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RNA. Aug 2007; 13(8): 1172–1178.
PMCID: PMC1924904

miR-200b mediates post-transcriptional repression of ZFHX1B


MicroRNAs have important functions during animal development and homeostasis through post-transcriptional regulation of their cognate mRNA targets. ZFHX1B is a transcriptional repressor involved in the TGFβ signaling pathway and in processes of epithelial to mesenchymal transition via regulation of E-cadherin. We show that Zfhx1b and miR-200b are regionally coexpressed in the adult mouse brain and that miR-200b represses the expression of Zfhx1b via multiple sequence elements present in the 3′-untranslated region. Overexpression of miR-200b leads to repression of endogenous ZFHX1B, and inhibition of miR-200b relieves the repression of ZFHX1B. In accordance with these findings, miR-200b regulates the activity of the E-cadherin promoter.

Keywords: microRNA, translational repression, E-cadherin, miR-200b, ZFHX1B


ZFHX1B is a transcriptional repressor of the ZFH-1 family that acts as a downstream mediator of TGFβ and BMP signaling (Verschueren et al. 1999; Postigo 2003). ZFHX1B is widely expressed in humans and mice, most prominently in the heart and in neural tissues (Yamada et al. 2001; Bassez et al. 2004). Mutations causing ZFHX1B haploinsufficiency during embryogenesis form the genetic basis of Mowat–Wilson syndrome, characterized by dysmorphic facial features, mental retardation, microcephaly, seizures, and variable organ malformations (Cacheux et al. 2001; Wakamatsu et al. 2001; Dastot-Le Moal et al. 2007). Genetic ablation of Zfhx1b in mice is embryonically lethal at E9.5–E10.5, and the mice exhibit developmental defects in the formation of the neural crest (Higashi et al. 2002; Van de Putte et al. 2003). The ZFHX1B protein has two zinc-finger clusters that bind to conserved E-boxes in promoter regions of its target genes, which include Brachyury, E-cadherin, and α4-integrin (Remacle et al. 1999; Verschueren et al. 1999). Dynamic down-regulation of E-cadherin expression leading to epithelial–mesenchymal transition and migratory cell behavior is essential to early development and tissue formation (Shook and Keller 2003), and aberrant E-cadherin expression caused by lack of Zfhx1b is thought to contribute to the developmental defects in the Zfhx1b −/− embryos (Van de Putte et al. 2003). Furthermore, elevated ZFHX1B expression correlates with reduced E-cadherin expression in certain cancers (Rosivatz et al. 2002; Elloul et al. 2005) and has been implicated in carcinoma progression by means of its ability to repress E-cadherin and induce epithelial–mesenchymal transition (Comijn et al. 2001; Rosivatz et al. 2002; Miyoshi et al. 2004; Maeda et al. 2005).

MicroRNAs (miRNAs) are an abundant class of small noncoding RNA molecules that regulate gene expression post-transcriptionally (Jackson and Standart 2007). miRNAs are transcribed by RNA polymerase II as long primary transcripts (Cai et al. 2004; Lee et al. 2004) that are processed in multiple steps into cytoplasmic ~22-nt duplexes (Hutvagner et al. 2001; Ketting et al. 2001; Lee et al. 2003; Yi et al. 2003; Bohnsack et al. 2004; Lund et al. 2004). The duplex is loaded into a ribonucleoprotein particle, miRISC, where unwinding of the duplex enables the mature single-stranded miRNA to base-pair with its target mRNA (Kim and Nam 2006). The majority of mammalian miRNAs are thought to recognize their targets by base-pairing of the miRNA seed region (bases 2–8 at the 5′ end) with one or more sites of perfect complementarity in the 3′ untranslated region (UTR) of the target mRNA (Doench and Sharp 2004; Wang et al. 2006).

miRNA-mediated repression appears to occur by at least two different mechanisms, resulting either in degradation of mRNA or inhibition of mRNA translation. miRNAs mediate RNAi-like cleavage of targets with perfect complementarity, as demonstrated for miR-196 and its endogenous HOXB8 target (Yekta et al. 2004). In addition, miRNAs with incomplete target complementarity can promote target degradation, albeit by different mechanisms (Bagga et al. 2005; Jing et al. 2005; Wu et al. 2006). Other studies indicate that miRNAs, rather than affecting mRNA stability, inhibit mRNA translation at the step of initiation (Humphreys et al. 2005; Pillai et al. 2005; Wang et al. 2006) or elongation (Petersen et al. 2006). The apparent inconsistency of these studies could reflect the existence of several mechanisms for miRNA-mediated repression, which may not be mutually exclusive (Pillai 2005; Petersen et al. 2006; Wu et al. 2006). Individual miRNAs are thought to influence signaling pathways to affect cell proliferation, differentiation, and death as well as developmental timing (Ambros 2004). In addition, miRNAs may function as a fine-tuning mechanism by dampening the translation of mRNAs outside their expression boundaries (Cohen et al. 2006; Hornstein and Shomron 2006).

As ZFHX1B has important functions during embryogenesis and in a number of human diseases, we set out to investigate a putative role for miRNAs in the regulation of ZFHX1B. In this study we identify miR-200b as a post-transcriptional regulator of ZFHX1B and demonstrate the ability of miR-200b to affect the promoter activity of the ZFHX1B target gene E-cadherin.


The ZFHX1B mRNA contains a 1.4-kb 3′-UTR with putative binding sites for many miRNAs. Sequence inspection revealed the presence of five sequence elements (S1–S5) complementary to the seed region of miR-200b (nt 2–8). Of these sites, S1 and S3–5 are predicted miR-200b binding sites by TargetScan (Lewis et al. 2005), S4 and S5 are predicted by the miRBase Targets database (Griffiths-Jones et al. 2006), whereas S2 are not predicted binding sites by these databases. We found all five miR-200b complementary sites in the Zfhx1b 3′ UTR to be highly conserved across several species, supporting a biological relevance of these sequence motifs (Fig. 1). To investigate if Zfhx1b and miR-200b are expressed in the same regions of the mouse brain we performed fluorescent in situ hybridization of miR-200b on sagittal sections of adult mouse brain to compare it with previous reports of Zfhx1b expression. Zfhx1b is strongly expressed in the pyramidal cell layer and dentate gyrus of the hippocampal formation as well as the cerebral and cerebellar cortex (Allen Brain Atlas 2004). As evident from Figure 2, the expression pattern of miR-200b overlaps that of Zfhx1b in several regions of the brain, including the hippocampus and the cerebellum. The observed regional coexpression of the Zfhx1b mRNA and miR-200b lends in vivo relevance to the predicted interaction between the molecules.

miR-200b binding sites in the Zfhx1b 3′-UTR are conserved across species. Schematic representation of the mouse Zfhx1b 3′-UTR containing five potential binding sites for miR-200b (S1–S5), based on occurrence of the complementary ...
Expression of miR-200b and Zfhx1b mRNA in the brain. (Top panel) Sagittal section of adult mouse brain hybridized with an antisense RNA probe for Zfhx1b (Allen Brain Atlas 2004). (Bottom panel) Sagittal section of adult mouse brain hybridized with an ...

The ability of miR-200b to bind and regulate the 3′-UTR of Zfhx1b was evaluated using luciferase reporter assays. A 1-kb fragment of the mouse Zfhx1b 3′-UTR was cloned into a pGL3-control vector, placing the 3′-UTR with the five potential miR-200b binding sites downstream of luciferase (pGL3 S1–5). Cotransfections of HEK293 cells with pGL3 S1–5 and concentrations of miR-200b ranging from 2.5 nM to 30 nM resulted in a dose-dependent repression of the reporter (Fig. 3A). Repression was specific to miR-200b as a mutated version of miR-200b containing two point mutations in the seed region failed to repress, as did a structurally unrelated miRNA, miR-10a (Fig. 3A). The repression was furthermore dependent on the 3′ UTR of Zfhx1b, as miR-200b did not affect the expression of the pGL3 control vector (Fig. 3B).

miR-200b regulates the 3′ UTR of Zfhx1b. (A) A luciferase reporter construct containing five miR-200b binding sites was cotransfected into HEK293 cells along with miR-200b, mutated miR-200b, or miR-10a. Five to 30 nM miR-200b represssed the reporter ...

To analyze the importance of individual miR-200b binding sites for the 3′ UTR-dependent regulation of luciferase, fragments containing S1–2 and S3–5 were subcloned in pGL3. HEK293 cells were cotransfected with each of the reporter constructs and 10 nM of miR-200b or mutated miR-200b (Fig. 3B). miR-200b, but not the mutated control, caused significant repression of both constructs (P < 0.001), indicating that the Zfhx1b 3′-UTR contains several biologically relevant miR-200b binding sites. In order to determine the mechanism for repression of the luciferase reporter, we quantified the mRNA levels (Fig. 3C). miR-200b caused a significant decrease in the amount of all three luciferase reporters (P < 0.001). As none of the putative binding sites predict annealing between miR-200b and the Zfhx1b mRNA around nucleotides 10–11 relative to the 5′ end of the miRNA, we speculate that the mRNA degradation involves mRNA deadenylation and destabilization rather than Ago2-mediated cleavage (Elbashir et al. 2001).

Having established the ability of miR-200b to mediate repression of luciferase via the Zfhx1b 3′-UTR, we next assayed its capacity to regulate the endogenous ZFHX1B protein. To this end, we transfected the human breast cancer cell line MDA-MB-435S, expressing high levels of ZFHX1B (Comijn et al. 2001), with miR-200b, control miRNA, or a pool of siRNAs directed against ZFHX1B and analyzed protein expression by Western blotting. As evident from Figure 3D,E, miR-200b markedly reduced ZFHX1B protein and mRNA levels compared to mock transfected cells. In contrast, none of the control miRNAs repressed ZFHX1B. Importantly, blocking endogenous miR-200b by transfection with a specific LNA-modified inhibitor resulted in an increase in the level of endogenous ZFHX1B (Fig. 3D), thereby supporting the notion that miR-200b regulates ZFHX1B in vivo.

To investigate the biological significance of ZFHX1B regulation by miR-200b we assayed the effect of miR-200b using an E-cadherin promoter construct, a well-established ZFHX1B target. We used a luciferase reporter construct containing 227 nt of the human E-cadherin promoter including the E-box binding sites for ZFHX1B (Grooteclaes and Frisch 2000) and tested the ability of miR-200b to cause derepression of the E-cadherin promoter. As expected, cotransfections of HEK293 cells with the E-cadherin promoter construct and a Zfhx1b expression vector caused repression of the E-cadherin promoter (Fig. 4). Conversely, siRNA-mediated knockdown of endogenous ZFHX1B relieved repression of the promoter construct. In agreement with a role for miR-200b in regulation of ZFHX1B, cotransfections of the E-cadherin reporter with miR-200b resulted in a significant derepression of the reporter (P < 0.001). This effect was caused specifically by miR-200b, since cotransfections with miR-10a did not influence the E-cadherin reporter. ZFHX1B has been shown to bind and regulate the E-cadherin promoter through the two E-boxes present in the proximal promoter (Remacle et al. 1999). Overexpression of Zfhx1b or transfection with miR-200b only marginally affected a mutant E-cadherin promoter in which both E-box motifs were altered by point mutation (Fig. 4). This result supports the involvement of miR-200b in the regulation of E-cadherin via ZFHX1B, although we cannot exclude that miR-200b may also affect E-cadherin through routes parallel to ZFHX1B repression.

miR-200b increases E-cadherin promoter activity. A luciferase construct containing part of the human E-cadherin promoter, including the E-boxes, was cotransfected into HEK293 cells with Zfhx1b, siRNA against ZFHX1B, miR-200b, or miR-10a. miR-200b derepressed ...

In conclusion, we find that miR-200b is a potent regulator of ZFHX1B expression via binding to multiple sites in the 3′-UTR. Consequently, exogenous miR-200b results in ZFHX1B repression, and inhibition of endogenous miR-200b results in increased levels of ZFHX1B protein. The significance of the miR-200b:ZFHX1B interaction can furthermore be demonstrated using a promoter construct of the bona fide ZFHX1B target gene E-cadherin. Interestingly, we note that ZFHX1a (δEF1), the other member of the ZFH-1 family, is also a predicted target of miR-200b. In addition, ZFHX1a and -1b are both predicted targets of miR-200c (Lewis et al. 2005; Griffiths-Jones et al. 2006). As ZFHX1a and -1b share several target genes, such as Brachyury and E-cadherin (Remacle et al. 1999), it is tempting to speculate that the miR-200 family has evolved to regulate the family of ZFH-1 genes, possibly during processes of mesenchymal to epithelial transition, and control the containment of ZFH-1 expression within specific cell types.


Cell culture and transfections

HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin and incubated at 5% CO2 and 37°C.

All transfections were carried out with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. For Zfhx1b luciferase assays, HEK293 cells were seeded 1 × 104 per 96-well the day prior to transfection with 0.15 μg luciferase expression construct, 2.5–30 nM miRNA duplex, and 0.015 μg Renilla luciferase vector pRL-TK (Promega) for normalization. For E-cadherin luciferase assays, HEK293 cells were transfected with 0.1 μg luciferase expression construct, 0.1 μg Zfhx1b expression construct, 50 nM siRNA, 5–25 nM miRNA duplex, and 0.015 μg pRL-TK for normalization. Mock transfected cells were transfected with the luciferase constructs alone. Luciferase activity was measured 48 h after transfection using the Dual-Glo Luciferase kit (Promega). For quantitative RT-PCR, HEK293 cells were seeded 400,000 per 6-well the day prior to transfection with 1.5 μg luciferase expression construct, 0.15 μg pRL-TK, and 10 nM miRNA duplex.

MDA-MB-435S cells were maintained in DMEM with 10% FBS, 10 μg/mL insulin, 100 U/mL penicillin, and 100 μg/mL streptomycin. For Western blotting and quantitative RT-PCR, MDA-MB-435S cells were seeded 400,000 per 6-well the day prior to transfection with 30 nM miRNA, LNA, or siRNA using Lipofectamine 2000 (Invitrogen). Mock transfected cells were treated with transfection reagent alone. Transfections were repeated after 24 h, and the cells were harvested 72 h after the first transfection. siGENOME SMARTpool siRNA against ZFHX1B were used (Dharmacon).

Plasmid vectors

A multiple cloning site was inserted into the pGL3 control vector (Invitrogen) at the XbaI site 3′ of the luciferase gene (hereafter pGL3). A 1-kb fragment of the Zfhx1b 3′ UTR (corresponding to positions 3979–5018 of the Ensembl transcript ENSMUST00000076836) was PCR amplified from mouse genomic DNA and cloned into pGL3. Four different constructs were made, each containing two or more of the five potential miR-200b binding sites present in the Zfhx1b 3′ UTR (the binding sites referred to as S1 to S5 as illustrated in Fig. 1). The primer sequences used for PCR amplification were as follows (restriction sites indicated in lower case):

  • pGL3 S3–5: 5′-GGgaattcagatctGGTTACCTTTGCACCAGCTTCAGTG-3′ and
  • pGL3 S4–5: 5′-GGgaattcagatctCAGTTCCTTAGTTTACATATGTTTGTGC-3′ and

For E-cadherin luciferase assays we used a pGL2 vector with an insert upstream of the luciferase gene corresponding to position −108 to +119 of the human E-cadherin gene (Ensembl gene ID ENSG00000039068) in which two E-boxes are located (CACCTG/CAGGTG). The mutated version of this construct carries point mutations in the E-box sequences (AACCTA/AAGGTA) (Grooteclaes and Frisch 2000).

miRNA duplexes and anti-miRNA oligonucleotides

  • miRNA duplexes with the following sequences were synthesized by CureVac:
  • miR-200b (sense): 5′- PO4-UAAUACUGCCUGGUAAUGAUGAC-3′,
  • miR-200b (guide): 5′- PO4-CAUCAUUACCAGGCAGUAUUAAA-3′,
  • mutated miR-200b (sense): 5′- PO4-UACUAGUGCCUGGUAAUGAUGAC-3′,
  • mutated miR-200b (guide): 5′- PO4-CAUCAUUACCAGGCAUUGGUAAA-3′,
  • miR10a (sense): 5′- PO4-UACCCUGUAGAUCCGAAUUUGUG-3′,
  • miR10a (guide): 5′- PO4-CAAAUUCGGAUCUACAAAGUAAA-3′.

The LNA-modified oligonucleotide used for miR-200b inhibition and the LNA probe for in situ hybridization was purchased from Exiqon.

Fluorescent in situ hybridization

In situ detection of miR-200b was performed on 10-μm frozen tissue sections from adult mouse brain. Sections were fixed in 4% paraformaldehyde and acetylated in acetic anhydride/triethanolamine, each followed by washes in PBS. Sections were then prehybridized in hybridization solution (50% formamide, 5× SSC, 0.5 mg/mL yeast tRNA, 1× Denhardt's solution) at 48°C for 30 min. Three-picomole probe (LNA-modified oligonucleotide, Exiqon) complementary to miR-200b was DIG-labeled (DIG Oligonucleotide 3′ Tailing Kit, Roche Applied Sciences) and hybridized to the sections for 1 h at 48°C. After post-hybridization washes in 0.1× SSC at 55°C, the in situ hybridization signals were detected using the tyramide signal amplification system (Perkin Elmer) according to the manufacturer's instructions. Slides were mounted in Prolong Gold containing DAPI (Invitrogen) and analyzed with an Olympus MVX10 microscope equipped with a CCD camera and Olympus CellP software.

Real-time quantitative PCR

Total RNA was Trizol purified, DNase digested, and reverse transcribed (TaqMan reverse transcription reagents) followed by quantitative PCR using Sybrgreen PCR Master Mix and the 7300 Real-Time PCR system (Applied Biosystems). The primer sequences used for human ZFHX1B and RPLP0 (ribosomal protein, large, P0) gene were


For the Renilla and firefly luciferase genes, the primer sequences were


Antibodies and Western blot analysis

A polyclonal antibody was raised against mouse and human ZFHX1B protein by immunization of rabbits with an oligopeptide (CDGHAVSIEEYLQRS) and affinity purified using Sulfolink Coupling Gel (Pierce).

MDA-MB-435S cells were harvested 72 h after the first transfection, washed once in PBS, and lysed in RIPA buffer (150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl at pH 8, 2 mM EDTA) containing 1 mM DTT and 1× Pefabloc (Roche). Twenty micrograms of protein per lane were separated on an 8% polyacrylamide gel and transferred to a nitrocellulose membrane. Anti-ZFHX1B was used at a 1:500 dilution, and the hVIN-1 vincullin antibody (Sigma) was used at 1:20,000.


We thank Lisa Frankel for critically reading the manuscript. Work in the authors' laboratories is supported by the Biotech Research and Innovation Centre, the Vilhelm Pedersen and Hustrus Foundation, The Danish Medical Research Council, the Danish Cancer Research Foundation, the Danish Cancer Society, the Association for International Cancer Research, the Lundbeck Foundation and the Danish National Advanced Technology Foundation.


Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.586807.


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