Figure 1

DHFR and TS are the direct targets of miR-215. (A) The 3'UTR of DHFR mRNA contains a putative binding site of miR-215. The 3'UTR of TS harbors two putative binding sites of miR-215. (B) The DHFR and TS protein levels were down-regulated in osteosarcoma U-2 OS and colon cancer HCT 116 (wt-p53) cells transfected with miR-215 (100 nM) by Western immunoblot analysis. Oligofectamine alone (vehicle control) and non-specific miRNA (miR control) were used as the negative controls. (C) The impacts of miR-215 on DHFR and TS expression by luciferase assays. HCT 116 (wt-p53) cells were cotransfected with 100 ng of pMIR-REPORT constructs (including wild-type DHFR or TS 3'UTRs and their corresponding mutant controls), 1 ng of Renilla luciferase plasmid phRL-SV40 and 100 nM of miR-215. Firefly luciferase activity for each condition was normalized by Renilla internal control. The value of relative luciferase activity for empty vector (control) was set as 1, the values for wild-type or mutant constructs were calculated as fold induction. ** P < 0.01, compared to the control, Student's t test (two-tailed). Each condition was repeated 3 times in triplicate and error bars represent standard deviations.