Figure 2

OCT4 is a target of miR-34a-3p. (a) Predicted potential binding site of miR-34a-3p at 3′UTR of OCT4. (b) Comparison of the levels of miR-34a-5p and miR-34a-3p in human transformed epithelial cells. As described in Figure 1c, the miR-34a-3p levels were measured from these cell lines using real-time PCR and normalized with the miR-34a-5p levels in non-transformed control cells. The data were normalized with an internal control RNA, RNU48. (c) Outline of the OCT4 constructs: plasmid encoding HA-OCT4 with (WT or mutant) or without 3′UTR of OCT4. (d) Immunoblots of whole-cell lysates from 293FT cells expressing HA-OCT4 with (WT) or without (d) the 3′UTR of OCT4 at 48 h after transfection. HA antibody was used to detect the HA-OCT4 levels and PCNA was used as an internal loading control. (e) Immunoblots of whole-cell lysates from 293FT cells expressing HA-OCT4 with WT or mutant 3′UTR that deleted the key binding site of miR-34a-3p (M) of OCT4 at 48 h after transfection. (f) MiR-34a-3p specifically represses OCT4 measured by luciferase assay in 293FT cells. WT, wild type of OCT4 3′UTR; DM, deletion mutation of OCT4 3′UTR without the binding site of miR-34a-3p (CTGGTTG) with the proper primers as described in Supplementary Table S2. The error bars presented as mean+S.D. from three independent experiments. **P<0.01