MiR-145, a new regulator of the DNA fragmentation factor-45 (DFF45)-mediated apoptotic network

Abstract

Background: MicroRNA-145 (miR-145) is considered to play key roles in many cellular processes, such as proliferation, differentiation and apoptosis, by inhibiting target gene expression. DNA Fragmentation Factor-45 (DFF45) has been found to be the substrate of Caspase-3, and the cleavage of DFF45 by caspase-3 during apoptosis releases DFF40 that degrades chromosomal DNA into nucleosomal fragments. There are currently no in-depth studies on the relationship between miR-145 and the DFF45 gene.

Results: In this study, we identified DFF45 as a novel target of miR-145. We demonstrated that miR-145 targets a putative binding site in the coding sequence (CDS) of DFF45, and its abundance is inversely associated with DFF45 expression in colon cancer cells. Using a luciferase reporter system, we found that miR-145 suppresses the expression of the luciferase reporter gene fused to the putative binding site of DFF45. The level of DFF45 protein, but not DFF45 mRNA, was decreased by miR-145, suggesting a mechanism of translational regulation. Furthermore, we demonstrate that this specific silencing of DFF45 by miR-145 accounts, at least in part, for the staurosporine-induced tumor cell apoptosis in vitro.

Conclusions: Our study reveals a previously unrecognized function of miR-145 in DFF45 processing, which may underlie crucial aspects of cancer biology.

Figure 1

Expression of endogenous miR-145 and DFF45 protein in colon cancer cells. A. The expression levels of primary, precursor and mature miR-145 were examined in normal colon cells (normal), Dukes' type B cells (SW480 and LS174T), Dukes' type C cells (SW620 and COLO320DM) and Dukes' type D cells (COLO205) by real time PCR. U6 RNA was used as the quantification control. B. Detection of mature miR-145 in LS174T cells transfected with the miR-145 mimic and inhibitor. Expression of mature miR-145 in LS174T cells was quantitated 48 hours after transfection of miR-145 mimic or inhibitor by Hairpin-it™miRNAs Real-Time PCR Quantitation Assay. Assays were performed in triplicate and are shown as the mean ± SD. *: P < 0.05. C. Deregulation of the DFF45 protein was identified by antibody microarray analysis in LS174T cells transfected with the miR-145 mimic. D. Expression of endogenous DFF45 or p53 protein (wild-type or matant). Western blotting analysis was performed with total protein isolated from normal colon cells and different colon tumor cells.

Figure 2

MiR-145 targets a putative binding site in the coding sequence of DFF45. A. Sequence alignment of miR-145 with the putative binding sites in the human DFF45 gene. The numbers are relative to the start codon site. B. Schematic diagram of the luciferase reporter construct. Putative binding sites (PBS) predicted by bioinformatics were cloned at the Xba1 site into the 3'UTR of the luciferase gene. C. Regulation of reporter gene expression by ectopic miR-145. LS174T cells were co-transfected with the miR-145 mimic and a luciferase reporter containing one putative binding site. D. Regulation of reporter gene expression by endogenous miR-145. Normal colon cells were transfected with the pGL3 vector or a luciferase reporter containing the 854-876 putative binding sites (luc-854). These experiments were performed in triplicate, and are shown as the mean ± SD, *: P < 0.05.

Figure 3

Specific targeting of the DFF45 putative binding site by miR-145. A. Dose-dependent suppression of luc-854 by miR-145. LS174T cells were co-transfected with luc-854 and various amounts of the miR-145 mimic. B. Effects of a miR-145 inhibitor on luciferase activity of luc-854. Normal colon cells were co-transfected with miR-145 inhibitor and luc-854. C. Schematic diagram of DFF45-854-Mutation. For the DFF45-854-Mutation, seven nucleotides (gagcGggaG) were changed with ctcgGcctG. D. Reporter mutation analysis. Downregulation of the reporter gene with the entire region (coding region plus 3'UTR) from DFF45 (DFF45-854-Wild) was apparent, whereas no effect on the DFF45-854-Mutation was detected. These experiments were performed in triplicate, and the results are shown as the mean ± SD, *: P < 0.05.

Figure 4

Regulation of endogenous DFF45 expression by miR-145. DFF45 mRNA levels in LS174T cells transfected with miR-145 mimic did not change significantly, by quantitative RT-PCR (A) or real-time PCR assay (B), at either 24 h or 48 h after transfection. (C) The DFF45 protein expression levels was affected significantly by miR-145. LS174T cells were transfected with the miR-145 mimic or miRNA control. DFF45 protein levels were analyzed by Western blotting at either 24 h or 48 h after transfection. All experiments were performed in triplicate and are shown as the mean ± SD.

Figure 5

Effects of miR-145 on staurosporine-induced DNA fragmentation. LS174T cells (A), or normal colon cells (B) were transfected with the miR-145 mimic or siRNA-DFF45 (50 nM), and then exposed to staurosporine. DNA ladders in samples were collected at various times after treatment with staurosporine and visualized on a 1.5% agarose gel. After transfection with the miR-145 mimic or siRNA-DFF45, down-regulation of DFF45 protein was detected by Western blotting at 6, 12 and 18 hours in LS174T cells (C), but not in normal colon cells (E). Values in D and F are the means of three separate experiments ± SD. *, P < 0.05.

Figure 6

Nuclear staining of LS174T cells with Hoechst 33258. After transfection with the miR-145 mimic or siRNA-DFF45 (50 nM), and subsequent exposure to staurosporine (STA) for 12 h, LS174T cells were stained with Hoechst 33258, and visualized using a fluorescence microscope. (A) MiRNA control-treated group. (B) MiRNA control with STA-treated group. (C) MiR-145 mimic-treated group. (D) MiR-145 mimic with STA-treated group. (E) siRNA control-treated group. (F) siRNA control with STA-treated group. (G) siRNA-DFF45-treated group. (H) siRNA-DFF45 with STA-treated group. (I) Untreated group. (J) STA-treated group. LS174T cells treated with the miR-145 mimic or siRNA-DFF45 showed apoptotic morphology: chromatin condensation. (K) The percentage of cell death was calculated by counting the number of cells with condensed chromatin over the total number of cells. The data represent the mean ± SD of three independent experiments. At least 300 cells were counted for each condition.