Pgl3 vector

A DNA fragment corresponding to the XIAP 3′UTR (which contains a miR-643 biding site) was synthesized and cloned into the XbaI site, downstream from the stop codon of the luciferase gene, in the pGL3 vector (GenScript) to obtain the pGL3-XIAP-3′UTR construct. SW-480 cells (40,000 cells/well) were transfected with the pGL3-XIAP-3′UTR plasmid (1 μg) using lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. A pGL3-SIAH1-3′UTR construct that contains the 3′UTR of SIAH1 gene and lack of a miR-643 biding site was used as control. At 24 h postransfection SW-480 cells were incubated with E. histolytica trophozoites for 45 min, and then transfected with anti-miR-643 or scramble as described above. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) at 24 h post transfection. Data were normalized against values corresponding to SW-480 cells transfected with pGL3-XIAP without interaction with E. histolytica. Each assay was repeated three times in duplicate, and data were expressed as mean ± standard deviation.

The 3′ -UTR of SAMD4, SAMD3, RB1 and EP300 containing the putative target site for miR-17-92 cluster were cloned into the pGL3 vector (Genscript). The plasmid was cotransfected with pRL-SV40 and miRNA mimics into HEK 293 T cells using Lipofectamine™ 2000 (Invitrogen). At 48 h post transfection, the luciferase activities were measured with the Dual-Luciferase Reporter Assay System (Promega). Relative luciferase activity was normalized with Renilla luciferase activity.