Identification of rat lung-specific microRNAs by micoRNA microarray: valuable discoveries for the facilitation of lung research

Abstract

Background: An important mechanism for gene regulation utilizes small non-coding RNAs called microRNAs (miRNAs). These small RNAs play important roles in tissue development, cell differentiation and proliferation, lipid and fat metabolism, stem cells, exocytosis, diseases and cancers. To date, relatively little is known about functions of miRNAs in the lung except lung cancer.

Results: In this study, we utilized a rat miRNA microarray containing 216 miRNA probes, printed in-house, to detect the expression of miRNAs in the rat lung compared to the rat heart, brain, liver, kidney and spleen. Statistical analysis using Significant Analysis of Microarray (SAM) and Tukey Honestly Significant Difference (HSD) revealed 2 miRNAs (miR-195 and miR-200c) expressed specifically in the lung and 9 miRNAs co-expressed in the lung and another organ. 12 selected miRNAs were verified by Northern blot analysis.

Conclusion: The identified lung-specific miRNAs from this work will facilitate functional studies of miRNAs during normal physiological and pathophysiological processes of the lung.

Figure 1

miRNA microarray fabrication and hybridization strategy. (A) The probes were two copies of antisense oligos of mature miRNA. The 5' ends of the probes were amino modified. The probes were linked to epoxy-coated slides covalently. miRNA samples were tagged and hybridized to the slides. (B) Each slide contained three identical blocks and each block was hybridized with two-colored paired samples. There were 6 duplicated probes for each miRNA as shown in block A. MIR xxxx, series number.

Figure 2

Specificity of miRNA microarray hybridization. The control oligos in the 3 DNA array900 microRNA direct kit were ligated with Cy5 capture sequence and hybridized onto one block. The mismatched probes had one (m1), two (m2) or three (m3) mismatched nucleotides with the corresponding control oligos. The signals from the slide were processed with the method described in Materials and Methods. Data shown were means ± S.E. from 3 hybridizations.

Figure 3

Organ-specific microRNAs. (A) Hot maps. The microRNA samples from 6 organs were co-hybridized with the common reference. The signals were processed as described in Materials and Methods. The normalized data were subjected to the SAM test [67]. The miRNAs that passed SAM were further tested by the HSD test (P < 0.05) to identify organ-specific miRNAs. Each column represents one hybridization and each row represents one miRNA. The figure was drawn by Treeview. Red represents positive values, green negative values, and black zero. The number of miRNAs for each organ is: Lung 2; heart 6; brain 13; liver 5; kidney 2 and spleen 18. (B) Summary of differently expressed miRNAs among 6 organs. The number beside each organ represents the number of miRNAs that are expressed significantly higher in this organ than any other organs (P < 0.05). The number on the line between any two organs is the quantity of miRNAs expressed significantly higher in these two organs than in other organs (P < 0.05).

Figure 4

Northern blot validation. 15 μg total RNA was separated on a denaturing 15% PAGE gel. The ³²P labeled probes for the microRNA were hybridized to the membrane overnight. The U6 snRNA was probed as the loading control after the hybridization of microRNA probes. (a) lung-specific miRNAs; (b) kidney-specific miRNAs; (c) co-expressed miRNAs in the lung and heart identified by HSD; (d) miRNAs highly expressed in the lung and/or the heart. (e) commonly expressed miRNAs.

Figure 5

Comparison between results from the microarray and Northern blots. The Northern blots in Figure 4 were quantitated by the Personal Molecular Imager^® FX and normalized to U6. Y axes represent the normalized intensities in the Northern blots or the normalized ratios in the microarrays divided by the average of those from 6 organs. The microarray data shown are means ± S.E. from 8 hybridizations. (a) lung-specific miRNAs; (b) kidney-specific miRNAs; (c) co-expressed miRNAs in the lung and heart identified by HSD; (d) miRNAs highly expressed in the lung and/or the heart. (e) commonly expressed miRNAs.