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Keywords:

  • microRNA;
  • lung;
  • mouse;
  • human;
  • postnatal;
  • neonatal;
  • fetal;
  • adult

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

MicroRNAs (miRNAs) are a recently discovered class of noncoding genes that regulate the translation of target mRNA. More than 300 miRNAs have now been discovered in humans, although the function of most is still unknown. A highly sensitive, semiquantitative real-time polymerase chain reaction method was used to reveal the differential expression of several miRNAs during the development of both mouse and human lung. Of note was the up-regulation in neonatal mouse and fetal human lung of a maternally imprinted miRNA cluster located at human chromosome 14q32.31 (mouse chromosome 12F2), which includes the miR-154 and miR-335 families and is situated within the Gtl2-Dio3 domain. Conversely, several miRNAs were up-regulated in adult compared with neonatal/fetal lung, including miR-29a and miR-29b. Differences in the spatial expression patterns of miR-154, miR-29a, and miR-26a was demonstrated using in situ hybridization of mouse neonatal and adult tissue using miRNA-specific locked nucleic acid (LNA) probes. Of interest, miR-154 appeared to be localized to the stroma of fetal but not adult lungs. The overall expression profile was similar for mouse and human tissue, suggesting evolutionary conservation of miRNA expression during lung development and demonstrating the importance of maternally imprinted miRNAs in the developmental process. Developmental Dynamics 236:572–580, 2007. © 2006 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

MicroRNAs (miRNAs) comprise a large family of small, noncoding genes that have been implicated in regulating the translation of many protein-coding genes (Ambros,2001). miRNAs are expressed as long primary-miRNAs in the nucleus where they undergo initial processing by the RNase III type enzyme Drosha to form pre-miRNAs approximately 70 nucleotides in length (Pillai,2005). Further processing in the cytoplasm by the enzyme Dicer results in the formation of mature 21- to 23-nucleotide length, double-stranded miRNAs, which are incorporated into an RNA-induced silencing complex (RISC). Following the loss of one strand of the miRNA, binding of a guide strand to a target mRNA occurs in a noncomplementary manner, resulting in repression of protein translation through an unknown mechanism (Pillai,2005).

The importance of miRNAs during vertebrate development was clearly demonstrated in zebrafish (Giraldez et al.,2005) and mice deficient in the enzyme Dicer (Bernstein et al.,2003; Harfe et al.,2005; Yang et al.,2005). Thus, loss of Dicer resulted in embryonic lethality in both species. Large-scale screening using miRNA-specific microarrays revealed the differential expression of many miRNAs in various animal tissues (Lagos-Quintana et al.,2002; Babak et al.,2004; Monticelli et al.,2005), whereas in situ hybridization studies have shown that many miRNAs are even cell-type specific within individual organs (Wienholds et al.,2005). Moreover, a single miRNA often dominates the population of all expressed miRNAs in a given tissue, suggesting a role in maintaining tissue and/or cell phenotype (Lagos-Quintana et al.,2002).

Although the function of most miRNAs is unknown, they do play a significant role during mammalian morphogenesis. Thus, limb development was shown to be affected by miR-196 (Hornstein et al.,2005); miR-1 and miR-133 have been implicated in cardiogenesis (Kwon et al.,2005; Zhao et al.,2005) and skeletal muscle development (Chen et al.,2006); while miR-181 expression enhanced the differentiation of myoblasts (Naguibneva et al.,2006). miRNAs have also been shown to regulate the differentiation of neuronal progenitor cells (Krichevsky et al.,2006), inhibit neuronal dendrite growth (Schratt et al.,2006), affect skin morphogenesis (Yi et al.,2006), hair follicle growth (Andl et al.,2006), and angiogenesis (Yang et al.,2005).

Significantly, miRNAs have been strongly implicated in the morphogenesis of embryonic lung tissue. Thus, a transgenic mouse model containing a conditional Dicer knockout exhibited defective development (Harris et al.,2006). In this study, we have attempted to build upon this observation by identifying individual miRNA that might be involved in lung development and morphogenesis. To this end, we have used a novel and highly sensitive real-time polymerase chain reaction (RT-PCR) approach to determine the differential miRNA expression profile in adult mouse and human lung compared with that in mouse neonates and human fetal lung. These data revealed remarkable similarities in the overall expression profile in adult mouse and human lung, as well as the differential expression of miRNAs during lung development. Notably, an miRNA cluster located within the maternally imprinted Gtl2-Dio3 region at human chromosome 14q32.31 was up-regulated in neonatal/fetal lung. In situ hybridization revealed miRNA expression predominantly in epithelia, although a member of the miRNA cluster miR-154 was predominantly localized in the stromal tissue in neonate lungs and suggested that differences in expression were partly due to changes in distribution of cell types within the lung.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Comparison of miRNA Expression Profile in Adult Mouse and Human Lung

Initial studies were undertaken to determine the relative miRNA quantities in adult mouse and human lungs using a novel RT-PCR based approach (Chen et al.,2005; Fig. 1). The relative expression (ΔCT) of individual miRNA was determined by normalization to 18S. The absolute CT values for 18S remained constant across samples and time points obtained from each species; therefore, the ΔCT values (miRNA assays vs. 18S) could be used to compare the relative miRNA expression in mouse and human lung samples (data not show). The relative levels (ΔCT) of all miRNAs expressed in adult mouse and human lungs are shown in Supplementary Table S1(which can be viewed at http://www.interscience.wiley.com/jpages/1058-8388/suppmat) and the 30 most expressed in both species shown in Figure 1 (only those miRNA that were most highly expressed are presented for purposes of clarity and physiological relevance). These data indicate that there is substantial similarity between species. For example, 23 of the 30 most highly expressed miRNAs are shared in adult lungs from mice (Fig. 1A) and humans (underlined in Fig. 1B), and the relative expression levels (ΔCT) are very similar. Of note, miR-26a is the most highly expressed individual miRNA, whereas the let-7 family, miR-29 family, miR-30 family, and miR-199 are also expressed highly in both species.

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Figure 1. MicroRNA (miRNA) expression profile in mouse and adult lung. Total RNA was extracted from the adult lung of mice and human. The relative expression of miRNA was determined by real-time polymerase chain reaction (RT-PCR), normalized to 18S and are, therefore, represented as the mean ΔCT ± SEM (n = 3–4). A,B: The top 30 most expressed miRNA in mouse (A) and human (B) lung are plotted. Those underlined in the human profile (B) show those that are also present in the mouse (A).

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Differential Expression of miRNAs in the Postnatal and Adult Lung of Mice

A detailed account of the miRNA expression profile during postnatal lung development was previously unreported. For this reason, the levels of miRNAs were measured (ΔCT) in whole lung tissue from 1- and 14-day-old neonatal mice (Supplementary TableS2) and 60-day-old adult mice (Supplementary Table S1). The fold-change in miRNA expression between neonatal and adult lung was also calculated (Supplementary Table S3). The differential miRNA expression in neonatal lung compared with adult lung (Fig. 2) revealed a group of 14 miRNAs that were more highly expressed in the neonatal lung (Fig. 2A). In particular miR-154, miR-323, miR-335, miR-337, and miR-370 were expressed more than 20-fold higher (an increase of > 4 CT units) compared with adult lung. In contrast, 30 miRNAs were up-regulated in adult lung compared with neonatal lung (Fig. 2B,C). In particular, miR-29a, miR-29b, miR-29c, miR-150, and miR-151 were expressed more than 50-fold higher in adult lung compared with neonatal lung. The miRNA levels at 14 days were often intermediate between 1 day and 60 days, which suggests a gradual developmental shift in the expression pattern.

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Figure 2. Differential miRNA expression profile during mouse lung development. All values were normalized against 18s CT values and are, therefore, represented as ΔCT (the lower the value the more highly expressed). A: The microRNAs (miRNAs) expressed more highly in 1 day neonatal lung compared with 14-day- and 2-month-old adult lung. B,C: The miRNAs that are more highly expressed in adult 2-month-old lung compared with 1-day-old neonatal lung. Only those miRNAs that were statistically significant (P < 0.05) are represented and are given as mean ± SEM (n = 3).

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Differential Expression Profile in Human Fetal and Adult Lung

To determine miRNAs that have an evolutionary conserved role in lung development, we examined the differential expression profile between fetal and adult human lung (Supplementary Table S4). In this case, 13 miRNAs were expressed more in fetal lung (Fig. 3A), while 8 miRNAs were up-regulated in adult lung (Fig. 3B). In particular, miR-299, miR-323, and miR-368 levels were more than 15-fold higher in fetal than adult lung. Conversely, miR-29a, miR-29b, and miR-29c were expressed the highest in adult lung compared with fetal human lung. Significantly, many of these changes in miRNA expression were common to mice. Specifically, the expression of miR-134, miR-154, miR-214, miR-296, miR-299, miR-323, miR-337, and miR-370 were highly expressed in both neonatal mouse and fetal human lung and subsequently down-regulated in adult lung. In contrast, miR-26b, miR-29a/b, miR-146-3p, and miR-187 were up-regulated in adult tissue in both mice and humans.

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Figure 3. Differential microRNA (miRNA) expression profile during human lung development. All values were normalized against 18s CT values and are, therefore, represented as ΔCT (the lower the value the more highly expressed). A: The miRNAs expressed more highly in fetal human lung compared with adult human lung. B: The miRNAs that are more highly expressed in adult human lung compared with fetal human lung. Only those miRNAs that were statistically significant (P < 0.05) are represented and are given as mean ± SEM (n = 3).

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Genomically Clustered miRNAs That Are Differentially Expressed During Lung Development

Genomic analysis of those miRNAs that were highly expressed during development, in both neonatal mouse and fetal human lung, showed that many are clustered at mouse chromosome 12F2 or the syntenic region at human chromosome 14q32.31 (Table 1). These miRNAs, which include miR-134, miR-154, miR-299, miR-323, miR-337, miR-368, and miR-370, might be coordinately expressed. Significantly, these regions on mouse chromosome 12F2 and human chromosome 14q32.31 incorporate the imprinted Gtl2-Dio3 domain, which extends over 1.1 Mb. Importantly two miRNA clusters within this domain are only expressed from the maternal chromosome: one cluster is located in a retrotransposon-like gene Rtl1 (miR-337 and miR-370), while the second cluster is located 150 kb upstream in the miRNA-containing gene Mirg (including miR-154). The remainder of the miRNAs up-regulated in fetal lung do not appear to be associated with any known genomic clusters (Table 1). Of the miRNAs whose expression is increased in adult lung, few are associated with known clusters (Table 2). In mice, the exceptions are miR-23a and miR-27a that cluster on mouse chromosome 8, and miR-34b and miR-34c that cluster on mouse chromosome 9. The miR-29a and miR-29b cluster on mouse chromosome 6/human chromosome 7 and were found to be up-regulated in both adult mouse and adult human lung.

Table 1. Genomic Location of miRNAs Down-Regulated During Lung Development
miRNAMouseHumanSpecies expression
miR-13412F214q32.31Both
miR-15412F214q32.31Both
miR-29912F214q32.31Both
miR-32312F214q32.31Both
miR-33712F214q32.31Both
miR-368Not found (12F)14q32.31Human
miR-37012F214q32.31Both
miR-122a18E118q21.31Mouse only
miR-135b1E41q32.1Both
miR-199b2B9q34.11Human only
miR-2141H11q24.3Human only
miR-2962H320q13.32Both
miR-30111C17q23.2Both
miR-302c3H14q25Mouse only
miR-3356A37q32.2Both
Table 2. Genomic Locations of miRNAs Up-Regulated During Lung Development
miRNAMouseHumanSpecies expression
miR-23a8C319p13.12Mouse only
miR-27a8C319p13.12Mouse only
miR-29a6A37q32.3Both
miR-29b6A37q32.3Both
miR-34b9B11q23.1Mouse only
miR-34c9B11q23.1Mouse only
let-7b15E322q13.31Mouse only
let-7d13B19q22.32Mouse only
let-7g9F13p21.2Mouse only
miR-15a14C313q14.2Mouse only
miR-26a9F43p22.3Mouse only
miR-26b1C32q32Both
miR-29c1E41q32.2Both
miR-30a-3p1A56q13Mouse only
miR-30d15D38q 24.22Mouse only
miR-30e4D11p34.2Mouse only
miR-99a16C3.121q21.1Mouse only
miR-1009B11q24.1Mouse only
miR-1262A39q34.3Mouse only
miR-142-3p11C17q23.2Both
miR-142-5p11C17q23.2Both
miR-14611B1.15q33.3Both
miR-1507B419q22.32Both
miR-15115E18q24.3Mouse only
miR-18718B118q12.2Both
miR-1909D15q22.2Mouse only
miR-19311B517q11.2Mouse only
miR-19511B417p13.1Mouse only
miR-20312F314q 32.33Mouse only
miR-222XA2Xp11.1Mouse only
miR-223XC2Xq12Human only
miR-33811E217q25.3Mouse only

In Situ Hybridization on Neonatal and Adult Mouse Lung Tissue

To support the semiquantitative RT-PCR data, the spatial expression of the most relevant miRNAs was examined by in situ hybridization using miRNA-specific locked nucleic acid (LNA) probes. Of the 156 miRNAs studied, 3 miRNAs that were either highly expressed or exhibited high levels of differentiated expression were analyzed from neonatal and adult mouse lung tissue. These included miR-26a as it is the most highly expressed miRNA in lung tissue, irrespective of age or species; miR-29a as this is the most up-regulated in adult tissue; and miR-154 as this is the most highly expressed miRNA in neonatal tissue compared with adult and is a member of the maternally imprinted miRNA cluster. In addition, a scrambled miRNA probe was used as a nonspecific hybridization control.

The general expression pattern for all three lung-expressed miRNAs was different in neonatal tissue compared with adult tissue (Fig. 4). In neonatal lung, miR-26a was predominantly expressed at the basal side in epithelia of bronchioles and larger airways, with diffuse expression throughout the stromal tissue. In adult lung tissue, miR-26a was highly expressed in the epithelia of large airways, endothelia of arterioles, and venules and less intensely within alveolar epithelium (Fig. 4A). A similar pattern was observed for miR-29a, which was predominantly expressed in adult airway and alveolar epithelium, while high expression in neonatal tissue was restricted to larger airway epithelium with a less intense, diffuse expression throughout the stroma (Fig. 4B). Both miR-26a and miR-29a showed higher staining intensities in adult tissue, although staining was more widely distributed in neonatal tissue. Conversely, miR-154 was highly expressed throughout neonatal lung tissue, including epithelia and stromal cells, while expression was most intense in the airway and alveolar epithelium of adult tissue (Fig. 4C). No expression was observed for the scrambled miRNA probe (Fig. 4D).

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Figure 4. In situ hybridization on mouse lung tissue. MicroRNA (miRNA) expression was compared between the lungs of neonatal (1 day) and adult (3 months) BALB/c mice. Locked nucleic acid (LNA) probes, specific for miR-26a, miR-29a, and miR-154 were labeled with digoxigenin (DIG) and expression visualized using alkaline phosphatase substrate. All slides were taken at an original magnification of ×400. Arrows indicate miR-26a expression at the basal surface of airway epithelial cells.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

At present, little is known of the role of miRNAs in lung physiology and development. For this reason, we used an RT-PCR–based approach to compare the expression of miRNAs in adult lungs obtained from mice and humans and investigated their role during lung development. This approach has several advantages compared with existing techniques. Principally, unlike DNA-based microarrays, it permits relative quantification of mature miRNAs over seven orders of magnitude. Furthermore, compared with the 5–50 μg of total RNA required to detect a single miRNA by Northern blotting; only 5 ng is required when using RT-PCR (Chen et al.,2005).

Using this approach, examination of the profile of miRNA expression in adult lung showed similarities between mouse and human. In both species, the most highly expressed miRNAs included miR-26a and members of the let-7, miR-29, miR-30 families. Significantly, this finding is similar to reports using microarrays (Babak et al.,2004) and Northern blotting (Sempere et al.,2004) that determined miRNA expression At present, there have been no reports of the functional roles of these miRNAs, although decreased let-7 expression has been associated with poor survival from lung cancer (Takamizawa et al.,2004; Yanaihara et al.,2006). Indeed, overexpression of let-7 in the human lung epithelial A549 cell line was shown to inhibit cell growth (Takamizawa et al.,2004).

The importance of miRNAs during lung development has been demonstrated using conditional Dicer knockout mice (Harris et al.,2006). Mice lacking the Dicer enzyme exhibit broad developmental abnormalities, including defective epithelial cell branching and disregulated programmed cell death (Harris et al.,2006). However, although several miRNAs have been shown to regulate the development of the heart (Zhao et al.,2005; Naguibneva et al.,2006) and brain and neuronal system (Krichevsky et al.,2003; Giraldez et al.,2005; Schratt et al.,2006) and be involved in embryonic stem cell differentiation (Houbaviy et al.,2003; Krichevsky et al.,2006), no information is available on the role that specific miRNAs play during lung development. To address this situation, we, therefore, compared the differential expression profile between mouse adult (60 days) and neonatal (1 and 14 days) lungs. The mouse is an ideal animal to study the latter stages of lung development as their lungs do not fully mature until approximately 30 days after birth, which coincides with the final processes of branching morphogenesis, vascularization, and alveolarization (Massaro and Massaro,1996). Because it is envisaged that those miRNA that are central to lung development might be evolutionarily conserved, we also examined samples obtained from adult and fetal human lungs. A direct comparison can be made with human development as the neonatal period of murine lung growth coincides with late human fetal and postnatal lung development (Thurlbeck,1975a,b).

Multiple miRNAs were shown to be differentially expressed during lung development. Interestingly, the increased expression of eight miRNAs in neonatal and fetal lung (miR-134, -154, -214, -296, -299, -323, -337, and -370) and the up-regulation of five miRNAs in adult mouse and human lung (miR-26b, -29a, -29b, -142-3p, and -187) were found to be conserved during development in both species. Although the functional role of these changes in miRNA expression is unknown, examination of their genomic localization shows that six of the miRNAs highly expressed in the developing lung of both species (miR-134, -154, -299, -323, -337, and -370), as well as miR-368 in humans, map to the Gtl2-Dio3 domain at human chromosome 14q32.31 (12F2 in mice), a region that is highly conserved between the two species. These miRNAs are contained within two separate clusters. miR-337 and miR-370 map to an intragenic region within the Rtl1 gene, while miR-134, miR-154, miR-299, miR-323, and miR-368 form part of a separate cluster that is located in the intergenic region known as Mirg (miRNA-containing gene; Seitz et al.,2004). Importantly, imprinting means that these miRNAs are only expressed from the maternally inherited chromosome and their expression is regulated by an intergenic germline-derived differentially methylated region (IG-DMG) located ∼200 kb upstream of the miRNA cluster (Seitz et al.,2004; Tierling et al.,2006). Imprinting also results in the Gtl-2 and Rian genes being expressed from the maternal chromosome, while Dio-3 and the retrotransposon-like gene Rtl-1 are expressed only from the paternal chromosome (Lin et al.,2003). This region also contains miR-127 and miR-136, and although their expression did not change during development, they are expressed in an antisense manner within the Rtl1 gene and actually inhibit its translation by means of an RNAi mechanism (Seitz et al.,2003; Davis et al.,2005). Overall, although members of this miRNA cluster were previously shown to be expressed in the trunk/head of the mouse embryo and in the adult brain (Seitz et al.,2004; Tierling et al.,2006), this is the first study to demonstrate their specific expression profile in the lung. Furthermore, aberrant regulation of imprinted genes has been associated with development of disease, for example Beckwith-Wiedemann syndrome is related to loss of proper control of the IGF2-H19 domain (Jouvenot et al.,2003). Differential expression and hence transcriptional control of the imprinted miRNAs at 14q32.31 suggests that manipulation of miRNA expression may influence disease outcome.

In a parallel situation, several miRNAs are up-regulated in the adult. For example, miR-29a and miR-29b are up-regulated in adult tissue, being expressed at a lower level in developing lungs in both mouse and human. These miRNA genes map to human chromosome 7q32.3 and the syntenic region at mouse chromosome 6A3. This region maps to a fragile site that has been associated with several cancers (Caldas and Brenton,2005). A deletion in such fragile sites, including that at chromosome 13q14.2 containing miR-15a and miR-16, often involves a deletion in one or more tumor suppressor genes. However, the fragile site FRA7H that includes miR-29a and miR-29b does not encode any tumor suppressor genes (Calin et al.,2004). This finding suggests that, if a deletion of these miRNAs were to occur, it may influence the progression of cancers by preventing the inhibition of genes involved in cell proliferation. A similar mechanism may exist during lung development, in that a low expression of these miRNAs may allow cell proliferation to take place in the neonate/fetus, while up-regulation in the mature adult lung may prevent further proliferation and maintain cellular homeostasis. Analysis of potential targets for these miRNAs and over- or underexpression studies will provide a clearer insight into the function of such genes.

In situ hybridization of neonatal and adult mouse lung tissue revealed a differential expression pattern dependent on age. The overall expression patterns for miR-26a, miR-29a, and miR-154 corresponded to the data obtained from the semiquantitative RT-PCR. Therefore, staining intensities for miR-26a and miR-29a were higher in adult tissue, while that for miR-154 was higher in neonatal tissue. Of interest, the expression of miR-26a appears to be localized to airway and alveolar epithelia in both adult and neonate and within the endothelium of adult lungs. Similarly, miR-29a is expressed in the airway epithelium of adult and neonate lung and dominantly expressed in the alveolar epithelium of adult lung. In contrast, although miR-154 is expressed in epithelium of adult and neonate, the expression is diminished in adult lung. This finding reflects the varied cell types found within the developing lung tissue, which differs to the epithelial-dominated adult lung. As the lung matures, it may be that expression levels alter as a result of loss, or gain, of specific cell types rather than changes within resident cells. For example, miR-154 was highly expressed within stromal cells of the neonatal lung, cell types that are largely absent from the mature lung. Therefore, miRNA expression may remain constant in epithelial cells throughout development as a consequence of retaining tissue homeostasis or cell phenotype. Maintenance of lung phenotype during development may require the subtle expression of tissue-specific miRNAs by migratory or transient cells, as well as resident epithelial cells.

In summary, a semiquantitative RT-PCR–based expression profile of mouse and human lung has been used to identify evolutionary conserved changes in miRNA expression during lung development. Importantly, we have shown for the first time that miRNAs expressed from a highly imprinted region at human chromosome 14q32.31 (12F2 in mice) are differentially down-regulated during lung development in both humans and mice. The identification of these miRNAs associated with lung development provides important information on the function of these noncoding genes. Furthermore, manipulation of these miRNAs may provide a novel therapeutic approach for the treatment of diseases that result in disregulation of normal development or homeostasis. For example, miRNAs implicated in epithelial cell proliferation may be manipulated to prevent the progression of lung cancer or, alternatively, to promote the regeneration of lung tissue after lung injury.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Animals and Experimental Design

Litters of 1-day-old BALB/c mice (Harlan, UK) were housed in specified pathogen-free conditions according to institutional and Home Office (UK) guidelines. Whole lungs were removed from pups and placed immediately in RNAse Later (Sigma), stored overnight at 4°C, and then frozen. Lungs from 1-day-old, 14-day-old, and 2-month-old mice were sampled. Total RNA was extracted using MirVana kits (Ambion) according to the manufacturer's guidelines and stored at −20°C. Human adult lung and fetal lung RNA, extracted using a modified guanidine thiocyanate technique (Chomczynski and Sacchi,1987), was supplied by two commercial sources. Adult lung total RNA was supplied by Stratagene (single donor, 49 years old), AMS Biotechnology (five donor pool from 23, 23, 26, 29, and 36 years old), or obtained from adult lung (26 years old), whereas fetal lung total RNA was supplied by Stratagene (two pooled from 18- and 20-week-old lung) and AMS Biotechnology (separate 26- and 29-week-old lung).

Semiquantitative RT-PCR

The miRNA expression profile for each lung sampled was analyzed using Applied Biosystems miRNA TaqMan Early Access panel, which consists of 156 individual miRNAs. The panel consists of reverse transcription primers and separate PCR primers for each miRNA and, therefore, uses a two-step reaction. A total of 2 ng of starting total RNA was used for each miRNA assay. The RT reaction per specific miRNA contained 0.0775 μl of dNTPs, 0.5 μl of Multiscribe, 0.75 μl of RT buffer, 0.094 μl of RNAse inhibitor, 2.081 μl of nuclease-free water (Promega), and 2.5 μl of total RNA (2 ng/μl) from TaqMan MicroRNA RT Kit (Applied Biosystems) plus 1.5 μl of RT primer (Applied Biosystems) or 1.5 μl of random hexamers (Applied Biosystems), and the reaction conditions were 30 min at 16°C, 30 min at 42°C, and 5 min at 85°C. The PCR reaction comprised 5 μl of TaqMan 2× Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems), 3.335 μl of RNAse-free water (Promega), 1 μl of TaqMan probe (Appled Biosystems) or 1 μl 18s probe (Applied Biosystems) plus 0.67 μl of RT product, and the reaction conditions were 30 min at 16°C, 20 min at 2°C, and 5 min at 85°C. The amount of RNA from each sample was calibrated to the expression of the ribosomal 18s house-keeping gene. This value then gave a delta CT (ΔCT) value for each miRNA (miRNA CT value − 18s CT value). As 18s expression was consistently higher than any miRNA, a higher miRNA expression level corresponded to a smaller ΔCT value. The ΔCT values for all miRNAs on the panel are shown in The Supplementary Materials. Those achieving significance <0.05, according to Student's t-test, after comparing mouse 1 day vs. 2 month or human fetal vs. adult samples are represented. Fold differences in miRNA expression were also calculated as 2-(ΔCT 1 day − ΔCT 2month) or 2-(ΔCT fetal − ΔCT adult) as an increase in 1 CT value was equivalent to a twofold increase in amount of starting cDNA (shown in the Supplementary Materials).

Genomic Analysis of miRNAs

Mouse and human miRNA sequences were analyzed using miRBase:Sequence Release 8.1 (Sanger Institute, Wellcome Trust, UK; http://microrna.sanger.ac.uk/sequences/). The genomic coordinates were determined for each miRNAs and mapped to a specific chromosomal region in both mouse and human genomes. Patterns of clustering of miRNA genes were determined. The synteny between the mouse and human genome was also established.

In Situ Hybridization

Lungs from 1-day-old or 3-month-old BALB/c mice were inflated and coated with O.C.T. compound (Tissue-Tek) and immediately frozen in liquid nitrogen. Cryosections were cut 12-μm-thick, and the sections fixed in 95% ethanol for 10 min and then dried in a desiccator for 20 min. Sections were fixed in 4% paraformaldehyde (Sigma) in phosphate buffered saline (PBS) for 10 min and then washed three times in PBS for 2 min each. Sections were incubated with 10 μg/ml proteinase K (Sigma) for 10 min at room temperature and immediately fixed again in 4% paraformaldehyde. After washing three times in PBS sections were prehybridized in hybridization buffer (50% formamide [Sigma], 5× standard saline citrate [SSC] buffer [Sigma], 250 μg/ml yeast RNA [Ambion], 1× Denhardt's solution [Fluka] in diethylpyrocarbonate [DEPC, Fluka) -treated water) for 1 hr at room temperature in a humidifying chamber. LNA probes were preincubated at 65°C for 5 min and immediately placed on ice. The prehybridization buffer was removed and replaced with 2.5 μM of miRNA-specific LNA probe or scrambled probe (Exiqon) labeling with digoxigenin (DIG) in hybridisation buffer in a humidifying chamber at 50°C for 18 hr. Sections were washed in stringency buffer (50% formamide, 5× SSC buffer in DEPC-treated water) that was warmed to 50°C twice for 45 min and once in PBS at room temperature. Sections were incubated in blocking buffer (10% sheep serum [Sigma] in PBS) for 1 hr at room temperature and then with sheep anti-DIG FAb fragments (Roche Diagnostics) labeled with alkaline phosphatase (AP) diluted 1:500 for 2 hr at room temperature. Sections were washed three times in PBS for 2 min and then incubated with AP-substrate nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (BCIP/NBT) substrate kit (Vector Laboratories) for 18 hr at room temperature in a humidifying chamber. Sections were washed twice in PBS and once in water and then mounted with aqueous mounting medium (DAKO) before microscopic examination.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

S.M. was supported by BBSRC, M.M.P. was supported by the Royal Brompton Clinical Research Committee, and M.A.L. and A.E.W. were supported by the Wellcome Trust.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

The Supplementary Material referred to in this article can be viewed at http://www.intersciece.wiley.com/pages/1058-8388/suppmat

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jws-dvdy.21047.doc792KSupporting Information file jws-dvdy.21047.doc

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