e149 Nucleic Acids Research, 2007, Vol. 35, No. 22 Table 3. Densitometric analysis of E2F1 protein expression in K562 cells transduced with anti-miR-20a antagomirs K562 ‘mock’ K562 ‘S-17/19b-IEW‘ K562 ‘mock’ dcH1-ctrl-antagomir-SEW dcH1-antagomir-20a-SEW K562 ‘mock’ control: S-mir30-ctrl-IEW S-mir30-20a-IEW Lane 1 2 3 4 5 6 7 8 miRNA miR-20a Lane 1 2 3 4 Table 5. Densitometric analysis of E2F1 protein expression (B) in K562 cells transduced with anti-miR-20a antagomirs after sorting according to EGFP-ﬂuorescence intensity Table 4. miRNA levels in K562 cells transduced with anti-miR-20a antagomirs after sorting according to EGFP-ﬂuorescence intensity High 4 anti-miR-20a Medium 3.3 Low 2.1 K562 ‘mock’ dcH1-antagomir-20a-SEW, high dcH1-antagomir-20a-SEW, medium dcH1-antagomir-20a-SEW, low in this study. Furthermore, EGFP ﬂuorescence of antagomir-transduced cells correlates directly with reduc- tion of miRNA function and concomitant increase of target protein expression in a semi-quantitative manner. Similar dose–response correlations have been observed for stable RNAi after lentivirus-mediated shRNA expres- sion (14). In our study, antagomir-mediated inhibition of miRNA function was ﬁrst demonstrated using miRNA-speciﬁc reporter assays. In these experiments, antagomirs speciﬁ- cally relieve miRNA-dependent repression of luciferase activity (Figure 1B). In addition, miRNAs levels are speciﬁcally reduced in the presence of the corresponding antagomirs if determined by miR-qRT-PCR (Figure 1C). In contrast, northern blot and miCHIP analyses do not detect reduced expression of miR-18a and miR-20a in these cells (Figure 1D and data not shown). Since the latter methods involve a denaturation step before probe hybridization, this diﬀerence may be due to hybridization of miRNAs with antagomirs interfering with or prevent- ing primer annealing for reverse transcription. To test this hypothesis in an in vitro model, miR-qRT-PCR was performed in the presence of oligonucleotides E2F1/a-tubulin expression [%] 100 53 100 93 199 100 85 42 E2F1/a-tubulin expression [%] 100 360 320 168 PAGE 8 OF 9 complementary to diﬀerent parts of the miRNA sequences (Figure 1E). We found that hybridization to the 30- but not 50-miRNA sequence in vitro reduces its detection by miR-qRT-PCR most probably by competition for binding of the looped RT-primer used for this speciﬁc reaction. Although the molecular mechanisms in vivo mediating reduced miRNA detection by miR-q-RT-PCR in the presence of intracellularly expressed antagomirs are currently not known, the data are in line with a model suggesting that miRNA–antagomir dimers may exist intracellularly for some period of time. Accordingly, miRNA levels detected by miR-qRT-PCR correlate to miRNA function but not to total miRNA levels detectable by northern blotting and/or miCHIP analyses (M. Castoldi and M. Muckenthaler, personal commu- nication). However, the mechanism of miRNA/antagomir duplex formation, their stability and intracellular traﬃck- ing, and the kinetic of individual miRNA/antagomir turnover remain to be elucidated. Target protein expression can be regulated by miRNAs as demonstrated for miR-20a and its experimentally validated target E2F-1 (16). Over-expression of both the miR-17-19b cluster and miR-20a individually decrease whereas lentivirus-mediated anti-miR-20a antagomir expression increases E2F-1 protein expression in K562 cells (Figure 3A and Table 3). The change in E2F-1 protein levels correlates inversely to (free) miR-20a levels as determined by miR-q-RT-PCR and directly to EGFP expression of K562 cells transduced with antagomir-encoding lentiviruses (Figure 3A and B and Tables 4 and 5). With these tools to modulate miRNA –function, we can assign speciﬁc functions to individual miRNAs encoded on the polycistronic miR-17-92 miRNA cluster. This cluster is over-expressed in hematological malignancies including early phase CML CD34+ cells (7) and in an erythroleukemia model as recently reported (18). By combined expression of miRNAs and the corresponding antagomirs, we have identiﬁed a positive role of miR-18a (and correspondingly a negative impact of anti-18a antagomirs) on proliferation of CML-derived K562 cells. In contrast, over-expression of miR-20a strongly inhibits proliferation of K562 cells, whereas no functional role of miR-19b for cell proliferation has been observed in these experiments. Interestingly, induction of apoptosis upon transient transfection of anti-miR-20a oligonucleotides has recently been described in lung cancer cell lines depending on miR-17-92 expression levels (19). These data may suggest some degree of tissue- or lineage-speciﬁcity of miRNA function. In summary, lentivurs-mediated expression of miRNAs and antagomirs can be used to generate and isolate stable gain- and loss-of-function phenotypes for individual miRNAs in a wide range of target cells. This strategy allows identiﬁcation of individual miRNA function of polycistronically encoded miRNAs. In addition, it will facilitate the identiﬁcation of miRNA targets and the sequences involved in regulating target gene expression. However, stable interference with endogenous miRNA expression and function may be accompanied by selection