Figure 3. eIF4GI participates in the miRNA-mediated gene silencing.

(A) Schematic diagrams of the reporter constructs for the investigation of the miRNA-mediated gene silencing. pcDNA3.1-FL-6×Bulge expresses a FL mRNA containing six imperfect repeats of the miCXCR4-binding site. pcDNA3.1-FL expresses a FL mRNA lacking the miCXCR4-binding site. Abbreviations: P_CMV, the CMV promoter; P_T7, the T7 promoter; FL, firefly luciferase. (B) HeLa cells cultivated on a 24-well plate were transfected with the reporter plasmids in the presence of miControl or miCXCR4. After 24 h of incubation, the luciferase activities were measured by a dual luciferase assay. The activity ratios of FL to RL (FL/RL) were calculated, and the relative values were determined by setting the value from miControl-treated cells to 100%. (C) Total RNAs from the cells described in panel B were extracted, and the levels of reporter mRNAs were analyzed by Northern blotting. The EtBr-staining of 28S rRNAs was assessed as a control for total RNA levels. (D) De-repression of the miRNA-mediated gene silencing by siRNAs against translation initiation factors. The overall experimental procedures and analyses were described in the Materials and Methods. (E) The relative amounts of the reporter mRNAs in panel D were measured by quantitative RT-PCR analysis. The ratios of FL/RL mRNAs obtained from miCXCR4-treated cells were first normalized by that of miControl-treated cells and then relatively presented by setting the value from miControl/si-Control-treated cells to 100% (lane 1). (F) The knock-down efficiency of each siRNA was examined by Western blotting using the indicated antibodies. For comparison, WCEs from mock-transfected HeLa cells (100–0%; denoted as Mock) were loaded in lanes 1–6. All experiments were performed in triplicate. In panels B and D, the P-values are described (Student t-test). Standard deviations are indicated by bars.