Figure 3. The chemokine CCL22 is a bona fide target of miR-34a.

(A) PVTT-1 cells were transfected with 80 nM of pre-miR-34a mimetic oligos and a RT-PCR-based expression array was carried out to determine the expression profile of 94 cytokines, chemokines and their receptors 6 hrs later. The expression pattern for 31 out of the 94 genes is presented as representatives.

(B) Putative miR-34a binding sites in the 3’-UTR of human CCL22.

(C) HEK293T cells were transiently transfected with pre-miR-34a mimetic or control oligos together with the pGL3 control plasmid, a modified pGL3 plasmid containing the wild type CCL22 3’UTR or a mutant CCL22 3’UTR with all three putative target sequences mutated. Pre-miR-34a mimetic or control oligos were added at the indicated concentrations and the luciferase activity was analyzed 24 hrs later. Data is presented as relative firefly luciferase units normalized with the value of renilla luciferase. N=3. ** p<0.01 (Student t-test).

(D) PVTT-1 cells were transfected with 80 nM of pre-miR-34a mimetic oligos and the CCL22 mRNA level was determined by qPCR 48 hrs later and normalized to GAPDH. **p<0.01 (Student t-test).

(E) PVTT-1 cells were treated as in panel D and CCL22 protein level in culture media was determined by Western blot. γ-tubulin in corresponding cell lysates was used as a loading control. N=3.

(F) PVTT-1 cells were transfected with AS-miR-34a or control oligos at the indicated concentrations and the CCL22 level in culture media was determined by Western blot 48 hrs later. γ-tubulin in corresponding cell lysates was used as a loading control. N=3.

All error bars indicate mean±SD. See also Figure S3.