Pediatric Pulmonology 49:863–872 (2014)

Regulation of Pulmonary Surfactant Synthesis in Fetal Rat Type II Alveolar Epithelial Cells by MicroRNA-26a Xiao-Qun Zhang, Pan Zhang, Yang Yang, Jie Qiu, Qin Kan, Hong-Lu Liang, Xiao-Yu Zhou,* and Xiao-Guang Zhou* Summary. Pulmonary surfactant, a unique developmentally regulated, phospholipid-rich lipoprotein, is synthesized by the type II epithelial cells (AECII) of the pulmonary alveolus, where it is stored in organelles termed lamellar bodies. The synthesis of pulmonary surfactant is under multifactorial control and is regulated by a number of hormones and factors, including glucocorticoids, prolactin, insulin, growth factors, estrogens, androgens, thyroid hormones, and catecholamines acting through beta-adrenergic receptors, and cAMP. While there is increasing evidence that microRNAs (miRNAs) are involved in the regulation of almost every cellular and physiological process, the potential role of miRNAs in the regulation of pulmonary surfactant synthesis remains unknown. miRNA-26a (miR-26a) has been predicted to target SMAD1, one of the bone morphogenetic protein (BMP) receptor downstream signaling proteins that plays a key role in differentiation of lung epithelial cells during lung development. In this study, we explored the regulation role of miR-26a in the synthesis of pulmonary surfactant. An adenoviral miR-26a overexpression vector was constructed and introduced into primary cultured fetal AECII. GFP fluorescence was observed to determinate the transfection efficiency and miR-26a levels were measured by RT-PCR. MTT was performed to analyze AECII viability. qRT-PCR and Western blotting were used to determine the mRNA and protein level of SMAD1 and surfactant-associated proteins. The results showed that miR-26a in fetal AECII was overexpressed after the transfection, and that the overexpression of miR-26a inhibited pulmonary surfactant synthesis in AECII. There was no significant change in cell proliferation. Our results further showed that overexpression of miR-26a reduced the SMAD1 expression both in mRNA and protein level in fetal AECII. These findings indicate that miR-26a regulates surfactant synthesis in fetal AECII through SMAD1. Pediatr Pulmonol. 2014; 49:863–872. ß 2014 Wiley Periodicals, Inc. Key words: miR-26a; pulmonary surfactant synthesis; fetal rat type II cells. Funding source: National Natural Science Foundation of China, Number: 81270725

INTRODUCTION

Pulmonary surfactant is a unique complex of phospholipids, neutral lipids, and proteins that maintains alveolar integrity by reducing surface tension at the alveolar air–liquid interface and participates in host defense and the control of inflammation in the lung.1,2 Lung alveolar type II epithelial cells (AECII) that are positioned in the corners of the alveoli have the

highly specialized functions of synthesizing, secreting, and reutilizing pulmonary surfactant.3 The fetal lung acquires the capacity for pulmonary surfactant synthesis in AECII relatively late in gestation; augmented surfactant synthesis and secretion are initiated after completion of 75–90% of gestation in all mammalian species thus far studied.4,5 Infants born before this period of gestation lack sufficient pulmonary surfactant, and are predisposed to develop respiratory distress syndrome

Department of Neonatology, Nanjing Children’s Hospital of Nanjing Medical University, Nanjing, P.R. China.

72 Guangzhou Road, Nanjing 210008, P.R. China. E-mail: [email protected]; [email protected]

Conflict of interest: None.

Received 7 December 2012; Accepted 23 August 2013.

Xiao-Qun Zhang and Pan Zhang contributed equally to the work as the first authors.

DOI 10.1002/ppul.22975 Published online 3 January 2014 in Wiley Online Library (wileyonlinelibrary.com).



Correspondence to: X.-Y. Zhou and X.-G. Zhou, Department of Neonatology, Nanjing Children’s Hospital of Nanjing Medical University,

ß 2014 Wiley Periodicals, Inc.

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(RDS), one of the leading causes of neonatal morbidity and mortality.6 The maturation of fetal lung and the synthesis of pulmonary surfactant in AECII are influenced and regulated by a variety of factors including cell-to-cell interactions and the action of local paracrine mediators and circulating hormones, such as glucocorticoids, prolactin, insulin, growth factors, estrogens, androgens, and thyroid hormones.3,4,7,8 In addition to these factors, recent studies have reported that microRNAs (miRNA), a class of endogenous 20–24 nucleotide non-proteincoding RNAs that regulate eukaryotic gene expression at the post-transcriptional level, are also involved in the regulation of fetal lung maturation and pulmonary surfactant metabolism.9–11 miRNAs are known to play multiple roles in carcinogenesis, immune responses, and organ development,12 and have been implicated in many critical cellular processes, including proliferation, differentiation, growth control, and apoptosis.13 They have direct effects on gene expression by inducing mRNA degradation or translation inhibition.12 To date, over 900 different miRNAs genes have been discovered in the human genome, and it has been speculated that miRNAs may be associated with the regulation of almost every aspect of cell physiology.13,14 A number of studies have highlighted the role of miRNA in the regulation of fetal lung development. By using a conditional deletion of Dicer in embryonic lung epithelium, Harris et al.15 reported that the resulting mutant lungs exhibited severe lung epithelial branching defects and increased epithelial apoptosis, implicating that miRNA is involved in the regulation of lung development. Lu et al.16 found that transgenic overexpression of miR17– 92 resulted in the promotion of proliferation and the inhibition of differentiation of epithelial progenitor cells in developing lungs, whereas Ventura et al.17 proved that deletion of miRNA17–92 lead to lung hypoplasia. In fetal lung organ culture, Bhaskaran et al.18 uncovered that overexpression of miR-127 significantly decreased the terminal bud count, increased terminal and internal bud sizes, and caused unevenness in bud sizes, indicating that miR-127 may have an important role in fetal lung development. In a current study, our group performed miRNA profiling at three time points of the developing rat lung and identified seven miRNAs that showed significant changes in expression.19 Synthesis and metabolism of pulmonary surfactant in AECII is closely associated with the state of maturation of the fetal lung. While more and more members of miRNA family are discovered to involve in the regulation of fetal lung maturation, information on the role of miRNAs in the regulation of pulmonary surfactant synthesis and metabolism remains very limited. To date, only two miRNA members, miR-15011 and miR-375,20 have been reported to be involved in the regulation of pulmonary surfactant Pediatric Pulmonology

secretion. In searching for more miRNA members that may be involved in the regulation of pulmonary surfactant synthesis and metabolism, in this study, we selected miR26a as a potential candidate. It has been shown that miR26a is selectively expressed within the bronchial and alveolar epithelial cells in murine lung,21 and upregulated in the adult lung following the postnatal period of lung development.22 Bioinformatic analysis of the target genes of miR-26a has demonstrated that SMAD1 is one of the target genes for miR-26a, which is in accordance with the findings reported by Luzi et al.23 and Leeper et al.24 SMAD1 is expressed in the developing lung epithelium cells as well as in the surrounding mesenchyme,25 and is known to be an important transcription factor for the regulation of BMP signaling during lung development and pulmonary vascular remodeling.26,27 Reduction of endogenous SMAD1 expression caused 18% reduction of lung epithelial branching.26 When endogenous SMAD1 expression was knocked down, proliferation and differentiation of peripheral lung epithelial cells were inhibited, and secretion of pulmonary surfactants into the airspace was barely detectable, thus leading to immaturity of lung, extensive atelectasis and the development of RDS at birth.25 Taken together, it is postulated that miR-26a might be important in controlling essential developmental and physiological events in the lung, including the synthesis and metabolism of pulmonary surfactant in AECII. In this study, we investigated for the first time the regulation role of miR-26a in the synthesis of pulmonary surfactant. Our results show that miR-26a in fetal AECII was overexpressed after the transfection, and that the overexpression of miR-26a inhibited pulmonary surfactant synthesis in AECII by targeting SMAD1. We provide the first evidence that dynamic regulation of miRNAs may play an important role in the synthesis of pulmonary surfactant in AECII. MATERIALS AND METHODS Animals Sprague–Dawley Rats. All healthy adult rats (five female rats, weight 220–280 g and five male rats, weight 250– 300 g in total) were maintained in a specific pathogen-free an animal facility at animal center of the Nanjing Medical University.

Reagents

Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Wisent Biotechnology (St. Bruno, QC, Canada). Enhanced chemiluminescence reagent was obtained from Biogenes Biotechnology Corp (Nanjing, China). Adenovial miR-26a overexpression vectors were purchased from GeneChem

Regulation of Surfactant Synthesis by miR-26a

(Shanghai, China). Mouse anti-GAPDH antibodies were from Bioss USA (Woburn, MA). Reagents and primers for miRNA real-time PCR were obtained from Applied Biosystem (Applied Biosystems Incorporated, Forest City, CA). Prediction of miR-26a Target Genes and Bioinformatics Analysis

The target genes of miR-26a were predicted using miRNA target gene database miRGen. TargetScan and PicTar were used to predict target genes of hsa-miR-26a. The intersection of the two results and validated targets from DIANA LAB-TarBase6.0 database was analyzed by gene ontology (GO) and pathway analysis. Construction of Adenovial miRNA Overexpression Vectors

Using the miRBase sequence database (http://www. mirbase.org/), we identified the nucleotide sequence for the pre-miR-26a gene for matching to the rat genome database. We used Primer 5.0 software (Premier Biosoft International, Palo Alto, CA) to design the following primers, which are complementary to the nucleotide sequences that flanked the miR-26a gene: forward primer: 50 -CGGCCGCGACTCTAGCCCCTTCTCTTTGACAGTAG-30 ; reverse primer: 50 -ATAAGCTTGATATCGGCAGCAAGCTTGGCTGCAT-30 . We amplified a 320-bp PCR product from rat genomic DNA. The digested PCR product was cloned into GV264 plasmid between EGFP and VS40 poly A terminal sequence. The empty vector without miRNA insert was used as a vector control. Connecting products were transformed into competent Escherichia coli DH5a and were screened using LB plate with 100 mg/ml ampicillin. Part of the clone was selected for PCR identification and part for further sequencing. The sequencing results were analyzed based on miRNA database. Adenovirus was produced by transfecting the recombinant plasmid that carried the foreign gene and auxiliary packaging plasmid that carried most of the adenovirus genome into HEK 293A cells, and amplified by transducing HEK 293A cells. Virus titer was assayed by making dilutions of viral stock, transducing HEK 293A cells and counting virus-infected cells. miRNA Real-Time PCR

Total RNAs were isolated by using TRIzol (Invitrogen, Carlsbad, CA) and the miRNeasy Mini kit (Qiagen, Valencia, CA) following manufacturer’s instructions. Of total RNAs, 100 ng were used in reverse transcription for miR-26a or U6 snRNA. miR-26a was measured by realtime PCR using the TaqMan microRNA assay kit (Applied Biosystems).

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Isolation, Purification, and Primary Culture of Fetal AECII

Animal use was approved by the Nanjing Medical University Animal Care and Use Committee. Pregnant Sprague–Dawley rats were sacrificed with CO2 on day 19 of gestation. Fetal AECII at 19 days gestation were isolated using trypsin and collagenase digestions, followed by different centrifugal force and different adherences to remove fibroblasts, as described by Zhu et al.28 The cells were plated in DMEM with 10% FBS on six-well plates. The nature of the cultures was identified by electron micrograph. Cell viability was >97% as determined by the trypan-blue exclusion assay. The purity was >90% as determined by fluorescent immuocytochemistry with SP-C. Bioinformatic Analysis of miR-26a Target Genes

Targetscan 5.1 and Pictar were used to identify putative miRNA gene targets. Enrichment for KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway categories and GO biological process categories were determined using DAVID, with P < 0.05 cutoff. Proliferation and Cell Survival Assays

We determined cell viability using 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT; BIOSHARP, Nanjing, China). After 24 hr of incubation in a humid incubator at 378C in 5% CO2, cells were washed once and further incubated in serum-free MEM containing 0.5 mg/ml MTT for 4 hr at 378C. After medium aspiration, the formazan dye was extracted with DMSO and the absorbance was read at 492 nm using a microplate reader (NDM-9602, Perlong, Beijing, China). Results were expressed as a percentage of the control measured under normoxic conditions after subtracting background absorbance from all values. The AECII viability at 48 and 72 hr were detected using the same method, and the experiment was repeated three times. RNA Extraction and Quantitative Real-Time RT-PCR Analysis

Total RNA was isolated from AECII using the TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA). Total RNA (500 ng) each was reverse transcribed using MMLv revertase with 2 ml reverse primer following the manufacturer’s protocol in a final volume of 18 ml. RTPCR was performed by using SYBR-Green (Roche, Shanghai, China) and gene-specific primers synthesized according to published cDNA sequences. PCR reactions were performed on an ABI 7500 thermal cycler (Applied Biosystems). Relative quantification of gene expression between multiple samples was achieved by normalization Pediatric Pulmonology

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against endogenous controls GAPDH using the 2DDCt method of quantification. Western Blotting Analysis

Briefly, AECIIs were lysed on ice by total protein extraction kit. Total cellular lysate proteins (50 mg) were loaded for gels, and the separated proteins were transferred to PVDF membrane (Millipore, Massachusetts). The membrane was then blocked for non-specific binding for 2 hr by incubating with 5% fat-free dry milk in 100 mM Tris-buffered saline plus 0.1% Tween-18 (TBS-T). Primary antibody directed against rat SMAD-1 (1:500, BioWorld, Nanjing, China) and rat SP-C (1:100, Santa Cruz, CA) were diluted in blocking buffer and incubated with membrane overnight at 48C. After being washed in TBST three times, the membrane was incubated with HRP-conjugated goat–anti-rabbit secondary antibody (1:2,000, ZSGB, Beijing, China) for 1.5 hr at room temperature. The antibody-detected protein bands were visualized by enhanced chemiluminescence detection system. Statistical Analysis

Experiments were performed in triplicate and repeated for at least three times, and the results were expressed as the mean  standard deviation (SD). Statistical analysis was performed using SPSS13.0 statistical software. Groups were compared using the one-way analysis of variance (ANOVA). P < 0.05 was considered to be statistically significant. RESULTS Prediction of miR-26a Target Genes and Bioinformatics Analysis

Target genes exist in all of the cell components, including cell membrane, cytoplasm, and nucleus. The functions of these target genes were enriched in translation regulation, the protein modification, the regulation of cellular proliferation, apoptosis, and differentiation process, and kinase activity regulation. The Wnt-signaling pathway, MAPK (mitogen-activated protein kinase) signaling pathway, TGF-beta (transforming growth factor beta), the pathway of tumor p53 signaling pathways, cell cycle and adherens junction pathways were significantly enriched (Fig. 1). Construction of Adenovial miRNA Overexpression Vectors

Length of miR-26a precursor sequence we cloned is about 320 bp in the human genome. PCR detection (Fig. 2A) and the sequencing of recombinant plasmid both showed that the insert fragment is miR-26a precursor Pediatric Pulmonology

Fig. 1. KEGG pathways of miR-26a target genes. Analysis of the conserved putative targets of miRNA-26a reveals several enriched KEGG pathways (P < 0.05). Number of target genes/ category shown.

sequence (Fig. 2B and C). Recombinant plasmid was transfected into HEK293 cells, green fluorescent protein expression was observed after 3 days under the fluorescence microscope (Fig. 2D) and showed typical cytopathic effect (Fig. 2E). The virus titer of recombinant adenovirus Ad-26a reached 4  109 PFU/ml by repeated amplification. miR-26a Overexpression in AECII

To investigate the roles of miRNA in surfactant synthesis, we constructed adenoviral vectors for the overexpression of miRNAs. This vector encodes enhanced green fluorescence protein (eGFP) between miRNA sequence and CMV promoter. As a result, transfection efficiency can be monitored by examination of expressed eGFP. Mature miRNAs and flanking sequences were amplified from rat genome. An adenovirus expressing miR-26a was used to transduce primary AECII. At 48 hr post transfection, almost 95% cell had positive expression of eGFP (Fig. 3A), indicating that the transduction of the adenovirus into AECII reached nearly 95%. To examine whether miR-26a was overexpressed in AECII, total RNAs were isolated and mature miR-26a level was measured by real-time PCR. As shown in Figure 3B, compared to virus control, adenovirus vector for overexpression of miR-26a gave a significant higher expression level of miR-26a. An adenoviral vector expressing rat mature miR-26a and flanking sequences had a comparable miR-26a expression as the rat miR-26a vector. The rat miRNA expression vectors were used in all of the subsequent experiments. miR-26a Has No Effect on Cell Proliferation

We used the MTT assay to detect proliferation ability of AECII overexpressed with miR-26a. There was no significant difference between the blank control group,

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Fig. 2. Identification of miR-26a overexpression vector. A: 1—Negative control (ddH2O); 2—blank vector control; 3—positive control (GAPDH); 4—marker; 5–12—miR-26a 1–8 converter. B: The results of sequencing. C: miR-26a precursor stem loop structure. D: The GFP expression of Ad-26a in HEK293 cells. E: Typical cytopathic effect of Ad-26a (CPE).

the virus control group, and the miR-26a overexpressed group in cell proliferation as determined by MTT assay (Fig. 4). miR-26a Reduced the Expression of SP-B and SP-C

To determine whether lung surfactant synthesis in AECII is affected by overexpression of miR-26a, SP-B, and SP-C were detected. By real-time quantitative PCR, SP-B mRNA, and SP-C mRNA levels were found to be significantly reduced by 80% and 70% in overexpressing miR-26a AECII (Fig. 5A and B), in comparison with the virus control-treated cells and the blank control cells. In addition, SP-C protein was found to be significantly reduced as well (Fig. 5C). miR-26a Targeted Expression of SMAD1

Because miR-26a has been predicted to target SMAD1, we then assessed the direct effects of miR-26a on the

expression of the pro-differentiation BMP cascade molecule. Overexpression of miR-26a significantly decreased SMAD-1 expression by an average of threefold in comparison with the negative control virus cells and the blank control cells (Fig. 6A, P < 0.05). To confirm this effect at the protein level, AECIIs were harvested for Western blot analysis. Overexpression of miRNA-26a lowered the expression of SMAD-1 compared with the virus control-treated cells and the blank control cells. Densitometric analysis of these blots confirmed a twofold lower expression of SMAD-1 protein (Fig. 6B, P < 0.05). DISCUSSION

Pulmonary surfactant, essential for normal breathing, alveolar stability, and host defense system in the lungs, is synthesized, processed, packaged, secreted, and recycled by AECII during the latter stages of gestation.3 Inadequate pulmonary surfactant synthesis by the structurally immature lung in premature infants can result in RDS, the Pediatric Pulmonology

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Fig. 3. Overexpression of miR-26a in fetal alveolar type II cells. Type II cells was transduced with none (blank control) or adenoviruses carrying an empty vector (virus control), a rat miR26a, at an MOI of 100. At 24 hr post-transduction, GFP fluorescence was observed (A, rmiR-26a) and miR-26a levels were measured (B). The miR-26a expression levels were normalized to U6 snRNA using equation 2DDCt. Data in the graph are mean  SE for N ¼ 3.  P < 0.05.

leading cause of neonatal morbidity and mortality in developed countries. The maturation of fetal lung and the synthesis of pulmonary surfactant by AECII have been known to be regulated by a number of hormones and factors, including glucocorticoids, prolactin, insulin, growth factors, estrogens, androgens, thyroid hormones,

Fig. 4. Effects of overexpression of miR-26a on cell proliferation. MTT result of three groups (A value). Data in the graph are mean  SE for N ¼ 3. P > 0.05.

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Fig. 5. Effects of overexpression of miR-26a on surfactant synthesis. AECII were seeded on plastic dishes and transduced with none (blank control) or adenoviruses carrying an empty vector (virus control), a rat miR-26a (miR-26a), then cultured for 2 days. Total RNA or protein was extracted. Real-time PCR was done to determine the mRNA abundance of SP-C and SP-B. Western blot was used to detect the abundance of SP-C protein. A: miR-26a inhibits the expression of SP-C mRNA ( P < 0.05). B: miR-26a inhibits the expression of SP-B mRNA ( P < 0.05). C: Western blots confirm decreased SP-C levels with overexpress of miRNA-26a in AEC II. Left lanes: blank control; middle lanes: virus control; right lanes: overexpressed-miRNA-26a.

and catecholamines acting through cyclic AMP.3,4,7,8 In addition to these factors, miRNAs have been increasingly recognized to be involved in the regulation of fetal lung maturation.9,10 However, currently very little is known about the role of miRNAs in the regulation of pulmonary surfactant synthesis and metabolism. A number of miRNA profiling studies evaluating mouse and rat lung development have demonstrated the differential expression of miRNAs during lung development.22 Our previous work performed miRNA profiling at three time points (E16, E17, and E21) of the developing fetal rat lung and demonstrated that miR-26a was upregulated during late lung development.19 In agreement with this finding, our current study found that miR-26a in fetal rat AECII was overexpressed after transfection. Our finding here is also consistent with the results reported by other research groups, where they demonstrated that miR26a was selectively expressed within the bronchial and alveolar epithelial cells in murine lung,21 and upregulated in the adult lung following the postnatal period of lung development.22 Bioinformatic analysis revealed that the

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TABLE 2— Analysis of miR-26a Conserved Sequences Among Different Species Species Human Pongo pygmaeus a Mus musculus a Rattus norvegicus Suss scrofa Bos taurus Canis familiaris Zebrafish Gallus gallus a Xenopus tropicalis

Fig. 6. Effects of overexpression of miR-26a on SMAD1 expression. miR-26a decreases the SMAD1 protein expression. AECII cells were treated with miR-26a overexpressing viruses (MOI ¼ 100). A virus overexpressing GFP was used as a virus control. Cells with none adenoviruses were used as blank control. Forty-eight hours after treatment, mRNA (A) and protein (B) levels were examined using real-time PCR and Western blot. GAPDH was used as an internal control. For panel B, data are mean  SE (n ¼ 3 cell preparations, assayed in duplicate).

putative targets of miR-26a were enriched for several pathways relevant to lung development, including Wnt, TGF-b pathways, and cell cycle (Fig. 1). The large number of predicted targets of miR-26a within lung development-related pathways, and its highly conserved status (Table 1), coupled with the facts that miR-26a is selectively expressed in AECII, implicate that miR-26a could plays an important regulation role in biologic process of fetal lung development (Table 2). miR-26a has been reported to be involved in various cell functions. It has been shown that miR-26a has dual effects on various tumor cells and functions either as tumor suppressors or oncogenes in different types of tumors. It has been reported that miR-26a presented an

miRNA

Conserved sequence

hsa-miR-26a ppy-miR-26 mmu-miR-26 rno-miR-26a ssc-miR-26a bta-miR-26a cfa-miR-26a dre-miR-26a gga-miR-26 xtr-miR-26a

10-uucaaguaauccaggauaggcu-31 10-uucaaguaauccaggauaggcu-31 16-uucaaguaauccaggauaggcu-37 16-uucaaguaauccaggauaggcu-37 10-uucaaguaauccaggauaggcu-31 14-uucaaguaauccaggauaggcuu-35 1-uucaaguaauccaggauaggcu-22 11-uucaaguaauccaggauaggcuu-32 10-uucaaguaauccaggauaggc-30 14-uucaaguaauccaggauaggc-34

anti-proliferative property that inhibited cancer cell growth in human liver cancer, nasopharyngeal carcinoma, and human breast carcinoma.29–31 As an oncogene, miR26a was found to be amplified and promote cell growth in glioblastoma and glioma.32,33 Interestingly, in human lung cancer, Dang et al.34 demonstrated that miR-26a acted as a tumor suppressor, whereas Liu et al.35 reported that miR-26a enhanced lung cancer cell metastasis potential via modulation of metastasis-related gene expression, and activation of AKT pathway by PTEN suppression. In addition to its roles in tumors, miR-26a was shown to regulate the hypertrophy in human airway smooth muscle cell,36 promote reactive oxygen speciesinduced apoptosis in cardiomyocytes,37 and regulate the proliferative phase of liver regeneration by repressing expressions of cell cycle proteins CCND2 and CCNE2.38 In the present study, miR-26a is found to have another role in regulating pulmonary surfactant synthesis in AECII. We demonstrated that overexpression of miR-26a inhibited pulmonary surfactant synthesis in AECII. SP-B mRNA, SP-C mRNA, and SP-C protein levels were found to be significantly reduced in overexpressing miR-26a AECII, in comparison with the virus controltreated cells and the blank control cells. The AECII in our experiment was from embryonic day of 19 fetal rats, similar to 27–34 weeks of human fetal lung development, the expression level of SP-B protein may be low relatively; therefore, we could not detect effect of overexpression of miR-26a on the expression of SP-B at the protein level.

TABLE 1— Sequences of Oligonucleotides Used as Forward and Reverse Primers for Real-Time RT-PCR Gene product SMAD1 SP-B SP-C GAPDH

Forward primer

Reverse primer

CACACCCCCGCCTGCTTACC ATGCACCAGGCCTGCCTTCG GCAGAAACCGCTGCGGGACA TCAGTGCCGGCCTCGTCTCAT

TTGATCTCCGCGGGCAGTGTC AGCTGGGGCATGTGCCGTTC CCCCGAGGCTGTAGGAGACACC TGACCAGGCGGCCAATACGG

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Emerging evidence suggests that miRNAs are functionally associated with their host target genes. To further explore the underlying mechanism behind the role of miR-26a in regulating pulmonary surfactant synthesis, we investigated the target of miR-26a. Bioinformatic analysis of the target genes of miR-26a has showed that SMAD1 is one of the miR-26a target genes. Several experimentally validated miR-26a targets have been reported, such as MTDH,31 GSK3b,39 interleukin-6,40 interleukin-2,41 SMAD1,23,24 EZH2,24,32,42 CCND2,29 CCNE2,29 PTEN,32,33,43 RB1,33 and MAP3K2.33 However, none of these targets were indicated to be related to the role of miR-26a in the regulation of pulmonary surfactant metabolism in AECII. In this study, we have found that SMAD1 is a potential target of miR-26a in AECII, which is supported by our observation that overexpression of miR-26a inhibited SMAD1 expression both in mRNA and protein level in the fetal AECII. Our observation is consistent with the findings reported by two other research groups.23,24 SMAD1 has been shown to be expressed in the developing lung epithelium cells as well as in the surrounding mesenchyme.25,44 Alejandre-Alca´zar et al.44 has demonstrated that both nuclear and cytosolic staining were detected for SMAD1 in AECII in the alveolar airspaces and no staining was detected in the more elongate, thinner, type I pneumocytes (AECI). As one of the BMP receptor downstream signaling proteins, SMAD1 has been implicated to play a key role in organogenesis including lung development and maturation. The signal transduction pathways of BMPs have been extensively studied in cultured cells. Studies in vitro have demonstrated that BMP ligands bind to heteromeric BMP receptor complexes and activate the receptor serine/ threonine kinases, which subsequently phosphorylate cytoplasmic SMAD1, SMAD5, and SMAD8.45 These phosphorylated SMADs then dissociate from the receptors, form complexes with SMAD4, translocate into the nucleus, and bind to BMP-responsive elements in their target genes to modulate gene expression.45 It has been reported that the BMP signaling system is active during late lung development.44 Several studies in SMAD1 knockout mice have evidenced abnormal lung development. Chen46 has demonstrated that lungs of SMAD1 knockout mice displayed reduced genes, such as Scd1, Pon1, Gdpd2, Fabp5, Soat1, and Ldlr, which are related with lipid metabolism, suggesting that SMAD1 may be involved in pulmonary surfactant phospholipid metabolism. Peca et al.47 found reduced TTF-1 and SP-C in distal epithelial cells in lungs of SMAD1 knockout mice. Xu et al.25 reported that knocking down endogenous SMAD1 expression inhibited proliferation and differentiation of peripheral lung epithelial cells, and the secretion of pulmonary surfactants into the airspace, leading to development of RDS at birth. Given its predicted gene Pediatric Pulmonology

target, miRNA-26a may alter AECII biology in part via an inhibitory effect on the signaling pathways downstream of the BMP superfamily of growth factors. Our observing that overexpression of miRNA-26a in AECII inhibited the synthesis of SP-B and SP-C, and suppressed the expression of SMAD-1 both at mRNA level and protein level supports this miR-26a target interaction in AECII. miR-26a has been reported to play an important role in cell proliferation, differentiation, and apoptosis. The overexpression of miR-26a in the human lung adenocarcinoma cell line dramatically inhibited cell proliferation, blocked G1/S phase transition, and induced apoptosis in vitro.36 However, miR-26a was also found to promote cholangiocarcinoma growth.34 miR-26a can promote the differentiation of skeletal muscle cells.42 Conversely, others reported that miRNA-26a can inhibit differentiation as adipose-derived stem cells evolve towards lineagecommitted osteoblasts.23 In this study, we found that the cells with overexpressed miR-26a displayed no significant difference in cell proliferation capacity compared with virus control-treated cells and blank control cells. Our result, coupled with the findings reported by other researchers, suggests that the role of miR-26a in cell proliferation, differentiation, and apoptosis may depend on tissue types to influence protein translation during various cellular processes. It may also depend on their target genes that affect different biological pathways with diverse functions. Our research preliminarily studied the effect of miR26a on pulmonary surfactant synthesis in AECII, but this research still has deficiencies. First of all, pulmonary surfactant is composed of phospholipids and pulmonary surfactant protein, because of the immaturity of technology, we failed to detect phospholipids synthesis, only detected the changes of SP-B and SP-C expression; secondly, in view of the limitation of time and technology, we failed to do further experiments such as functional inhibitory effect of miR-26a on the expression of pulmonary surfactant; and finally, because of the limitation of time and technology, we are not able to do the luciferase reporter gene method to determine the direct regulation of miR-26a to SMAD1. In conclusion, the current study demonstrates that the overexpression of miR-26a in AECII inhibits pulmonary surfactant synthesis by regulating SMAD1-related BMP signal pathways. Our results suggest an important role for miR-26a in the molecular biology of pulmonary surfactant metabolism and implicate the potential application of miR-26a in the therapy of pulmonary surfactant-related lung disorder—RDS. REFERENCES 1. Andreeva AV, Kutuzov MA, Voyno-Yasenetskaya TA. Regulation of surfactant secretion in alveolar type II cells. Am J Physiol Lung Cell Mol Physiol 2007;293:L259–L271.

Regulation of Surfactant Synthesis by miR-26a 2. Crouch E, Wright JR. Surfactant proteins A and D and pulmonary host defense. Annu Rev Physiol 2001;63:521–554. 3. Mulugeta S, Beers MF. Surfactant protein C: Its unique properties and emerging immunomodulatory role in the lung. Microbes Infect 2006;8:2317–2323. 4. McDonald JA, editor. Lung growth and development. New York: Dekker Press; 1997. pp 495–575. 5. Whitsett JA, Stahlman MT. Impact of advances in physiology, biochemistry, and molecular biology on pulmonary disease in neonates. Am J Respir Crit Care Med 1998;157:S67–S71. 6. Rodriguez RJ. Management of respiratory distress syndrome: An update. Respir Care 2003;48:279–286. 7. Ballard PL. Hormonal regulation of pulmonary surfactant. Endocr Rev 1989;10:165–181. 8. Cross I. Regulation of fetal lung maturation. Am J Physiol 1990;259:L337–L344. 9. Sayed D, Abdellatif M. MicroRNAs in development and disease. Physiol Rev 2011;91:827–887. 10. Nana-Sinkam SP, Karsies T, Riscili B, Ezzie M, Piper M. Lung microRNA: From development to disease. Expert Rev Respir Med 2009;3:373–385. 11. Weng T, Mishra A, Guo Y, Wang Y, Su L, Huang C, Zhao C, Xiao X, Liu L. Regulation of lung surfactant secretion by microRNA150. Biochem Biophys Res Commun 2012;422:586–589. 12. Bartel DP. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 2004;116:281–297. 13. Croce CM, Calin GA. miRNAs, cancer, and stem cell division. Cell 2005;122:6–7. 14. Sonkoly E, Pivarcsi A. Advances in microRNAs: Implications for immunity and inflammatory diseases. J Cell Mol Med 2009;13: 24–38. 15. Harris KS, Zhang Z, McManus MT, Harfe BD, Sun X. Dicer function is essential for lung epithelium morphogenesis. Proc Natl Acad Sci USA 2006;103:2208–2213. 16. Lu Y, Thomson JM, Wong HY, Hammond SM, Hogan BL. Transgenic over expression of the microRNA miR-17–92 cluster promotes proliferation and inhibits differentiation of lung epithelial progenitor cells. Dev Biol 2007;310:442–453. 17. Ventura A, Young AG, Winslow MM, Lintault L, Meissner A, Erkeland SJ, Newman J, Bronson RT, Crowley D, Stone JR. Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters. Cell 2008;32: 875–886. 18. Bhaskaran M, Wang Y, Zhang H, Weng T, Baviskar P, Guo Y, Gou D, Liu L. MicroRNA-127 modulates fetal lung development. Physiol Genomics 2009;37:268–278. 19. Yang Y, Kai G, Pu XD, Qing K, Guo XR, Zhou XY. Expression profile of microRNAs in fetal lung development of Sprague– Dawley rats. Int J Mol Med 2012;29:393–402. 20. Zhang H, Mishra A, Chintagari NR, Gou D, Liu L. MicroRNA375 inhibits lung surfactant secretion by altering cytoskeleton reorganization. IUBMB Life 2010;62:78–83. 21. Moschos SA, Williams AE, Perry MM, Birrell MA, Belvisi MG, Lindsay MA. Expression profiling in vivo demonstrates rapid changes in lung microRNA levels following lipopolysaccharideinduced inflammation but not in the anti-inflammatory action of glucocorticoids. BMC Genomics 2007;8:240. 22. Williams AE, Moschos SA, Perry MM, Barnes PJ, Lindsay MA. Maternally imprinted microRNAs are differentially expressed during mouse and human lung development. Dev Dyn 2006;236: 572–580. 23. Luzi E, Marini F, Sala SC, Tognarini I, Galli G, Brandi ML. Osteogenic differentiation of human adipose tissue-derived stem cells is modulated by the miR-26a targeting of the SMAD1 transcription factor. J Bone Miner Res 2008;23:287–295.

871

24. Leeper NJ, Raiesdana A, Kojima Y, Chun HJ, Azuma J, Maegdefessel L, Kundu RK, Quertermous T, Tsao PS, Spin JM. MicroRNA-26a is a novel regulator of vascular smooth muscle cell function. J Cell Physiol 2011;226:1035–1043. 25. Xu B, Chen C, Chen H, Zheng SG, Bringas P Jr, Xu M, Zhou X, Chen D, Umans L, Zwijsen A, Shi W. SMAD1 and its target gene Wif1 coordinate BMP and Wnt signaling activities to regulate fetal lung development. Development 2011;138:925–935. 26. Chen C, Chen H, Sun J, Bringas P Jr, Chen Y, Warburton D, Shi W. SMAD1 expression and function during mouse embryonic lung branching morphogenesis. Am J Physiol Lung Cell Mol Physiol 2005;288:L1033–L1039. 27. Frank DB, Abtahi A, Yamaguchi DJ, Manning S, Shyr Y, Pozzi A, Baldwin HS, Johnson JE, de Caestecker MP. Bone morphogenetic protein 4 promotes pulmonary vascular remodeling in hypoxic pulmonary hypertension. Circ Res 2005;97:496–504. 28. Zhu H, Chang L, Li W, Zhang S. Isolation, purification and primary culture of fetal lung cells from rats. J Huazhong Univ Sci Technol Med Sci 2003;32:597–600. 29. Kota J, Chivukula RR, O’Donnell KA, Wentzel EA, Montgomery CL, Hwang HW, Chang TC, Vivekanandan P, Torbenson M, Clark KR, Mendell JR, Mendell JT. Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model. Cell 2009;137:1005–1017. 30. Lu J, He ML, Wang L, Chen Y, Liu X, Dong Q, Chen YC, Peng Y, Yao KT, Kung HF, Li XP. miR-26a inhibits cell growth and tumorigenesis of nasopharyngeal carcinoma through repression of EZH2. Cancer Res 2011;71:225–233. 31. Zhang B, Liu XX, He JR, Zhou CX, Guo M, He M, Li MF, Chen GQ, Zhao Q. Pathologically decreased miR-26a antagonizes apoptosis and facilitates carcinogenesis by targeting MTDH and EZH2 in breast cancer. Carcinogenesis 2011;32:2–9. 32. Huse JT, Brennan C, Hambardzumyan D, Wee B, Pena J, Rouhanifard SH, Sohn-Lee C, le Sage C, Agami R, Tuschl T, Holland EC. The PTEN-regulating microRNA miR-26a is amplified in high-grade glioma and facilitates gliomagenesis in vivo. Genes Dev 2009;23:1327–1337. 33. Kim H, Huang W, Jiang X, Pennicooke B, Park PJ, Johnson MD. Integrative genome analysis reveals an oncomir/oncogene cluster regulating glioblastoma survivorship. Proc Natl Acad Sci USA 2010;107:2183–2188. 34. Dang X, Ma A, Yang L, Hu H, Zhu B, Shang D, Chen T, Luo Y. MicroRNA-26a regulates tumorigenic properties of EZH2 in human lung carcinoma cells. Cancer Genet 2012;205:113–123. 35. Liu B, Wu X, Liu B, Wang C, Liu Y, Zhou Q, Xu K. miR-26a enhances metastasis potential of lung cancer cells via AKT pathway by targeting PTEN. Biochim Biophys Acta 2012;1822: 1692–1704. 36. Mohamed JS, Lopez MA, Boriek AM. Mechanical stretch upregulates microRNA-26a and induces human airway smooth muscle hypertrophy by suppressing glycogen synthase kinase3beta. J Biol Chem 2010;285:29336–29347. 37. Suh JH, Choi E, Cha MJ, Song BW, Ham O, Lee SY, Yoon C, Lee CY, Park JH, Lee SH, Hwang KC. Up-regulation of miR-26a promotes apoptosis of hypoxic rat neonatal cardiomyocytes by repressing GSK-3b protein expression. Biochem Biophys Res Commun 2012;423:404–410. 38. Zhou J, Ju W, Wang D, Wu L, Zhu X, Guo Z, He X. Down-regulation of microRNA-26a promotes mouse hepatocyte proliferation during liver regeneration. PLoS ONE 2012;7: e33577. 39. Stein U, Arlt F, Smith J, Sack U, Herrmann P, Walther W, Lemm M, Fichtner I, Shoemaker RH, Schlag PM. Intervening in betacatenin signaling by sulindac inhibits S100A4-dependent colon cancer metastasis. Neoplasia 2011;13:131–144.

Pediatric Pulmonology

872

Zhang et al.

40. Jones MR, Quinton LJ, Blahna MT, Neilson JR, Fu S, Ivanov AR, Wolf DA, Mizgerd JP. Zcchc11-dependent uridylation of microRNA directs cytokine expression. Nat Cell Biol 2009;11:1157–1163. 41. Xu H, Yao Y, Smith LP, Nair V. MicroRNA-26a-mediated regulation of interleukin-2 expression in transformed avian lymphocyte lines. Cancer Cell Int 2010;10:15. 42. Wong CF, Tellam RL. MicroRNA-26a targets the histone methyltransferase: Enhancer of Zeste homolog 2 during myogenesis. J Biol Chem 2008;283:9836–9843. 43. Sander S, Bullinger L, Klapproth K, Fiedler K, Kestler HA, Barth TF, Moller P, Stilgenbauer S, Pollack JR, Wirth T. MYC stimulates EZH2 expression by repression of its negative regulator miR-26a. Blood 2008;112:4202–4212.

Pediatric Pulmonology

44. Alejandre-Alca´zar MA, Shalamanov PD, Amarie OV, SevillaPerez J, Seeger W, Eickelberg O, Morty RE. Temporal and spatial regulation of bone morphogenetic protein signaling in late lung development. Dev Dyn 2007;236:2825–2835. 45. Massague J, Chen YG. Controlling TGF-beta signaling. Genes Dev 2000;14:627–644. 46. Chen C. The function of BMP4 downstream signal molecular SMAD1 in lung development. Chin Med Univ 2005. 47. Peca D, Petrini S, Tzialla C, Boldrini R, Morini F, Stronati M, Carnielli VP, Cogo PE, Danhaive O. Altered surfactant homeostasis and recurrent respiratory failure secondary to TTF-1 nuclear targeting defect. Respir Res 2011;12:115.

Regulation of pulmonary surfactant synthesis in fetal rat type II alveolar epithelial cells by microRNA-26a.

Pulmonary surfactant, a unique developmentally regulated, phospholipid-rich lipoprotein, is synthesized by the type II epithelial cells (AECII) of the...
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