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. 2008 Mar;18(3):404-11.
doi: 10.1101/gr.6587008. Epub 2008 Jan 29.

MicroRNA-target pairs in the rat kidney identified by microRNA microarray, proteomic, and bioinformatic analysis

Zhongmin Tian  1 , Andrew S Greene, Jennifer L Pietrusz, Isaac R Matus, Mingyu Liang
Affiliations

MicroRNA-target pairs in the rat kidney identified by microRNA microarray, proteomic, and bioinformatic analysis

Zhongmin Tian et al. Genome Res. 2008 Mar.

Abstract

Mammalian genomes contain several hundred highly conserved genes encoding microRNAs. In silico analysis has predicted that a typical microRNA may regulate the expression of hundreds of target genes, suggesting miRNAs might have broad biological significance. A major challenge is to obtain experimental evidence for predicted microRNA-target pairs. We reasoned that reciprocal expression of a microRNA and a predicted target within a physiological context would support the presence and relevance of a microRNA-target pair. We used microRNA microarray and proteomic techniques to analyze the cortex and the medulla of rat kidneys. Of the 377 microRNAs analyzed, we identified 6 as enriched in the renal cortex and 11 in the renal medulla. From approximately 2100 detectable protein spots in two-dimensional gels, we identified 58 proteins as more abundant in the renal cortex and 72 in the renal medulla. The differential expression of several microRNAs and proteins was verified by real-time PCR and Western blot analyses, respectively. Several pairs of reciprocally expressed microRNAs and proteins were predicted to be microRNA-target pairs by TargetScan, PicTar, or miRanda. Seven pairs were predicted by two algorithms and two pairs by all three algorithms. The identification of reciprocal expression of microRNAs and their computationally predicted targets in the rat kidney provides a unique molecular basis for further exploring the biological role of microRNA. In addition, this study establishes a differential profile of microRNA expression between the renal cortex and the renal medulla and greatly expands the known differential proteome profiles between the two kidney regions.

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Figures

Figure 1.
Figure 1.
MicroRNAs differentially expressed between the renal cortex and the renal medulla in Sprague-Dawley rats. Expression levels of 377 microRNAs were measured by microRNA microarrays (n = 4). Differentially expressed microRNAs, six enriched in the cortex and 11 in the medulla, are shown. MicroRNAs are ranked, from left to right, by cortex/medulla ratios.
Figure 2.
Figure 2.
Verification of microRNA differential expression by real-time PCR. Fold differences between the renal cortex and the renal medulla measured by microarray (array) and real-time PCR (qPCR) are shown. n = 6; (*) significantly different from the other kidney region.
Figure 3.
Figure 3.
Verification of protein differential expression by Western blotting. Western blots and fold differences between the renal cortex and the renal medulla measured by proteomic techniques (2D/MS) and Western blotting (Western) are shown. Band densities in Western blots were normalized to Coomassie blue staining. Note that the differences found in the proteomic analysis were statistically significant based on four rats, although error bars are not provided. (Sod1) Copper-zinc containing superoxide dismutase; (Hnrpk) heterogeneous nuclear ribonucleoprotein K. n = 3–4; (*) P < 0.05 vs. the other kidney region.
Figure 4.
Figure 4.
Analysis of reciprocal expression of microRNAs and computationally predicted targets. (A) Proportions of reciprocally expressed or coexpressed microRNA and protein pairs, or pairs of randomly selected microRNAs and differentially expressed proteins, that matched computational prediction using the indicated algorithm are shown. Details of the calculation are described in Methods. (*) P < 0.05 (Z-test). (B) Anti-miR-450 and pre-miR-450 (50 nM) significantly increased and decreased, respectively, the protein expression level of HNRPK in HK-2 cells. n = 4; (*) P < 0.05 vs. control.
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