In fact, the miRNA378/378* selleck locus represents the head-to-head insertion of two common vertebrate genomic repeats, that form a very long, very stable, and perfectly complementary hairpin loop. Thus we evaluated expression of miRNA378 and miRNA378* in 3T3-L1 adipocytes where Ago2 or Dicer had been knocked down. As expected, miRNA103 and miRNA 21 levels are significantly reduced in cells with decreased expression of Ago2 or Dicer (Fig. 7D). Surprisingly, levels of miRNA378 or miRNA378* are unchanged when expression of Ago2 or Dicer is deficient (Fig. 7D), suggesting that a different processing mechanism is involved in the formation of these miRNAs. It should be noted that genetic inactivation of Dicer in other cellular systems does not completely abolish the generation of mature miRNAs (2).

Similarly, Ago2 is not required for the generation of all miRNAs (3). Transactivation of C/EBP transcription factors by miRNA378/378*. As part of our efforts to better understand the mechanism of miRNA378/378* action, we investigated other potential activities for these miRNAs. Interestingly, we observed that cotransfection of a miRNA378/378* expression vector increased transactivation of the GLUT4 promoter by C/EBP�� and C/EBP�� (Fig. 8, A and B). Although expression of miRNA378/378* also increases transactivation of the GLUT4 promoter by C/EBP�� (Fig. 8B), coactivation properties appear specific to the C/EBPs since we did not observe interaction between miRNA378/378* and PPAR�� on a PPRE reporter gene (data not shown). Also, no activation of the GLUT4 promoter by miRNA378/378* was observed in the absence of cotransfected C/EBP.

These data suggest that effects of miRNA378/378* on lipid synthesis and adipocyte gene expression are mediated, at least in part, through transcriptional coactivation of C/EBP transcription factors. Fig. 8. Overexpression of miRNA378/378* regulates activation Glut4 promoter. 3T3-L1 cells were transfected with 500 ng luciferase reporter genes Glut4 promoters, along with the indicated amounts of expression vector for wild-type C/EBP�� (A) or wild-type … DISCUSSION There are a number of published reports asserting that miRNAs modulate adipogenesis (5, 12, 16, 18, 24�C26). Several miRNAs have been reproducibly observed to be altered during adipogenesis in vitro and in vivo both in human and mouse systems (such as miRNA103, miRNA107, and miRNA378/378*).

Although prior data have suggested that some of these miRNAs play a role in adipocyte differentiation, our data suggest that miRNAs are in addition likely to play a critical regulatory role in adipocyte metabolism. For example, knocking down Ago2 leads to slight increases in adipocyte gene expression but greatly impairs Drug_discovery conversion of glucose or acetate to triacylglyerol (Fig. 1). In addition, among several miRNAs that are increased during adipocyte differentiation, we provide evidence that miRNA378/378* may play a regulatory role in lipid metabolism.