Beta-catenin/TCF4 transactivates miR-30e during intestinal cell differentiation

Abstract

The Wnt/beta-catenin/TCF4 pathway plays critical roles in the maintenance of small intestinal epithelium; however, downstream targets of the beta-catenin/TCF4 complex are not extensively characterized. We identified miR-30e as an immediate target activated by the beta-catenin/TCF4 complex. miR-30e was detected in the peri-nuclear region of the intestinal crypt IEC-6 cells. Bioinformatics analysis revealed clustered beta-catenin/TCF4 binding sites within the miR-30e promoter region. This promoter region was cloned into pGL3-control luciferase reporter vector, with the enhancer region removed. Transfection of pCMV-SPORT6-beta-catenin expression vector dose-dependently increased luciferase activity, and co-transfection of pCMV-SPORT6-TCF4 expression vector further enhanced the promoter activity. Dexamethasone-induced IEC-6 cells differentiation caused a 2.5-fold increase in miR-30e expression, and upon beta-catenin siRNA transfection, miR-30e increased 1.3-fold. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirmed the binding between beta-catenin/TCF4 complexes from IEC-6 nuclear extracts and the putative sequences in the miR-30e promoter. These results demonstrate that beta-catenin/TCF4 transactivates miR-30e during intestinal cell differentiation.

Fig. 1

Subcellular localization of miR-30e in IEC-6 cells. miR-30e distribution in IEC-6 cells was examined by confocal microscopy. Paraformaldehyde-fixed cells were probed with LNA DIG-miR-30e probe. Reactivities were visualized by TSA Cy5 system (red a), and nuclei were stained with DAPI nucleic acid dye (blue b). c The peri-nuclear localization of miR-30e. Magnification of ×60 under oil. Scale bar 10 μm

Fig. 2

Comparative sequence analysis of miR-30e gene promoter from human, mouse, and rat. The promoter sequences were retrieved from miRBase Sequences Database, and the analysis was performed at TESS and TFSEARCH. The binding domains for two types of beta-catenin/TCF4 complex are shown with black and empty arrowheads, and for TCF4 with hatched arrowheads

Fig. 3

Effect of beta-catenin/TCF4 overexpression on the miR-30e gene promoter activity. IEC-6 cells were co-transfected with 3.5 μg/ml luciferase expression vector containing wild-type and mutated miR-30e promoter and increasing concentrations of pCMV-SPORT6-beta-catenin expression vector without (a,c) or with (b,d) 1.2 μg/ml pCMV-SPORT6-TCF4 expression vector. Luciferase activity was corrected for transfection efficiency based on pGL4.73[hRluc/SV40] vector transfection. Beta-catenin overexpression dose-dependently increased the luciferase activity (P < 0.05) (a) and overexpresison of TCF4 further enhanced the effect from beta-catenin (P < 0.05) (b). When the TTTTGTT sites were mutated, overexpression of TCF4 did not further enhance the effects of beta-catenin (c,d). Values are means ± SEM run in triplicates; asterisks indicate significant differences between treatment and control groups

Fig. 4

Effects of beta-catenin and TCF4 knock-down on miR-30e expression during dexamethasone induced cell differentiation. IEC-6 cells were treated with 100 nM dexamethasone for 48 h with or without 75 nM beta-catenin siRNA, 100 nM TCF4 siRNA. Total RNA was extracted and analyzed for miR-30 expression by real-time Q-PCR. miR-30e significantly increased upon dexamethasone treatment (P < 0.05), beta-catenin siRNA and TCF4 siRNA both blocked the upregulation. Values are means ± SEM run in triplicates, asterisk indicates significant differences between dexamethasone treatment alone and control group, letters indicate significant differences between dexamethasone treatments without (a) and with (b) siRNA treatment

Fig. 5

Identification of beta-catenin/TCF4 binding sites within the 2,000-bp miR-30e promoter region. a 3 μl of IEC-6 cell NE-PER nuclear extracts were incubated with 2 nM biotin-labeled DNA probe corresponding to the human miR-30e promoter sequence −1,239/−1,214 bp (lane 1). For binding competetion, 50-fold excess of unlabeled DNA probe was included in the reaction (lane 2). For supershift assay, 0.5 μg beta-catenin antibody (lane 3) or an irrelevant antibody (antibody for alkaline phosphatase, lane 4) were included. Supershift was also observed for probe corresponding to −272/−250 bp region of the miR-30e promoter (data not shown). b IEC-6 cells treated with dexamethasone, the precipitated DNA, along with the DNA isolated before immunoprecipitation (Input), were analyzed by PCR amplication with specific primers corresponding to the region1 (−1,348/−1,103), region2 (−386/−149), region3 (−1,535 to −1,328), region4 (−1,991 to −1,787) and negative control (+927/+1,168)