PAGE 7 OF 9 miR-20a Table 2. miRNA levels in K562 cells transduced with anti-miR-20a antagomirs anti-miR-20a 4.2 miR30-20a 2.2 controls (Figure 2E, upper panel). Expression of anti- miR-18a antagomirs in K562 cells inhibits cell prolifera- tion as seen before (Figure 1F, compare controls in Figure 2E, upper and middle panel). However, over- expression of miR-18a and the miR-17-19b polycistron, but not miR-20a miRNAs speciﬁcally rescues the growth inhibition mediated by anti-miR-18a antagomirs as compared to controls (Figure 2E, middle panel). These data suggest that over-expression of miR-18a and the polycistron functionally out-competes anti-miR-18a antagomirs in these cells. Finally, lentiviral expression of anti-miR-20a antagomirs can at least partially prevent the growth-inhibitory eﬀect of miR-20a (compare Figure 2E, upper and lower panel). Furthermore, anti-miR-20a antagomirs enhance proliferation of control K562 cells similar to that of K562 cells expressing miR-18a (Figure 2E, lower panel). In summary, miRNA-mediated eﬀects on cell proliferation can be identiﬁed and antag- onized by combined over-expression of miRNAs and corresponding antagomirs, respectively. Dose-dependent increase of E2F-1 protein expression by anti-miR-20a antagomirs E2F-1 is a conﬁrmed cellular target of miR-20a (16). To study the impact of lentivirally encoded antagomirs on miRNA-regulated protein expression, K562 cells were transduced with either anti-miR-20a antagomirs or miR- 20a and the miR-17-19b polycistron, respectively. Expression of antagomirs and miR-20a demonstrates a 4.2- and a +2.2-fold induction of miR-20a levels detected by miR-qRT-PCR, respectively, as compared to control cells (Table 2). Whole cell lysates of transduced K562 cells were analyzed for E2F-1 expression by immunoblotting. As shown in Figure 3A and Table 3, expression of both miR-20a (lane 8) and the miR-17-19b polycistron (lane 2) reduce, whereas anti-miR-20a antag- omirs (lane 5) increase E2F-1 levels. To analyze dose–response relations between miRNA and target protein expression, K562 cells were lentivirally transduced with anti-miR-20a antagomirs and subse- quently sorted according to the EGFP-ﬂuorescence intensity. As shown in Figure 3B and Table 5, three populations of transduced cells with diﬀerent mean ﬂuorescence intensities (MFI) could be isolated. The reduction of miR-20a levels as detected by miR-qRT- PCR ( 2.1, 3.3 and 4.0-fold expression as compared to control K562 cells) correlates with the MFI of the respective populations (350, 1250 and 2500). Interestingly, E2F-1 protein expression in these sorted populations increases in parallel with increasing MFI and decreasing miR-20a levels as detected by miR-qRT-PCR (Table 4). These data demonstrate that speciﬁc manipulation of Nucleic Acids Research, 2007, Vol. 35, No. 22 e149 Figure 3. Eﬀects of lentivirally encoded miR-20a and anti-miR-20a antagomir expression on E2F-1 expression. (A) K562 cells transduced with S-17/19b-IEW (lane 2), control dcH1-ctrl-antagomir-SEW (lane 4), dcH1-antagomir-20a-SEW (lane 5), S-mir30-ctrl-IEW (lane 7) and S-mir30-20a-IEW (lane 8), respectively were lyzed 4 days after transduction. Lanes 1, 3 and 6 show untransduced K562 cells. The immunoblots were probed with anti-E2F-1 and reprobed with anti-a- tubulin as loading control. (B) Histogram of EGFP ﬂuorescence of untransduced K562 cells (left) and cells sorted according to their EGFP ﬂuorescence after lentiviral transduction with anti-miR-20a antagomirs. MFI: mean ﬂuorescence intensity. Below: cellular lysates from the three sorted K562-anti-miR-20a antagomir cell populations shown in Figure 3B were subjected to immunoblotting with anti-E2F1 and anti-a-tubulin antibodies. Lane 1 shows untransduced K562 cells, lane 2, 3 and 4 K562 cells sorted according to high, medium and low green ﬂuorescence intensity, respectively. miRNA expression can mediate target protein expression in a dose-dependent manner. DISCUSSION Our data demonstrate that lentivirus-mediated expression of miRNAs and miRNA-speciﬁc antagomirs can induce stable gain- and loss-of-function phenotypes for individual miRNAs, respectively. In addition, lentiviral transduction allows identiﬁcation and puriﬁcation of transduced cells with modiﬁed miRNA expression based on lentivirally encoded reporter gene expression such as EGFP used