A microarray-based approach identifies ADP ribosylation factor-like protein 2 as a target of microRNA-16.

microRNAs (miRNAs) are generally thought to negatively regulate the expression of their target genes by mRNA degradation or by translation repression. Here we show an efficient way to identify miRNA target genes by screening alterations in global mRNA levels following changes in miRNA levels. In this study, we used mRNA microarrays to measure global mRNA expression in three cell lines with increased or decreased levels of miR-16 and performed bioinformatics analysis based on multiple target prediction algorithms. For further investigation among the predicted miR-16 target genes, we selected genes that show an expression pattern opposite to that of miR-16. One of the candidate target genes that may interact with miR-16, ADP-ribosylation factor-like protein 2 (ARL2), was further investigated. First, ARL2 was deduced to be an ideal miR-16 target by computational predictions. Second, ARL2 mRNA and protein levels were significantly abolished by treatment with miR-16 precursors, whereas a miR-16 inhibitor increased ARL2 mRNA and protein levels. Third, a luciferase reporter assay confirmed that miR-16 directly recognizes the 3'-untranslated region (3'-UTR) of ARL2. Finally, we showed that miR-16 could regulate proliferation and induce a significant G0/G1 cell cycle arrest, which was due at least in part, to the down-regulation of ARL2. In summary, the present study suggests that integrating global mRNA profiling and bioinformatics tools may provide the basis for further investigation of the potential targets of a given miRNA. These results also illustrate a novel function of miR-16 targeting ARL2 in modulating proliferation and cell cycle progression.

Identification and characterization of microRNAs in raw milk during different periods of lactation, commercial fluid, and powdered milk products.

Recent baby formula milk powder contamination incidents have shown that the classic markers or standards in milk quality control are insufficient in identifying "manipulated" poor-quality milk. In the present study, we demonstrated for the first time that cow milk contains large amounts of microRNAs (miRNAs) and that the unique expression profile of milk-specific miRNAs can serve as a novel indicator and possible new standard for the quality control of raw milk and milk-related commercial products, such as fluid milk and powdered formula milk. First, using Solexa sequencing, we systematically screened miRNA expression in raw milk and identified a total of 245 miRNAs in raw milk. Unlike other classic biomarkers whose expression levels are nearly identical at different periods of lactation, individual miRNAs can be significantly altered during lactation process, implicating that miRNAs may be a more accurate indicator to reflect the quality alteration of milk. Second, using TaqMan probe-based miRNA quantitative RT-PCR, we further identified seven miRNAs that have a relatively consistent expression throughout the lactation process, and more importantly, the expression profile of these seven milk-specific miRNAs can serve as an ideal biomarker for discriminating poor-quality or "manipulated" milk from pure raw milk, as well as for the quality control of commercial milk products, such as fluid milk and powdered formula milk. Together, our findings provide a basis for understanding the physiological role of milk miRNAs and a new potential standard for determining the quality of raw milk or milk-related commercial products.