miR-155 down-regulates HGAL expression by binding to specific sites in HGAL 3′-UTR. (A) Effect of hsa-miR-155 overexpression on native HGAL protein levels in Raji, MC116, and VAL cell lines at 48 hours after transfection, assessed by Western blot. Actin levels were used as loading control in all cases. (B-C) Effect of hsa-miR-155 overexpression on the mRNA levels of HGAL in Raji (B) and MCL116 (C) cell lines measured by real-time PCR using TaqMan Gene Expression Assays (Applied Biosystems) at 24, 48, and 72 hours after transfection. Values of triplicate wells are represented as fold expression with respect to the nontargeting control transfection. Overexpression of hsa-miR-155 was confirmed by TaqMan MicroRNA Assays (C right panel and D right panel), expressed as fold increase over the control transfection. Error bars represent SEM. (D) Dual luciferase activity of reporter plasmids with the wild-type or mutated 3′-UTR of HGAL fused to the luciferase gene following hsa-miR-155 cotransfection in HeLa cells. The black columns represent cotransfections with hsa-miR-155; and the gray columns, the cotransfection of the same reporter vector with the nontargeting control. Mutations of the putative binding sites are represented as M1 and M2 3′-UTR. Values are normalized to the value of each control, which is noted as 100%. *Significant difference (P < .05). Error bars represent SEM. (A-D) Results are representative of 3 independent experiments.