To confirm that miRNA-23 is directly responsible for suppression of HES1 expression, two approaches were taken. In one, synthetic miRNA-23 was introduced into undifferentiated NT2 cells (with their high HES1 levels and low miRNA-23 levels). This resulted in a decrease in expression levels of HES1 protein. In the other, siRNAs targeting miRNA-23 were introduced into differentiated NT2 cells (with their low HES-1 levels and high miRNA-23 levels). This led to a decrease in miRNA-23 levels and an increase in HES-1 levels.
The Role of miRNA-23 in Differentiation of NT2 Cells
The authors then showed that expression of miRNA-23 is responsible for the differentiation of NT2 cells (presumably via a reduction in HES1 protein levels). To this end, the authors made use of two protein markers--SSEA-3, which is only expressed in undifferentiated NT2 cells, and MAP2, which is only expressed in differentiated NT2 cells. Using these markers, the authors showed that addition of siRNAs targeting miRNA-23 prevents the differentiation of NT2 cells upon retinoic acid treatment. This blockage, however, is reversed by addition of synthetic miRNA-23.
Discussion
The results by Kawasaki and Taira suggest that suppression of HES1 expression by miRNA-23 is necessary for the differentiation of NT2 cells. It is interesting to note that in addition to miRNA-23, several worm and fruit fly miRNAs have been shown to play roles in development. It is therefore possible that other mammalian, worm, or fruit fly miRNAs also play roles in various developmental processes. This premise is supported by the differential spatial and temporal pattern of miRNA expression in various organisms.
To date, over 150 miRNAs have been cloned. These miRNAs have been identified based on their size and not their function. One way to uncover the function of miRNAs is to identify their mRNA target(s). This task has proved difficult because 1) many miRNAs may be only partially complementary to target transcripts and 2) lack of complete homology to target genes makes it is difficult to identify them via alignment programs. In this regard, it is interesting to note that database searches were used to identify Hesl as a miRNA-23 target. These results suggest that database searches may lead to the identification of new miRNA targets, thus paving the way for the identification of novel functions of other miRNAs.
References
1.Pasquinelli AE. (2002). MicroRNAs: deviants no longer. Trends in Genet. 18(4): 171-173.
2.Ambros V (2003). MicroRNA pathways in flies and worms: growth, death, fat, stress, and timing. Cell 113:673-676.
3.Kawasaki H and Taira K. (2003). Hes1 is a target of microRNA-23 during retinoic-acid-induced neuronal differentiation of NT2 cells. Nature 423:838-842.
4.Ishibashi M, Moriyoshi K, Sasai Y, Shiota K, Nakanishi S, and Kageyama R (1994). Persistent expression of helix-loop-helix factor HES-1 prevents mammalian neural differentiation in the central nervous system. EMBO J. 13:1799-1805.
