Background

MicroRNAs (miRNAs) include a large group of regulatory, non-coding small RNAs that measure ~22 nucleotides (nt) in length [1, 2]. In animal cells, miRNAs are first transcribed from genes by the RNA polymerase, pol II, as primary miRNAs, which are then cleaved by an RNase III enzyme, Drosha, into hairpin-like precursor miRNA (pre-miRNA) [3]. The pre-miRNAs are transported into the cytoplasm with the help of a protein called Exportin 5 [4] In the cytoplasm, the pre-miRNAs are cut into double stranded RNA duplexes by another RNase III enzyme, Dicer [5]. Typically, one of the strands becomes mature miRNA and is incorporated into a RISC complex with other components for target recognition [6]. The RISC complex then binds to its target mRNA through base pairing and carries out its functions. Customarily, in the target mRNAs of an animal miRNA, there are multiple complementary sites, which correspond to the same miRNA. This cooperative action makes inhibition more efficient [7, 8]. On the other hand, one miRNA usually inhibits multiple target mRNAs [9, 10]. This property enables miRNA to regulate many genes in a pathway or physiological process at the same time. The action of miRNAs includes cleavage of target mRNA, translational inhibition, and mRNA deadenylation [11–15]. Several studies have shown that the degree of complementarity between the miRNA and its target determines the mode of how a miRNA works [11, 56]. In the lung, the alveolar epithelial type II cells secrete surfactant through exocytosis, which helps to reduce the surface tension of the alveolar sacs and facilitate the normal function of gas exchange. The mechanism of the secretion of surfactant in the lung is similar to that of the secretion of insulin in the pancreas. We suspect that mmu-miR-375 works in both of these exocytosis processes. We may find some hints as to the mechanism of exocytosis in the lung if we find more targets of miR-375 or any of the components that interact with these targets.

Two well-known miRNAs, miR-1 and miR-133, have highly specific expression in cardiac and skeletal muscle tissue [55]. In our study, we also identified these miRNAs as having heart-specific expression. These two miRNAs are clustered together in the mouse genome and both of them modulate muscle proliferation and differentiation. miR-1 promotes myogenesis by targeting histone deacetylase 4 (HDAC4), while miR-133 promotes myoblast proliferation by inhibiting serum response factor (SRF) [57].

A brain-specific miRNA, miR-9, has been identified by our microarray as well as by microarrays from other groups. It has been shown to affect neural lineage differentiation in ES cells. STAT3, which is a member of the signal transducer and activator of transcription family, is believed to be involved in this function [27]. In presenilin-1 null mice, miR-9 has been shown to be down-regulated, leading to severe brain developmental defects [38].

The liver-specific miRNA, miR-122, likely modulates the hepatitis C virus by facilitating replication of the viral RNA. Mutational analysis and ectopic expression studies have revealed that miR-122 interacts with the 5' non-coding region of the viral genome [58]. This suggests that miR-122 may be a target for antiviral interaction [58]. In addition, miR-122 is a key regulator of cholesterol and fatty-acid metabolism in the adult liver by regulating plasma cholesterol levels, fatty-acid oxidation, hepatic fatty-acid synthesis as well as cholesterol synthesis [22].

Among the spleen-specific miRNAs identified, five of them belong to the mir17 miRNA cluster, which comprise miR-17, miR-18, miR-19a, miR-19b, miR-20, miR-25, miR-92, miR-93, miR-106a, and miR-106b [59]. Among these 5 miRNAs, miR-17-5p, miR-17 and miR-20 belong to one of the mir17 microRNA clusters, the mir-17–92 cistron, which is one with well characterized cancer association. The mir-17–92 polycistron is located at 13q31, a genomic locus that is often amplified in cancers. The substantial increase in the expression of microRNAs from this cistron has been reported in human B-cell lymphomas and human lung cancers [60, 61]. However, the prominent expression and function of these miRNAs in the spleen are not known.

There are few studies concerning the functions of miRNAs in the lung. Several recent studies have given rise to a great interest in this field of research. The reduction in the expression of let-7 in human lung cancers is correlated to increased death rates in patients [62]. Experimentally, over-expression of let-7 can inhibit lung cancer cell growth in vitro. This discovery shows that let-7 may have potential clinical value in treating lung cancers. Inactivation of Dicer results in the defect of epithelial branching [63]. This defect is independent of the requirement for Dicer in cell survival and does not stop the epithelial growth [63]. In the E11.5 lung, Ago1 and Ago2 are enriched in the branching regions, which undergo the most dynamic changes during lung remodeling. This discovery suggests that miRNAs regulate processes responsible for the biogenesis of the lung [64]. Another study shows that the decrease in Dicer expression is associated with the poor prognosis in lung cancer patients [65]. The miRNA expression profiles in lung cancers correlate with the prognosis of lung adenocarcinoma patients [66].

In summary, we designed a reliable miRNA microarray platform that is low in cost and easy to update with highly reproducible results. The expression profiling of microRNAs in 6 rat organs was detected with this platform. The expression patterns of lung-specific and lung co-expressed microRNAs were confirmed by Northern blot analysis. Our platform adds to the implementation of detecting microRNA profiles, as no other microarray platform has been made for the detection of rat microRNA profiles. Furthermore, our microarray platform contains several recently discovered miRNAs, making it supplementary to other platforms. When applied, our study of the expression patterns of miRNAs in the lung should shed light on the functions of miRNAs in lung physiology as well as lung pathophysiology.

Methods

Microarray fabrication

217 mature miRNA sequences were downloaded from the miRNA registry (Wellcome Trust Sanger Institute). 177 of these were from rat, and 40 non-redundant conservative ones were from human or mouse. Sequences of some of these human and mouse miRNAs did not match their corresponding sequence in the rat genome exactly and were modified in accordance with those in the rat genome. The probes for the miRNAs had two copies of antisense sequences (34–50 nt) (Figure 1A). Probe sets, which contained 5' amino modified C6 oligos were synthesized by Sigma-Genosys (Woodlands, TX) at 100 μM concentration and suspended in 3 × SSC buffer. The oligos were diluted to 25 μM with 3 × SSC prior to use. The probes were then printed onto epoxy-coated slides (CEL Associates, Pearland, TX) with an OmniGrid 100 array (GeneMachine, San Carlos, CA) at 65% humidity and then incubated for 48 hours at the same humidity. Each slide contained three identical blocks in a landscape orientation. Within each block every probe was printed 6 times in 3 separate pairs (Figure 1B). The oligo set also contained one probe for U6 and 3 probes for tRNAs as positive controls as well as one probe for a plant miRNA as a negative control.

Tissue sample and small RNA extraction

Four male Sprague-Dawley rats (200 g, Charles River Laboratories, Inc., Wilmington, MA) were anaesthetized. The six organs (lung, heart, brain, kidney, liver and spleen) were collected and powdered in liquid nitrogen. Small RNA was enriched from the powdered samples from the 6 rat organs using the mir Vana™ microRNA isolation kit from Ambion (Austin, TX), according to the manufacturer's protocol. First, 200 mg powder was homogenized in 2 ml Lysis/Binding Buffer. Then, one-tenth of the volume of miRNA homogenate additive was added to the homogenate and a volume of acid-phenol: chloroform was used to extract RNA. One-third volume of 100% ethanol was added to the aqueous phase, and the sample was passed through a filter cartridge. Two-thirds volume of 100% ethanol was then added to the filtrate, and the sample was passed through a second filter cartridge. The second filter cartridge was subsequently washed once with wash solution 1 and then twice with Wash Solutions 2 and 3. Afterwards, the small RNA was eluted with 95°C nuclease-free water. Total RNA for Northern blots was also extracted from these organs by the aforementioned protocol. Following organic extraction, one and one-forth volumes of 100% ethanol was added to the aqueous phase. The lysate/ethanol mixture was then passed through a filter cartridge. The cartridge was washed, and the total RNA was eluted with water as described above. The concentration of RNA was determined by a NanoDrop ND-1000 Spectrophotometer (NanoDrop Tech., Rockland, DE). The quality of enriched small RNA was determined on a denaturing 15% polyacrylamide gel, and the quality of total RNA was tested on a 1% agarose gel.

miRNA labeling and microarray hybridization

The labeling and hybridization of miRNA were performed with the 3 DNA array900 miRNA direct kit (Genisphere, Hatfield, PA), according to the manufacturer's protocol. Enriched small RNA (120 ng) was used for each hybridization. First, the miRNA was tailed with poly A by PAP enzyme (poly (A) polymerase). Then the capture sequence was ligated to the tailed miRNA. Tagged miRNA was purified with the MinElute PCR Purification Kit (Qiagen, Valencia, CA). All of the small RNA samples were separately labeled with Cy3 or Cy5 capture sequence. After labeling and purification, equal amounts of small RNA from all the samples, labeled with the same dye, were pooled together as a common reference. The hybridization was performed as previously described [54]. To each block, one labeled sample was hybridized along with a common reference labeled with the other dye. Dye-swap was performed to eliminate dye bias. Tagged miRNA hybridizations were performed at 52°C overnight, and then the slide was washed 15 min in pre-warmed 2 × SSC, 0.2% SDS, followed by 12 min in 2 × SSC at room temperature and finally for 12 min in 0.2 × SSC at room temperate. The 3 DNA hybridization was performed at 62°C for 4 h, and then the slide was washed and dried.

Microarray data analysis

The hybridized slides were scanned with ScanArray Express (PerkinElmer Life and Analytical Sciences, Boston, MA), and the images were analyzed with GenePix 5.0 pro (Axon Instruments, Inc. Union City, CA). The signal from each spot was normalized to the average signal of the whole block. The highest and lowest signals from the 6 identical probes in the same block were excluded from the data analysis. The geometric average of the other 4 signals was considered to be the signal of that particular miRNA. The ratio of the sample signal to the reference signal was log2 transformed. A quality test was performed with the software, Realspot, developed in our laboratory [52]. The miRNAs with an average quality index of <1 were filtered. The miRNAs that passed the quality test were analyzed with SAM (Significant Analysis of Microarray) in order to choose miRNAs that were significantly changed between different organs (q < 0.01) [53]. These miRNAs were then subject to the Tukey Honestly Significant Difference (HSD) test (p < 0.05) [54]. The organ specificity index (OSI) was also used to determine the relative specificity of miRNA in organs. The OSI was defined as the correlation coefficient of miRNA expression between a miRNA and a putative miRNA whose expression levels were given the value of 1,000 in prominent organs and a value of 0 in other organs [54].

Northern blot analysis

Total RNA from the same organ was pooled together. The probe sequences were exactly the same as the antisense sequences to miRNAs except that those with sequences which started with C were capped with G or T at the 5' end to increase 32P labeling efficiency. RNA samples were denatured at 95°C for 4 minutes. 15 μg total RNA was separated on a 15% denaturing PAGE gel at 100 V for 2 h in 1 × TBE buffer. The RNA was then transferred to a Hybond-N+ membrane (Amersham, Piscataway, NJ) using a Trans-blot SD semi-dry transfer cell (Bio-Rad, Hercules, CA) at 20 ~ 25 V for 1 hour using 0.25 × TBE as a transfer buffer. Membranes were UV crosslinked with a 120 mJ burst and then baked at 80°C for 1 hour. For each sample, 20 pmol antisense oligonucleotide probes were end labeled by γ32P dATP (>7000 Ci/mmol, MP Biomedical, Irvine, CA) with T4 polynucleotide kinase (NEB, Ipswich, MA) for 4 hours at 37°C. The reactions were stopped with 2 μl 0.5 M EDTA. The probes were then purified with a G-25 MicroSpin column (Amersham). Pre-hybridization and hybridization were carried out at 30°C using ULTRAhyb-Oligo hybridization buffer (Ambion), according to the manufacturer's manual. After hybridization the membranes were washed twice with 2 × SSC 0.5% SDS for 30 minutes at 30°C. The membranes were then exposed to a phosphor screen overnight and scanned with the Personal Molecular Imager® FX (Bio-Rad). U6 was probed as a loading control and only exposed to the phosphor screen for 5 to 10 minutes.