RNAi-Based Strategies for Cyclooxygenase-2 Inhibition in Cancer
Figure 2
Invasive tkColi infect CRC HT-29 cells and promote high COX-2 silencing associated with a reduced invasive behavior. E. coli was cotransformed with pGB2-Ω-inv-hly plasmid and pSUPER.retro vectors to obtain E. coli invasive strains carrying the shCOX-2 expression vector tkColi-pSTBE (in which shCOX-2 expression is controlled by TBE promoter carrying Tcf Binding Elements). The negative control was tkColi-pS-(not expressing shCOX2 but containing the original empty pSUPER.retro vector). GFP protein expression (a) was used to evaluate the efficiency of tkColi infection of HT-29 cells (bar = 30 m). 72 hours after the infection, the efficiency of infection was higher than 75%. The expressions of COX-2 protein and COX-2 mRNA were analyzed in HT-29 cells, 72 hours after tkColi infection, by Western blot and real-time PCR (c). COX-2 protein and COX-2 mRNA expressions were normalized against -actin protein and GUSB (-glucuronidase) mRNA levels, respectively. Relative expression of COX-2 protein and COX-2 mRNA refers to tkColi-pS-infected sample. The invasive behavior of tkColi infected CRC HT-29 cells was evaluated by using Boyden chambers and 8-m polycarbonate membranes coated with Matrigel. Samples were tested in the absence (dark bars) and in the presence (light bars) of PMA 40 nM. Relative invasion index refers to HT-29 cells infected with tkColi-pS-, not treated with PMA. Data reported in (b)–(d) represent the mean SEM of three independent experiments; .
