Putative tumor suppressor miR-145 inhibits colon cancer cell growth by targeting oncogene Friend leukemia virus integration 1 gene

Abstract

Background: Tumor suppressor microRNA miR-145 is commonly down-regulated in colon carcinoma tissues, but its specific role in tumors remains unknown.

Methods: In this study, the authors identified the Friend leukemia virus integration 1 gene (FLI1) as a novel target of miR-145. FLI1 is involved in t(11;22)(q24:q12) reciprocal chromosomal translocation in Ewing sarcoma, and its expression appears to be associated with biologically more aggressive tumors.

Results: The authors demonstrated that miR-145 targets a putative microRNA regulatory element in the 3'-untranslated region (UTR) of FLI1, and its abundance is reversely associated with FLI1 expression in colon cancer tissues and cell lines. By using a luciferase/FLI1 3'-UTR reporter system, they found that miR-145 down-regulated the reporter activity, and this down-regulation was reversed by anti-miR-145. Mutation of the miR-145 microRNA regulatory element sequence in the FLI1 3'-UTR abolished the activity of miR-145. miR-145 decreased FLI1 protein but not FLI1 mRNA, suggesting a mechanism of translational regulation. Furthermore, the authors demonstrated that miR-145 inhibited cell proliferation and sensitized LS174T cells to 5-fluorouracil-induced apoptosis.

Conclusions: Taken together, these results suggest that miR-145 functions as a tumor suppressor by down-regulating oncogenic FLI1 in colon cancer.

Figure 1

Identification of Fli-1 as a novel gene target of miR-145. A. Schematic diagram of luciferase/Fli-1 reporter construct. Putative microRNA regulatory elements (MREs) predicted by bioinformatics were cloned into the 3′UTR of luciferase gene at Xba1 site. B. Regulation of reporter gene expression by miR-145 MRE. 293T cells were cotransfected with a luciferase reporter containing miR-145 MRE. Twenty-four hours later, luciferase activity was measured. Renilla luciferase was used as an internal control. The experiments were performed in triplicates and are shown as mean ± SD, *: P < 0.05.

Figure 2

Specificity of Fli-1 microRNA regulatory element for miR-145. A. Dose-dependent suppression of Luc-Fli-1-519 by miR-145. 293T cells were transfected with various amounts of miR-145 precursor. B. Effectiveness of miR-145 inhibitor on luciferase activity of Luc-Fli-1-519. As show, miR-145 inhibitor increased the luciferase activity. C. Schematic diagram of Luc-Fli-1-3′UTR-Mutation. For Luc-Fli-1-3′UTR-M, eight nucleotides (AACUGGAA) were changed with GGTGATCG. W: Wild; M: Mutation. D. Reporter mutation analysis. Downregulation of reporter gene with full-length 3′UTR from Fli-1 was apparent, whereas no effect on the Luc-Fli-1-3′UTR-Mutation was detected. All experiments were performed in triplicates and are shown as mean ± SD. *: P<0.05.

Figure 3

Reverse expression of endogenous miR-145 (A) and Fli-1 (B) in colon tumor tissues and colon cancer cell lines. Northern blotting was performed to quantitate miR-145 from seven pairs of adjacent colon and colon tumor tissues and three human colon cancer cell lines: LS174T, SW620 and HCT116 cells. The abundance of Fli-1 mRNA was measured by RT-PCR. S = samples, T = tumors, N = adjacent non-tumor tissues. GAPDH and U2snRNA were used as the quantitation control.

Figure 3

Reverse expression of endogenous miR-145 (A) and Fli-1 (B) in colon tumor tissues and colon cancer cell lines. Northern blotting was performed to quantitate miR-145 from seven pairs of adjacent colon and colon tumor tissues and three human colon cancer cell lines: LS174T, SW620 and HCT116 cells. The abundance of Fli-1 mRNA was measured by RT-PCR. S = samples, T = tumors, N = adjacent non-tumor tissues. GAPDH and U2snRNA were used as the quantitation control.

Figure 4

Regulation of endogenous Fli-1 expression by miR-145 A-B: Detection of mature miR-145 in LS174T cells transfected with miR-145 precursor and inhibitor. Expression of mature miR-145 in LS174T cells was quantitated 48 hours after transfection of miR-145 precursor (A) or inhibitor (B) by Hairpin-it™ miRNAs Real-Time PCR Quantitation Assay. Assays were performed in triplicates and are shown as mean ± SD. *: P<0.05. C. The Fli-1 expression in protein levels is changed significantly by miR-145. LS174T cells were transfected with miR-145 precursor or inhibitor. Fli-1 protein levels were analyzed by western blotting. At 48 hours after transfection, Fli-1 protein level was significantly decreased in cells transfected with miR-145 precursor. D-E. The Fli-1 mRNA levels in LS174T cells transfected with miR-145 precursor (D) or inhibitor (E). Fli-1 mRNA levels were analyzed by real-time PCR. Transcript levels did not change significantly at either 24 h or 48 h after transfection. All experiments were performed in triplicates and are shown as mean ± SD.

Figure 4

Regulation of endogenous Fli-1 expression by miR-145 A-B: Detection of mature miR-145 in LS174T cells transfected with miR-145 precursor and inhibitor. Expression of mature miR-145 in LS174T cells was quantitated 48 hours after transfection of miR-145 precursor (A) or inhibitor (B) by Hairpin-it™ miRNAs Real-Time PCR Quantitation Assay. Assays were performed in triplicates and are shown as mean ± SD. *: P<0.05. C. The Fli-1 expression in protein levels is changed significantly by miR-145. LS174T cells were transfected with miR-145 precursor or inhibitor. Fli-1 protein levels were analyzed by western blotting. At 48 hours after transfection, Fli-1 protein level was significantly decreased in cells transfected with miR-145 precursor. D-E. The Fli-1 mRNA levels in LS174T cells transfected with miR-145 precursor (D) or inhibitor (E). Fli-1 mRNA levels were analyzed by real-time PCR. Transcript levels did not change significantly at either 24 h or 48 h after transfection. All experiments were performed in triplicates and are shown as mean ± SD.

Figure 5

Effect of miR-145 and siRNA/Fli-1 on the Growth of LS174T Cells. A. Growth inhibition of LS174T cells cultured under miR-145 precursor or siRNA/Fli-1–deprived conditions in vitro. Experiments were performed in triplicates and data are shown as mean ± SD. The cell growth was determined at different time point after transfection by WST-8 assays. As show, miR-145 was even better than the siRNA in inhibiting cell proliferation. No tx: non transfection. B. Western blotting showing that Rb and Bcl-2 protein level is changed by transfecting LS174T cells with miR-145 precursor or siRNA for Fli-1 (siRNA/Fli-1) as compared with control precursor or control siRNA. C. Effect of miR-145 on sensitizing LS174T cells to 5-FU-induced apoptosis. Enhanced apoptosis were assessed by FACS at 48 hours. Note that miR-145 was as effectively as siRNA in sensitizing cells to 5-FU-induced apoptosis. Blank: only with DMEM medium containing 10% FBS; Ctrl pre: control precursor; miR-145 pre: miR-145 precursor; Ctrl siRNA: control siRNA; siRNA: siRNA/Fli-1. Assays were performed in triplicates and are shown as mean ± SD. *: P<0.05.