Development of a micro-array to detect human and mouse microRNAs and characterization of expression in human organs

Abstract

MicroRNAs (miRNAs) are believed to play important roles in developmental and other cellular processes by hybridizing to complementary target mRNA transcripts. This results in either cleavage of the hybridized transcript or negative regulation of translation. Little is known about the regulation or pattern of miRNA expression. The predicted presence of numerous miRNA sequences in higher eukaryotes makes it highly likely that the expression levels of individual miRNA molecules themselves should play an important role in regulating multiple cellular processes. Therefore, determining the pattern of global miRNA expression levels in mammals and other higher eukaryotes is essential to help understand both the mechanism of miRNA transcriptional regulation as well as to help identify miRNA regulated gene expression. Here, we describe a novel method to detect global processed miRNA expression levels in higher eukaryotes, including human, mouse and rats, by using a high-density oligonucleotide array. Array results have been validated by subsequent confirmation of mir expression using northern-blot analysis. Major differences in mir expression have been detected in samples from diverse sources, suggesting highly regulated mir expression, and specific gene regulatory functions for individual miRNA transcripts. For example, five different miRNAs were found to be preferentially expressed in human kidney compared with other human tissues. Comparative analysis of surrounding genomic sequences of the kidney-specific miRNA clusters revealed the occurrence of specific transcription factor binding sites located in conserved phylogenetic foot prints, suggesting that these may be involved in regulating mir expression in kidney.

Figure 1

miRNA array specificity. The array was probed with a Let-7a transcript and detection was performed as described in Materials and Methods. Signal intensity values of the different Let-7 mirs are plotted in (A). Sequences of Let-7 mirs are seen in (B).

Figure 2

Human heart and skeletal muscle enriched mir cluster. Each bar represents the expression of an average signal intensity value of a given mir. Expression of miR-133a, miR-133b and miR-1d are higher in skeletal muscle compared with that in heart. There is no or very little expression of these mirs in other tested organs.

Figure 3

Kidney enrich mir cluster. This cluster contains five mirs (miR-192, miR-194, miR-204, miR-215 and miR-216).

Figure 4

Northern blotting of human tissue RNA (Ambion) for kidney enrich mir cluster. U6 snRNA was used to verify the equal RNA loading.

Figure 5

Cluster W alignment of miR-192 and miR-215. There is an 18 bp conserved DNA sequence (UGACCUAUGAAUUGACAG) common to each of these mirs in their processed mir sequence region.

Figure 6

Cross-species conservation of proto-oncogene ets-1 binding site located 2158 bp upstream of miR-192/miR-194-2 complex and 1155 bp upstream of miR-204.