This study was conducted in strict accordance with the recommendations outlined in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the Kaohsiung Chang Gung Memorial Hospital (Kaohsiung, Taiwan). Virgin Sprague-Dawley (SD) rats (12–16 weeks old) were obtained (BioLASCO Taiwan Co., Ltd., Taipei, Taiwan), and housed and maintained in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Virgin SD female rats were allowed to mate with male rats for 24 h, and were separated from the male rats and housed individually in a standard plastic home cage. Following confirmation of pregnancy on day 14 following mating, pregnant females were randomly assigned into prenatal steroid treatment or untreated until delivery groups. Pregnant rats were checked for litters daily at 10 a.m. The day of birth was designated postnatal day 0 (D0) and rat pups were weaned on postnatal day 21 (D21), with ad libitum access to standard chow and water. Only male rat pups were used in the present study. In the short-term prenatal steroid group, pregnant rats were administered i.p. DEX (0.1 mg/kg/day; 026804; Taiwan Biotech Co., Ltd., Taoyuan, Taiwan) at gestational day 19–20 (DEX2 group); whereas pregnant rats in the long-term antenatal steroid group were administered i.p. DEX (0.1 mg/kg/day) at gestational day 14–20 (DEX7 group). The early effects of prenatal programming using glucocorticoids were assessed on postnatal day 7 (D7), whereas the late programming effects were assessed on postnatal day 120 (D120). The vehicle group comprised of pregnant SD rats at gestational day 14–20 that received i.p. normal saline.

Rats were anesthetized by an intramuscular injection of a 1:1 mixture (100 mg/kg) of ketamin (#003542; United Biomedical, Inc., Hsinchu, Taiwan) and rompun (#06713; Bayer Taiwan Co., Ltd., Taipei, Taiwan), followed by cardiac puncture and perfusion. Immediately after the rats were sacrificed as described previously (14), the lungs were harvested and stored in saline on ice and were subsequently dissected from the surrounding tissues. Lung tissue was then fixed in 10% formalin neutral buffer solution, pH 7.4 (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Subsequently, the tissues were cut into 4-µm sections and stained with hematoxylin (Merck, Darmstadt, Germany) and eosin Y (HT110180; Sigma-Aldrich, St. Louis, MO, USA) for morphometric analysis. Images were captured using a digital camera (CTR 5000; Leica) and a Nikon Eclipse E600 microscope (magnification, ×10).

A 50-mg lung tissue sample was homogenized using 500 µl PRO-PREP Protein Extraction Solution (#17081; iNtRON Biotechnology, Inc., Seongnam, Korea). Cell lysis was induced by incubation for 30 min on ice and the samples was centrifuged at 14,000 × g for 20 min at 4°C. Protein concentrations were determined using a Bio-Rad Protein Assay kit II (#5000002; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein samples (50 µg) were boiled with gel-loading buffer (10 mM Tris-HCl, 1% sodium dodecyl sulfate (SDS), 25% glycerol, 0.1 mM β-mercaptoethanol and 0.03% bromophenol blue; pH 6.8) for 5 min and separated by 10% SDS-polyacrylamide gel electrophoresis. Following transfer to a polyvinylidene fluoride membrane (Roche Diagnostics, Basel, Switzerland) and blocking with phosphate-buffered saline-Tween containing 5% dry milk, the membranes were incubated for 2 h with anti-rat lamin A/C antibody (#4777; Cell Signaling Technology, Inc., Danvers, MA, USA) diluted 1:200 in Tris-buffered saline (TBS) containing 1.21 g Tris (#640310, Merck) and 8.76 g NaCl (#106404; Merck) in 1 L ddH₂O (pH 7.6), supplemented with 1% skimmed milk. Following five washes with 0.1% Tween-TBS (T-TBS), the membranes were incubated for 1 h with horseradish peroxidase-labeled secondary antibody (#7076; Cell Signaling Technology, Inc.) diluted 1:1,000 in T-TBS. Bands of interest were visualized using Western Lightning Plus enhanced chemiluminescence reagents (NEL105001EA; PerkinElmer, Inc., Waltham, MA, USA) and quantified using densitometry (Quantity One Analysis software, version 4.6.7; Bio-Rad Laboratories, Inc.) as integrated optical density after subtraction of background. The integrated optical density was factored for Ponceau S staining (PonS; P7170; Sigma-Aldrich) to correct for any variations in total protein loading. The protein abundance was represented as integrated optical density/PonS.

Total RNA was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. Purified RNA was quantified at an optical density of 260 nm using a ND-1000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc., Wilmington, DE, USA) and measured using a Bioanalyzer 2100 with an RNA 6000 LabChip kit (both Agilent Technologies, Inc., Santa Clara, CA, USA).

Small RNA library construction and deep sequencing were performed by Welgene Biotech, Co., Ltd. (Taipei, Taiwan). Samples were prepared using an Illumina Sample Preparation kit according to the TruSeq® Small RNA Sample Preparation Guide (Illumina, Inc., San Diego, CA, USA). Subsequently, 5′ and 3′ adaptors (Illumina, Inc.) were ligated to total RNA, followed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) amplification (described below). Enriched cDNA constructs were size-fractionated and purified by electrophoresis in 6% polyacrylamide gel with 30% acrylamide/Bis solution (#1610156) at a ratio of 29:1, and using a Mini-PROTEAN Tetra Vertical Electrophoresis System (Bio-Rad Laboratories, Inc.). Bands containing 18–40 nucleotide RNA fragments, 140–155 nucleotides in length with both adapters, were harvested. Libraries were sequenced using an Genome Analyzer IIx (50 cycle single read; Illumina, Inc.).

According to the manufacturer's protocol, the generated NGS data were analyzed using the miRSeq software package (21), which enables the acquisition of miRNA expression profiles and the evaluation of the overall quality of the NGS libraries. Using the readPro tool in miRSeq, a 15–27 length parameter was specified. Each sample used 1 ng RNA in the NGS experiment.

Targets of miRNAs were predicted using an online target prediction tool; TargetScan 6.2 (http:www.targetscan.org/). A prediction score of >0.5 was selected as the criterion for target genes with each miRNA.

In order to validate the miRNA profiling results, RT-qPCR was conducted on all samples using a SYBR® Green miRNA-based assay including the Universal cDNA Synthesis Kit (#203301) and an ExiLENT SYBR® Green Master Mix Kit (#203420; Exiqon, Inc., Woburn, MA, USA). Total RNA was extracted from the lung tissue using TRIzol® reagent. A Universal cDNA Synthesis kit (#203301; Exiqon, Inc.) was used to reverse transcribe 20 ng RNA from the tissues using the 2X ExiLENT SYBR® Green Master Mix, according to the manufacturer's protocol. A total of 50 ng RNA was reverse transcribed to cDNA in qPCR experiment. The mixture was incubated at 42°C for 60 min, followed by 95°C for 5 min. RT-qPCR reactions were subsequently performed using a total reaction volume of 10 µl containing 4 µl cDNA, 1 µl PCR primer mix and 5 µl ExiLENT SYBR® Green master mix (203420; Exiqon, Inc.). The PCR cycling was conducted as follows: Pre-incubation at 95°C for 10 min; then amplification for 45 cycles of 95°C for 10 sec and 60°C for 10 min; then melting cycle of 95°C for 5 sec, 65°C for 1 min and 97°C with continuous per 5°C acquisition of fluorescence; and then cooling at 40°C for 10 sec. All RT-qPCR reactions, including the controls, were performed in triplicate using a LightCycler® 480 system (Roche Diagnostics) (14). Relative miRNA expression levels were analyzed using the Cq method and normalized against U6 snRNA (203907; Exiqon, Inc.) expression levels. Target miRNA primer sequences were as follows: Rno-let-7b-5p, 5′-UGAGGUAGUAGGUUGUGUGGUU-3′; rno-miR-26a-5p, 5′-UUCAAGUAAUCCAGGAUAGGCU-3′; rno-miR-27a-3p, 5′-UUCACAGUGGCUAAGUUCCGC-3′; rno-miR-101a-3p, 5′-UACAGUACUGUGAUAACUGAA-3′; rno-miR-375–3p, 5′-UUUGUUCGUUCGGCUCGCGUGA-3′. Data were analyzed as previously described (22).

Data are expressed as mean ± standard error of the mean. Mann-Whitney U test was used when two groups were analyzed. All statistical tests were performed using SPSS software, version 19.0 (IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistical significant difference.

Histological examination of the prenatal DEX treatment group using hematoxylin and eosin staining demonstrated obvious dysplasia of the lung alveolar tissue at D7 and D120, as compared with the vehicle group (Fig. 1). The greatest dysplastic change was detected in the rat lungs of the DEX7 group, which were administered prenatal DEX for 7 days. Since lamin A/C level has been demonstrated to be an important determinant for lineage-specific differentiation during embryonic development (23), the abundance of lamin A/C was determined using western blotting. A reduction in lamin A/C was detected in the lung alveolar tissue of the prenatal DEX group at D7 and D120, as compared with the vehicle group (Fig. 2). The lowest lamin A/C expression levels were detected in the rat lungs of the DEX2 group, which were administered prenatal DEX for 2 days.

In the present study, six libraries were used from rat lung tissues sequenced using the NGS platform and the generated NGS data were analyzed using the miRSeq software package. The results demonstrated that the six libraries had similar performances regarding self-ligation reads, which are the reads generated from 5′ and 3′ adaptor self-ligation at the sample preparation step, and clean reads, which are the reads with the 3′ adaptor identified and trimmed. However, further investigation demonstrated that the clean reads of the D7 libraries tended to be less enriched in 22-nt length (Fig. 3A). The length distribution patterns of the D120 libraries were more homologous to the rat miRNAs in miRBase 20; therefore, these had a higher likelihood of being miRNAs (Fig. 2B). The D120 libraries accounted for a significantly higher proportion of the miRNA reads (P=0.015 pair-wise t-test) (Fig. 3B). Furthermore, among the three D120 libraries, a peak at 24 nt was detected at D120 in the DEX7 group, resulting in a higher proportion of non-coding RNA reads. Therefore, the sample preparation steps performed better in the D120 libraries, among which the D120 V and D120 DEX2 groups were particularly successful.

Following miRSeq analysis, miRNA expression profiles in six libraries were acquired. In order to compare the overall miRNA expression profiles among the six libraries, respective heat maps were plotted using the Partek® analysis toolkit (Partek, Inc., St. Louis, MO, USA). The six libraries were first classified into two clusters belonging to D7 and D120, respectively (Fig. 4). Therefore, age was the predominant factor for the observed differences in the miRNA expression profiles. In the D7 libraries, the vehicle and DEX7 groups demonstrated more homologous patterns; whereas, in the D120 libraries, the vehicle group was most homologous to the DEX2 group. Therefore, the results of the present study suggest that DEX treatment produced inconsistent effects on the miRNA expression profiles between the D7 and D120 libraries.

Since age was associated with notable differences, alterations in the miRNA expression profiles of rat lung tissues were compared between D7 and D120. miRNA profiles were grouped according to D7 or D120, regardless of prenatal DEX treatment. In this NGS profile, 167 differentially expressed miRNAs were detected between the D7 and D120 groups, and 12 of these miRNAs demonstrated a >2-fold change, including six upregulated miRNAs: miR-101a-3p; let-7b-5p; let-7c-5p, miR-23b-3p; miR-27a-3p; and miR30b-5p, and six downregulated miRNAs: miR-99b-5p; miR-146b-5p; miR-181b-5p; miR-182; miR-199a-3p; and mir-351-5p. (Table I). When the fold-change cutoff was set to 1.5–2.0, seven upregulated miRNAs and nine downregulated miRNAs were detected at D120 (Table II).

miRNA expression profiles of the rat lungs were compared between the vehicle and prenatal DEX treatment groups, including day 7 in the vehicle group (D7 V), prenatal DEX treatment for 2 days (D7 DEX2) and prenatal DEX treatment for 7 days (D7 DEX7). Since prenatal DEX treatment did not cause a > 2-fold increase in the miRNA expression levels, as compared with the vehicle, the cutoff value was set as a 1.2-fold change between the D7 DEX2 and D7 V groups or the D7 DEX7 and D7 V groups. Following the implementation of the 1.2-fold change cutoff, 12 upregulated and 20 downregulated miRNAs were detected following prenatal DEX treatment at D7 (Table III).

Differences in the miRNA expression profiles of the rat lungs at D120 were compared among the vehicle (D120 V), prenatal DEX treatment for 2 days (D120 DEX2) and D120 DEX7) groups. Following the implementation of the 1.2-fold change cutoff, four upregulated and five downregulated miRNAs were detected following prenatal DEX treatment at D120 (Table IV). These results suggest that the efficacy of prenatal treatment with DEX on the miRNA profiles in rat lung tissues is markedly decreased in the chronic stage.

The following differentially expressed miRNAs were selected to perform RT-qPCR, based on the NGS data: let-7b-5p, miR-26a-5p, miR-27a-3p, miR-101a-3p, and miR-375. The relative expression of these five miRNAs are shown in Fig. 5. These five miRNAs were selected for further study as, among the consistently upregulated miRNAs, let-7b-5p, miR-27a-3p, and miR-101a-3p demonstrated the greatest fold changes between the D7 and D120 groups. Furthermore, let-7b-5p, miR-27a-3p, and miR-101a-3p are reportedly associated with lung tumor development (24), and miR-26a and miR-375 have been associated with the regulation of pulmonary surfactant secretion. The alterations in miRNAs following prenatal DEX treatment were not validated by RT-qPCR as the effects of prenatal DEX treatment appear to be limited.