Fig. 3.

DHA blocks miR-21 expression. MDA-MB-231 (A, C and E) and MCF-7 (B, D and F) breast cancer cells were incubated with 152nM DHA for 24h. Total RNAs were extracted and used in qRT–PCR to detect pre-miR-21 (panels A and B), mature miR-21 (panels C and D) and pri-miR-21 (panels E and F) as described in Materials and methods. Mean ± SE of triplicate measurement is shown. *P = 0.002 and 0.003 versus control in panels A and B, respectively; *P = 0.029 and 0.0057 versus control in panels C and D, respectively; *P = 0.018 and 0.0034 versus control in panels E and F, respectively. NFκB regulates DHA-induced inhibition of miR-21 transcription (panels G to L). (G and H) MDA-MB-231 (panel G) and MCF-7 (panel H) breast tumor cells were transfected with miR-21-Luc reporter plasmids along with p65 expression vector or vector alone. The cell lysates were used for luciferase activity, 48h post-transfection, as described in Materials and methods. Mean ± SE of triplicate measurements is shown. *P = 0.016 and 0.011 versus vector in panels G and H, respectively. (I and J) MDA-MB-231 (panel I) and MCF-7 (panel J) breast tumor cells were transfected with miR-21-Luc reporter plasmid followed by incubation with 152nM DHA 24h post-transfection for additional 24h. Luciferase activity was determined in the cell lysates as described in Materials and methods. Mean ± SE of triplicate measurements is shown. *P = 0.049 and 0.048 versus vector in panels I and J, respectively. (K and L) MDA-MB-231 (panel K) and MCF-7 (panel L) breast tumor cells were transfected with miR-21-Luc reporter plasmids along with p65 expression vector or vector alone. Transfected cells were incubated with 152nM DHA for 24h. The cell lysates were used for luciferase activity as described in Materials and methods. Mean ± SE of triplicate measurements is shown. *P < 0.01 versus control; **P < 0.001 versus DHA in panels K and L. Bottom parts in panels G, H, K and L show expression of p65 and actin by immunoblotting.