D) Receiver operating characteristic curves were estimated to determine the extent to which miR concentrations predicted BPD at 36 weeks’ postmenstrual age.