hMLH1, hMSH2, and hMSH6 are direct targets of miR-155. (A) Locations of the target sites of miR-155 in the 3′ UTRs and/or the CDS of the indicated genes (see also Fig. S1). The base position is counted from the first nucleotide in the CDS. (B) Colo-320 DM cells were transfected with the phRL-SV40 construct as a control and either the luciferase construct WT or MUT-155 and with premiR-155 or premiR control. After 24 h, dual luciferase assays were performed. *P < 0.005 relative to premiR control.

Overexpression of miR-155 decreases the expression of MLH1, MSH2, and MSH6 in CRC cells. Colo-320 DM cells were transfected with premiR-155, premiR control, or siRNA against selected genes for 48 h. (A) Transfection efficiency was confirmed by real-time PCR. miR-155 expression was normalized to the expression of RNU44. Error bars represent SEM. *P < 0.001 (n = 3). (B) miR-155 exerts a posttranscriptional effect on MMR core proteins. mRNA expression of indicated genes was normalized to that of vinculin. Error bars represent SEM.*P < 0.05 compared with control premiR (n = 3). (C) Representative blot of Western blot analysis along with the mean and SEM of three independent experiments. *P < 0.005 compared with control premiR.

Stable clones with overexpression of miR-155, indicating the functional effect of miR-155 overexpression on CRC cell lines. MMR proficient Colo-320 DM cells were stably infected with a lentiviral vector overexpressing miR-155 (Colo-155) or an empty vector (Colo-empty). (A) miR-155 expression assessed by Northern blot analysis, with MV-411 cells used as positive controls. (B) Representative Western blot analysis results, along with mean ± SEM of three independent experiments. *P < 0.05 relative to controls. (C) mRNA expression of selected genes assessed by real-time PCR (normalized to vinculin). Error bars represent SEM. *P < 0.05 (n = 3). (D) Microsatellite analysis of Colo-155 and Colo-empty cells using the BAT-26 and BAT 25 (mononucleotide repeats) and D17S250 (dinucleotide repeat) markers. Size markers are given at the top of the panel.

miR-155 expression is inversely related to MLH1 and MSH2 in CRC tissues. (A) Paraffin-embedded, formalin-fixed CRC tissues were incubated with LNA-probe anti–miR-155 or scrambled probe and IHC antibodies against MSH2 and MLH1. Representative photographs were captured with the Nuance system software (Ventana Medical System Inc.), with staining positive for both miR-155 and MSH2 or MLH1 shown. Blue and red staining identifies miR-155 and the target protein, respectively. No colocalization of miR-155 and MLH1 or MSH2 in the same cell nest is seen. (B) RNA and proteins extracted from fresh frozen human colorectal tissue. miR-155 expression was assessed by real-time PCR, and MMR protein expression was assessed by Western blot analysis. CRC with up-regulation (>2-fold) of miR-155 expression and down-regulation of MMR protein expression are shown. A strong correlation between the loss of hMLH1 and hMSH2 expression and miR-155 expression was increased by >3-fold in tumor compared with adjacent normal tissue (red line). Samples with an miR-155 cancer/normal ratio <3-fold displayed an uncertain effect on MMR expression.

miR-155 expression in hMLH1-negative tumors. miR-155 expression was assessed by in situ hybridization on paraffin-embedded tissues. (A) CR-78 cancer tissue (with unknown causes of MLH1 loss) showing strong miR-155 expression (large arrow), with stroma (small arrow) negative for miR-155 expression. (B) CR-79 tissue (MLH1 loss due to promoter methylation) showing faint expression of miR-155 only in inflammatory cells (large arrow), with no signal detected in cancer tissue (small arrow).