Abstract
Maintenance of the small intestinal epithelium is based on a supply of specialized villous cell lineages differentiated from progenitors in the crypts. The Wnt/beta‐catenin/TCF4 signaling pathway plays critical roles in this process, but little is known about its downstream targets. MicroRNAs (miRNAs) are endogenous, non‐coding RNAs ~22 nt in size, down‐regulating protein translation by binding to their target mRNA 3'UTRs. We identified mir‐30e as an immediate downstream target activated by beta‐catenin/TCF4. Bioinformatics analysis revealed clustered beta‐catenin/TCF4 binding sites within a 2000 bp promoter region of mir‐30e gene for human, mice, and rats. 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 further enhanced promoter activity. Dexamethasone induced differentiation of IEC‐6 cells caused a 2‐fold increase in mir‐30e expression, and upon beta‐catenin siRNA transfection, mir‐30e increased 1.3‐fold. Binding of beta‐catenin/TCF4 complexes from IEC‐6 nuclear extracts to the 4 putative sequences in the human mir‐30e promoter was confirmed by EMSA. These results demonstrate that beta‐catenin/TCF4 transactivates mir‐30e during intestinal cell differentiation.Grant Funding SourceInternal
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