e149 Nucleic Acids Research, 2007, Vol. 35, No. 22 functional antagomir expression cassettes in both proviral LTRs (called dc: double copy vector). In addition, the lentiviral transgenic plasmid drives EGFP expression under control of the SFFV-LTR located within the lentiviral backbone. We initially analyzed the function of antagomirs against miR-18a, miR-19b and miR-20a which are all encoded on the polycistronic and oncogenic miR-17-92 cluster (15). We used luciferase-based reporter assays with speciﬁc miRNA- binding sites in the 30untranslated region of the luciferase gene using K562 cells as previously described (7,16). In these cells, expression of the miR-17-92 cluster depends on the presence of both the BCR-ABL onco-protein and c-MYC. In addition, transient transfection of chemically modiﬁed antagomirs can speciﬁcally enhance luciferase activity in these cells (7). To study the function of lentivirally encoded antagomirs, K562 were transduced to more than 95% EGFP-positive cells (data not shown). As shown in Figure 1B, lentiviral transduction with anti- miR-18a, anti-miR-19b and anti-miR-20a antagomirs speciﬁcally induces relief of miRNA-dependent luciferase repression, which is further enhanced upon silencing of c-MYC-expression by lentivirus-mediated RNAi. Impor- tantly, this luciferase induction upon lentivirus-mediated antagomir expression corresponds very closely to that observed upon transient transfection of chemical antag- omirs as previously described (7). These data demonstrate that lentivirally encoded antagomirs can speciﬁcally relief miRNA-dependent reporter gene repression. miRNA expression in the presence of lentivirally encoded antagomirs We next analyzed miRNA expression by miRNA-speciﬁc quantitative RT-PCR (miR-qRT-PCR) after transduction with lentivirally encoded antagomirs. miRNA levels are speciﬁcally reduced upon lentivirus-mediated antagomir expression whereas the polycistronic pri-miRNA level remains unchanged in transduced K562 cells (Figure 1C). In contrast to miR-qRT-PCR, northern blotting and miCHIP analyses do not detect reduced miR-18a and miR-20a expression under those conditions (Figure 1D and M. Castoldi and M. Muckenthaler, personal communica- tion). Since the latter methods, but not miR-qRT-PCR, include denaturing steps before probe hybridization we asked whether blocking of the 30-miRNA end may interfere with miR-qRT-PCR. We annealed oligonucleotides com- plementary to diﬀerent regions of the miRNA target before reverse transcription as schematically shown in Figure 1E. Hybridization of speciﬁc oligonucleotides to the entire (A) and in particular to the 30-(C) but not the 50-sequence (B) of individual miRNAs speciﬁcally inhibits miR-qRT-PCR for all three miRNAs tested. Furthermore, re-denaturation after addition of oligonucleotides complementary to the 30miRNA sequence by heating up to 708C relieves the inhibition of miR-qRT-PCR as shown in both bottom panels (without and with re-denaturation). These data demonstrate that the addition of oligonucleotides comple- mentary to the 30miRNA sequence can interfere with miR- qRT-PCR in vitro, most probably by preventing the PAGE 6 OF 9 binding of the miRNA-speciﬁc looped RT-primer and subsequent inhibition of reverse transcription. Impact of lentivirally encoded antagomirs on cell proliferation To study miRNA function on cell proliferation, K562 cells were transduced with lentiviruses encoding speciﬁc antagomirs to >95% based on virally encoded EGFP expression (data not shown). Anti-miR-18a, but not anti-miR-19b, anti-miR-20a, or control antagomirs inhibit proliferation of K562 cells (Figure 1F). Interestingly, retroviral over-expression of miR-17-19b, a variant of the miR-17-92 cluster (15) with concomitant elevation of miRNA expression (7) prevents inhibition of cell prolif- eration by anti-miR-18a speciﬁc antagomirs (Figure 1G). Similarly, anti-miR-18a-speciﬁc antagomirs have no detectable impact on cell proliferation in K562 cells over-expressing c-MYC (Figure 1H), which induces miR-17-92 expression and enhances proliferation of K562 cells (7). Taken together miRNA-speciﬁc antago- mirs can speciﬁcally modulate proliferation of K562 cells. In addition, enhanced miR-17-92 expression either by retroviral over-expression or by known positive regulators such as c-Myc can overcome the growth inhibitory eﬀects of speciﬁc antagomirs. Combined over-expression of antagomirs and miRNAs Having demonstrated that lentivirus-mediated antagomir expression can speciﬁcally inhibit miRNA function, we cloned lentiviral vectors for expression of individual miRNAs by embedding heterologous miRNA sequences into a miR-30 backbone as previously described (Figure 2A) (17). Increased expression of miR-18a, miR- 19b and miR-20a could be demonstrated by miR-qRT- PCR ranging between 2- and about 6-fold (Figure 2B). To study the function of lentivirally expressed miRNAs, luciferase reporter assays were performed similarly as described above. In these experiments, we used K562 cells with reduced c-MYC expression (due to anti c-MYC- RNAi), which have reduced miR17-92 expression (7) and enhanced luciferase activity (Figure 1B). As expected, miRNA-speciﬁc repression of luciferase activity is observed for all three miRNAs including miR-18a in these cells (Figure 2C). In addition, over-expression of individual miRNAs in control shRNA transduced K562 and in K562 cells with reduced c-MYC expression reveals enhanced miRNA expression as determined by miR-qRT- PCR (data not shown). In these cells, a speciﬁc and strong inhibition of cell proliferation by miR-20a can be detected [Figure 2D, K562 controls (left) and cells with reduced c-MYC expression (right)]. To study the function of speciﬁc miRNAs for cell proliferation in more detail, we individually over- expressed miR-30-controls, the polycistronic miR17-19b cluster, miR-18a and miR-20a in K562 cells expressing either control antagomirs (Figure 2E, upper panel), or anti-miR-18a- (Figure 2E, middle panel), or anti-miR-20a antagomirs (Figure 2E, lower panel), respectively. In control-antagomir K562 cells, miR-18a and the polycis- tronic miR17-19b miRNA increase, whereas miR-20a decreases the number of viable cells as compared to