Figure 8.
Importance of an hsa-mir-29b-DNMT3b-tumor suppressor gene axis with treatment of necitumumab with cisplatin/gemcitabine. Up-regulation of hsa-miR-29b with necitumumab with cisplatin/gemcitabine was associated with down-regulation of DNMT3b expression in A549 (A) but not NCI-H1650 (B) xenograft tumors. Re-activation of potentially methylation-silenced tumor suppressor genes is caused by necitumumab with cisplatin/gemcitabine in A549 tumors (A), but not in NCI-H1650 tumors (B). Effects of necitumumab with cisplatin/gemcitabine on DNMT3b and hsa-miR-29b expression in A549 (C) cells in vitro were similar to those found in vivo. Transient transfection of hsa-miR-29b mimic (M) in A549 cells (D) was shown to reduce DNMT3b expression and increase tumor suppressor gene expression compared to mock transfected cells (Mc). Co-transfection with an hsa-miR-29b inhibitor (Inh) reduces these effects (n=2-3 replicates). (E) The in vitro effect of necitumumab with cisplatin/gemcitabine is down-regulation of DNMT3b, as shown by western blot, and up-regulation of hsa-miR-29b expression, as shown by real-time PCR, in NCI-H1975, HCC827, EKVX-P2, NCI-H358, NCI-H441 and Calu6 cells. 1, control (saline); 2, cisplatin plus gemcitabine; 3, necitumumab; 4, cisplatin plus gemcitabine plus necitumumab.