Distinctive patterns of microRNA expression in extraocular muscles

Abstract

The extraocular muscles (EOMs) are a unique group of muscles that are anatomically and physiologically distinct from other muscles. We and others have shown that EOMs have a unique transcriptome and proteome. Here we investigated the expression pattern of microRNAs (miRNAs), as they may play a role in generating the unique EOM allotype. We isolated RNA and screened LC Sciences miRNA microarrays covering the sequences of miRBase 10.0 to define the microRNAome of normal mouse EOM and tibialis anterior (TA) limb muscle. Seventy-four miRNAs were found to be differentially regulated (P value <0.05) of which 31 (14 upregulated, 17 downregulated) were differentially regulated at signal strength >500. Muscle-specific miRNAs miR-206 and miR-499 were upregulated and miR-1, miR-133a, and miR-133b were downregulated in EOM. Quantitative PCR (qPCR) analysis was used to validate the differential expression. Bioinformatic tools were used to identify potential miRNA-mRNA-protein interactions and integrate data with previous transcriptome and proteomic profiling data. Luciferase assays using cotransfection of precursor miRNAs with reporter constructs containing the 3'-untranslated region of predicted target genes were used to validate targeting by identified miRNAs. The definition of the EOM microRNAome complements existing transcriptome and proteome data about the molecular makeup of EOM and provides further insight into regulation of muscle genes. These data will also help to further explain the unique EOM muscle allotype and its differential sensitivity to diseases such as Duchenne muscular dystrophy and may assist in development of therapeutic strategies.

Keywords: miR-206; miR-499; microarray profiling; muscle-specific microRNAs.

Fig. 1.

Unsupervised hierarchical clustering and heat maps of extraocular muscle (EOM) and tibialis anterior (TA) microRNA (miRNA) microarrays. Gene tree representation (left) and heat maps (right) of 8 experiments (4 EOM and 4 TA) demonstrate that the EOM and TA miRNA microarrays cluster into 2 distinct sets based on correlation of gene expression patterns. Relative expression levels of 74 statistically significant miRNAs (P value of <0.05) are shown in horizontal rows. Sample names are indicated at top. Each horizontal colored bar represents 1 probe set, and the color of the bar determines the degree of expression (red = upregulated genes; green = downregulated genes).

Fig. 2.

Differentially expressed miRNAs in EOM vs. TA. Bar graph shows log₂ fold changes of 31 differentially expressed miRNAs that were analyzed in detail after using a signal > 500 cutoff above the statistical cutoff (P < 0.05; means ± SD). Fourteen miRNAs were upregulated and 17 miRNAs were downregulated in EOM compared with TA. Sixteen of the 31 miRNAs showed a difference of under 1 log₂-fold, suggesting subtle differences in posttranscriptional regulation by these miRNAs in EOM vs. TA. The muscle-specific miRNAs, miR-499, miR-206, miR-133a, miR-133b, and miR-1, were differentially expressed and are shown by black bars.

Fig. 3.

Quantitative PCR (qPCR) validation of miRNA microarray data. The expression of selected miRNAs was verified with real-time PCR. A: relative fold changes (log₂ EOM/TA). B: comparison of fold changes seen in the microarrays and by qPCR. Changes were correlated, significantly different, and concordant in terms of expression levels. Means ± SE; n = 3. P < 0.05.

Fig. 4.

Validation of predicted miRNA-target gene interaction with 3′-untranslated region (UTR)-luciferase reporter systems. A, C, and E: alignments of the respective miRNAs with the target 3′-UTR as indicated. miR-128a and miR-125a-5p both have 2 predicted binding sites in Mapk14 3′-UTR, but in each case only 1 is shown as an example. Seed regions are in bold. B, D, and F: results of cotransfection assays in which precursor miRNA (pre-miRNA) and predicted target 3′-UTR-containing luciferase construct were cotransfected into C2C12 cells. While miR-128a has no significant inhibitory effect on Mapk14 (B), miR-125a-5p suppressed luciferase activity of the Mapk14 target clone (D). miR-128a inhibited luciferase activity of Mybpc2 (F). Negative controls are marked. Means ± SE; n = 3. *P < 0.05 for all assays compared with vector with no pre-miRNA. Luc, firefly luciferase; Ren, Renilla luciferase.