Figure 3

miR-143 downregulates HK2. A, Sequence alignment of the miR-143 seed region and HK2 3′UTR. (ENSG00000159399) B, Luciferase assay with pGL3+ constructs holding a 789 bp 3′UTR fragment of HK2 downstream to the firefly luciferase gene. DLD-1 cells were co-transfected with firefly luciferase reporters along with a Renilla luciferase transfection control plasmid either alone (mock) or with miR-143 duplex and scrambled duplex as a negative control. Data are shown as the mean ± S.D. of four replicates. *, p < 0.005 using a two-tailed t-test. C, Western blot analysis of DLD-1 and HCT116 cells transfected with miR-143 duplex or mock transfected cells blotted for HK2. Tubulin was used as loading control. The bands were normalized relative to the tubulin loading control and quantified relative to the HK2 protein level in mock transfected cells. D, miR-143 and HK2 are negatively correlated in TCGA colorectal adenocarcinoma. Linear Pearson correlation between miR-143 expression and HK2 expression in 184 colon and rectum adenocarcinoma samples.