Knockdown of miRs fails to suppress translational activation of TOP mRNAs.
(A) MDA-MB-231 cells were infected with lentivirus expressing either anti-miR 10b sponge or anti-miR-BART 1–5p sponge (control). The fluorescent signals of MICB and GFP were analyzed by FACS. The mean intensity of MICB or GFP in the control miR–transduced cells was arbitrarily set up to be 1, and the relative increase in the MICB expression or the decrease in the GFP fluorescence in sponge-10b–transduced cells was calculated accordingly (individual numbers are presented within the bars). (B) MDA-MB-231 cells infected with lentiviruses described in (A) were transiently transfected with Dual luciferase PsiCheck2 reporter vectors. These vectors contained within the 3′ UTR of the Renilla luciferase either a fragment from the 3′-UTR of NCOR2 that bears miR-10a/10b binding site (designated miR-10), or a negative control with a mutated miR-10a/10b seed region (designated miR-10mut). The Renilla to Firefly activity ratio (R/FF) was calculated for each sample and the average obtained for the miR-10b sponge-infected cells was normalized to that obtained for the control sponge-infected cells, which arbitrarily was set at 1.0. (*) p<0.001 versus miR-10a transfected cells (n = 8). (C) MDA-MB-231 cells that were either kept uninfected (None), expressed anti-miR 10b sponge or control sponge (Control) were kept untreated and cytoplasmic extracts from these cells were subjected to polysomal analysis. (D) and (F) RKO cells infected with Sin 18, an empty lentiviral vector (EV), or by lentivirus expressing shDrosha RNA. Cells were either untreated [Control in (F)], starved for serum for 19 h and during the last 3 h also for amino acids and then either kept without serum and amino acids [− in (D); −AA in (F)] or refed for just amino acid for additional 2 h [+ in (D); −AA→+AA in (F)]. Similarly infected cells were serum starved for 48 h [−in (D); –Serum in (F)] or serum starved for 48 h and then serum refed for 3 h [+ in (D); –Ser →+Ser in (F)]. Cells were harvested and subjected to Western blot analysis with the indicated antibodies (D) or subjected to polysomal analysis (F). (E) Total RNA was prepared from RKO cells infected with either empty lentiviral vector (EV) or lentivirus expressing shDrosha RNA. The abundance of each of the indicated miRs in Drosha knockdown cells was normalized to that in cells infected with empty vector, which was arbitrarily set at one.