Figure 4.

XIAP 3′UTR is a target of miR-643. (A) Western blot assays for XIAP expression. SW-480 cells were incubated with trophozoites for 0, 15, 30, and 45 min. Total protein were extracted and XIAP expression was determined by Western blot assays using XIAP antibody (Cell signaling). Actin antibodies (Santa Cruz) were used as control. Numbers represents the fold change in XIAP expression quantified by densitometry and normalized against actin. Values are expressed relative to the control group (time 0 h). (B) Schematic representation of pGL3-XIAP construct containing the 3′UTR of XIAP gene cloned at the XbaI site downstream of firefly luciferase gene (Luc+) of pGL3 Luciferase Reporter Vector. The miR-643 seed sequence is indicated in the colored box. (C) qRT-PCR assays for expression analysis of miR643. SW-480 cells were incubated with trophozoites for 45 min and transfected with miR-643 inhibitor at 30 nM and 50 nM or with scramble sequence (50 nM). (D) Luciferase reporter gene assays. SW-480 cells transfected with the pGL3-XIAP were incubated with trophozoites for 45 min and co-transfected with anti-miR-643 (30 and 50 nm) or scramble (50 nM). Luciferase activity was measured after 24 h. SW-480 cells transfected with pGL3-XIAP but not exposed to parasites were used as control. (E) Western blot assays showing cropped images for XIAP expression. SW-480 cells transfected with anti-miR-643, scramble or not transfected were incubated with trophozoites for 45 min and then submitted to immunoblotting with XIAP and actin antibodies. Bands intensity was quantified by densitometry and normalized to actin. In all cases, data are representative of three independent experiments by duplicate. Error bars represent S.D. The unpaired Student t-test was used to compare each condition with control cells that were not exposed to E. histolytica. *p < 0.05; **p < 0.01; ***p < 0.001.