increasing evidence indicates that aberrant activation of the embryonic programme  epithelial  mesenchymal transition ' ( emt ) promotes tumour cell invasion and metastasis ( berx et al , 2007 ) . emt allows detachment of cells from each other and increases cell mobility , both of which are necessary for tumour cell dissemination . metastases often recapitulate the differentiated phenotype of the primary tumour ; therefore , emt seems to be transiently activated by the inductive tumour environment at the invasive tumour edge , but is reversed in growing metastases ( brabletz et al , 2001 , 2005 ) . activators of emt , such as transforming growth factor ( tgf) , tumour necrosis factor  ( tnf ) and hepatocyte growth factor , are produced by infiltrating cells or the tumour cells themselves , and trigger expression of emt - inducing transcriptional repressors ( thiery & sleeman , 2006 ) . helix family , goosecoid and members of the zfh family ( zinc - finger e - box binding homeobox ( zeb)1 and zeb2 ; barrallo - gimeno & nieto , 2005 ; hugo et al , 2007 ; peinado et al , 2007 ) . recently , we described that zeb1 is a crucial emt activator in human colorectal and breast cancer , and suppresses expression of basement membrane components ( spaderna et al , 2006 ) and cell polarity factors ( aigner et al , 2007 ; spaderna et al , 2008 ) . expression of zeb1 promotes metastasis of tumour cells in a mouse xenograft model , indicating a role of zeb1 in invasion and metastasis of human tumours ( spaderna et al , 2008 ) . micrornas ( mirnas ) are small non - coding rnas that can silence their cognate target genes by specifically binding and cleaving messenger rnas or inhibiting their translation ( bartel , 2004 ) . mirnas regulate diverse cellular processes and some mirnas have been shown to function as either tumour suppressors or oncogenes ( esquela - kerscher & slack , 2006 ) . recent important examples are the oncogenic mir-10b , which promotes metastasis ( ma et al , 2007 ) , and mir-335/mir-126 , which suppress metastasis in breast cancer ( tavazoie et al , 2008 ) . owing to these important regulatory functions of mirnas , it is of prime interest to know how their expression is regulated by upstream factors . here , we address this point and focus on the activation and stabilization of emt in cancer cells . we investigated whether aberrant expression of the crucial emt activator zeb1 and the control of potential emt - regulatory mirnas are linked and can synergize to promote malignant tumour progression . tgf and tnf have been shown to induce an emt in differentiated colorectal cancer cells ( bates et al , 2005 ) . we observed activation of emt in differentiated pancreatic ( hpaf2 ) , colorectal ( dld1 ) and breast cancer ( mcf7 ) cell lines by tgf/tnf. thus , zeb1 was a crucial intracellular transmitter of this emt , as its expression was upregulated by both cytokines and its knockdown partly prevented emt ( supplementary fig 1a , b online ) . direct epithelial target genes suppressed by zeb1 , such as e - cadherin , cell polarity factors and basement membrane components , have been described previously ( grooteclaes & frisch , 2000 ; aigner et al , 2007 ; spaderna et al , 2008 ) . here , we investigated whether zeb1 also affects expression of mirnas and if detected mirnas themselves are candidate regulators of emt . a mirna expression microarray screen was used to analyse sw480 and hct116 colorectal and mda - mb231 breast cancer cell clones with stable short hairpin rna - mediated knockdown of zeb1 ( shzeb clones ) in comparison with control knockdown ( shctl ) clones . we have shown previously that stable knockdown of zeb1 in these clones led to a reversal of the fibroblastoid phenotype towards an epithelial differentiation in the sense of a mesenchymal  epithelial transition ( spaderna et al , 2008 ) . out of 743 human , rat and mouse mirnas included on the microarray screen , we detected average upregulation ( > 1.75-fold ) after zeb1 knockdown of 79 mirnas ( 10.59% ) in at least one cell line , 17 mirnas ( 2.27% ) in two cell lines and only 4 mirnas ( 0.53% ) in all three cell lines ( fig 1 ; supplementary table 1 online ) . the strongest effect was observed for the undifferentiated breast cancer cell line mda - mb231 . notably , three of the four mirnas upregulated in all cell lines ( mir-141 , mir-200b and mir-200c ) belong to the highly conserved mirna-200 family , which was recently linked to the induction of epithelial differentiation ( hurteau et al , 2007 ; gregory et al , 2008 ; park et al , 2008 ) . further analyses focused on mir-141 and mir-200c , which showed strongest upregulation after knockdown of zeb1 in all three cancer cell lines . the array data were validated by using real - time pcr for mir-141 and mir-200c , which showed a strong increase after knockdown of zeb1 in undifferentiated pancreatic , colorectal and breast cancer cell lines ( supplementary fig 1c online ) . next , we investigated whether zeb1 directly suppresses transcription of the mir-141 and mir-200c mirna genes . both mirnas map closely on human chromosome 12p13.31 and the stem loop sequences are separated by only a 338-base - pair spacer sequence ( fig 2a ) . this spacer and the putative promoter 600 bp upstream from the hsa - mir-200c stem loop contain six putative binding sequences for zeb1 , two of which were restricted to zeb factors ( z - box 1 and 2 , caggta ) . the remaining four were perfect e - boxes ( e - box 14 , caggtg ) , which , in addition to zeb1 , represent putative binding sites for other emt activators , such as snail factors . note that the overall mirna gene structure and the two z - boxes , as well as e - box 2 , are highly conserved in evolution from zebrafish to human ( fig 2a ) . all the conserved zeb1-binding sequences are located within the putative promoter , whereas the two e - boxes in the spacer sequence are not conserved . after cloning of the putative promoter ( 683 to 67 relative to first nucleotide of the mir-200c stem  loop ) and the spacer ( + 66 to + 338 ) into a luciferase reporter vector , zeb1-dependent activity was assessed . shrna - mediated knockdown of zeb1 in all undifferentiated colorectal , breast and pancreatic cancer cell lines tested resulted in an enhanced promoter activity when compared with shctl cell clones ( fig 2b , left ) . on the contrary , the promoter activity was suppressed by zeb1 overexpression in a dose - dependent manner . similar effects , although to a lesser extent , were also observed after snail1 overexpression , which can bind to only two of the four potential zeb1-binding sites within the putative promoter ( fig 2b , middle ) . the expression level of zeb1 had no significant effect on the transcriptional activity of the spacer sequence . mutation of the two highly conserved z - boxes showed that z - box 2 confers the strongest repressive function by zeb1 ( fig 2b , right ) and also made the promoter activity insensitive to stable knockdown of zeb1 ( supplementary fig 1d online ) . a direct binding of zeb1 to the two conserved zeb1 sites ( z - boxes ) was shown by electromobility shift assay by using recombinant dna - binding domain of zeb1 and nuclear extracts from sw480 colorectal cancer cells ( fig 2c ) . by applying chromatin immunoprecipitation ( chip ) with chromatin from sw480 or hct116 colorectal cancer cells , we could show that endogenous zeb1 binds to the native promoter region ( fig 2d ) . these data indicate that the transcriptional repressor zeb1 can directly suppress expression of both mir-141 and mir-200c by binding to their putative common promoter . next , we investigated whether the two mirnas suppressed by zeb1 represent direct emt regulators . we showed that mir-200c and mir-141 are strong inducers of an epithelial phenotype , which was also recently reported by other groups during the course of our work ( hurteau et al , 2007 ; gregory et al , 2008 ; park et al , 2008 ) . transient overexpression of both mir-141 and mir-200c resulted in the induction of epithelial differentiation of undifferentiated cancer cells ( fig 3a ; supplementary fig 2a , b online ) . in particular , the overall effect of mir-200c was comparable with the strong effect of zeb1 knockdown . notably , mir-200c , and to a lesser extent mir-141 , also led to increased e - cadherin expression and cell  cell adhesion , as well as reduced spreading of normal human fibroblasts ( supplementary fig 2c online ) . overexpression of mir-200c strongly reduced cancer cell migration and invasion into matrigel , indicating a potential inhibitory role in malignant tumour progression ( fig 3b ; supplementary fig 2d online ) . on the contrary , treatment of differentiated cancer cell lines hpaf2 and dld1 with inhibitors of mir-200c , and to a lesser extent of mir-141 , resulted in a mesenchymal transition as indicated by cell scattering , upregulation of vimentin and reduced expression of e - cadherin ( fig 3c ) . knowing that the breast cancer cell line mda - mb231 , which almost completely lost both mirnas , is derived from a basal type of breast cancer , we analysed various types of human breast cancer . as predicted from using cancer cell lines , we confirmed that basal types of breast cancers , characterized by an undifferentiated phenotype and poor clinical prognosis , show reduced expression of both mirnas compared with the common ductal invasive type of breast cancer ( supplementary fig 2e online ) . to explain further the mechanisms by which both mirnas induce epithelial differentiation , we searched for putative target genes on the basis of the predicted mrna recognition sequence of the conserved stem  loop sequences by using the targetscan search programme ( lewis et al , 2003 ) . notably , among the highest scored target genes for mir-200c were zeb1 and zeb2 , which confirms recent findings published during the course of our work ( hurteau et al , 2007 ; gregory et al , 2008 ; park et al , 2008 ) . in addition , a putative target of mir-141 is tgf2 , indicating that both mirnas are functionally linked by affecting different members of the same emt - inducing pathway . we further selected the putative target factors leptin receptor and cofilin 2 because they are known promoters of malignant tumour progression ( attoub et al , 2000 ; wang et al , 2007 ) . the predicted mirna binding sites in the 3 untranslated region ( utr ) were highly conserved during evolution ( fig 4a ; supplementary fig 3a online ) . we could show that overexpression of mirnas led to reduced expression of zeb1 , tgf2 and the other candidate genes in undifferentiated cancer cells ( fig 4b , c ; supplementary fig 3b online ) . as predicted , mir-141 had the strongest inhibitory effect on tgf2 and mir-200c on zeb1 expression , as shown by rna and protein levels . on the contrary , treatment of differentiated cancer cell lines hpaf2 and mcf7 with inhibitors of mir-200c or mir-141 resulted in a change in the expression of characteristic genes , including an increase in zeb1 and tgf2 expression ( supplementary fig 3c online ) . a negative regulation of zeb1 and tgf2 expression by mir-200c and mir-141 was indicated after cloning of their highly conserved , putative mrna 3utr target sequences in a luciferase reporter vector . after transfection into sw480 shzeb1 clones with reduced zeb1 and enhanced expression of both mirnas , both reporters showed reduced luciferase activity in two independent shzeb1 clones compared with shctl clones ( fig 4d ) . overexpression of mir-141 and mir-200c in sw480 cells led to a reduced activity of tgf2 - 3utr and zeb1 - 3utr constructs , respectively ( fig 4e ) . on the contrary , selective inhibition of mir-141 and mir-200c in sw480 shzeb1 cells by specific anti - mirnas resulted in a selective increase in the activity of tgf2 - 3utr and zeb1 - 3utr constructs , respectively ( supplementary fig 3d online ) . tgf and tnf have been shown to induce an emt in differentiated colorectal cancer cells ( bates et al , 2005 ) . we observed activation of emt in differentiated pancreatic ( hpaf2 ) , colorectal ( dld1 ) and breast cancer ( mcf7 ) cell lines by tgf/tnf. thus , zeb1 was a crucial intracellular transmitter of this emt , as its expression was upregulated by both cytokines and its knockdown partly prevented emt ( supplementary fig 1a , b online ) . direct epithelial target genes suppressed by zeb1 , such as e - cadherin , cell polarity factors and basement membrane components , have been described previously ( grooteclaes & frisch , 2000 ; aigner et al , 2007 ; spaderna et al , 2008 ) . here , we investigated whether zeb1 also affects expression of mirnas and if detected mirnas themselves are candidate regulators of emt . a mirna expression microarray screen was used to analyse sw480 and hct116 colorectal and mda - mb231 breast cancer cell clones with stable short hairpin rna - mediated knockdown of zeb1 ( shzeb clones ) in comparison with control knockdown ( shctl ) clones . we have shown previously that stable knockdown of zeb1 in these clones led to a reversal of the fibroblastoid phenotype towards an epithelial differentiation in the sense of a mesenchymal  epithelial transition ( spaderna et al , 2008 ) . out of 743 human , rat and mouse mirnas included on the microarray screen , we detected average upregulation ( > 1.75-fold ) after zeb1 knockdown of 79 mirnas ( 10.59% ) in at least one cell line , 17 mirnas ( 2.27% ) in two cell lines and only 4 mirnas ( 0.53% ) in all three cell lines ( fig 1 ; supplementary table 1 online ) . the strongest effect was observed for the undifferentiated breast cancer cell line mda - mb231 . notably , three of the four mirnas upregulated in all cell lines ( mir-141 , mir-200b and mir-200c ) belong to the highly conserved mirna-200 family , which was recently linked to the induction of epithelial differentiation ( hurteau et al , 2007 ; gregory et al , 2008 ; park et al , 2008 ) . further analyses focused on mir-141 and mir-200c , which showed strongest upregulation after knockdown of zeb1 in all three cancer cell lines . the array data were validated by using real - time pcr for mir-141 and mir-200c , which showed a strong increase after knockdown of zeb1 in undifferentiated pancreatic , colorectal and breast cancer cell lines ( supplementary fig 1c online ) . next , we investigated whether zeb1 directly suppresses transcription of the mir-141 and mir-200c mirna genes . both mirnas map closely on human chromosome 12p13.31 and the stem loop sequences are separated by only a 338-base - pair spacer sequence ( fig 2a ) . this spacer and the putative promoter 600 bp upstream from the hsa - mir-200c stem loop contain six putative binding sequences for zeb1 , two of which were restricted to zeb factors ( z - box 1 and 2 , caggta ) . the remaining four were perfect e - boxes ( e - box 14 , caggtg ) , which , in addition to zeb1 , represent putative binding sites for other emt activators , such as snail factors . note that the overall mirna gene structure and the two z - boxes , as well as e - box 2 , are highly conserved in evolution from zebrafish to human ( fig 2a ) . all the conserved zeb1-binding sequences are located within the putative promoter , whereas the two e - boxes in the spacer sequence are not conserved . after cloning of the putative promoter ( 683 to 67 relative to first nucleotide of the mir-200c stem  loop ) and the spacer ( + 66 to + 338 ) into a luciferase reporter vector , zeb1-dependent activity was assessed . shrna - mediated knockdown of zeb1 in all undifferentiated colorectal , breast and pancreatic cancer cell lines tested resulted in an enhanced promoter activity when compared with shctl cell clones ( fig 2b , left ) . on the contrary , the promoter activity was suppressed by zeb1 overexpression in a dose - dependent manner . similar effects , although to a lesser extent , were also observed after snail1 overexpression , which can bind to only two of the four potential zeb1-binding sites within the putative promoter ( fig 2b , middle ) . the expression level of zeb1 had no significant effect on the transcriptional activity of the spacer sequence . mutation of the two highly conserved z - boxes showed that z - box 2 confers the strongest repressive function by zeb1 ( fig 2b , right ) and also made the promoter activity insensitive to stable knockdown of zeb1 ( supplementary fig 1d online ) . a direct binding of zeb1 to the two conserved zeb1 sites ( z - boxes ) was shown by electromobility shift assay by using recombinant dna - binding domain of zeb1 and nuclear extracts from sw480 colorectal cancer cells ( fig 2c ) . by applying chromatin immunoprecipitation ( chip ) with chromatin from sw480 or hct116 colorectal cancer cells , we could show that endogenous zeb1 binds to the native promoter region ( fig 2d ) . these data indicate that the transcriptional repressor zeb1 can directly suppress expression of both mir-141 and mir-200c by binding to their putative common promoter . next , we investigated whether the two mirnas suppressed by zeb1 represent direct emt regulators . we showed that mir-200c and mir-141 are strong inducers of an epithelial phenotype , which was also recently reported by other groups during the course of our work ( hurteau et al , 2007 ; gregory et al , 2008 ; park et al , 2008 ) . transient overexpression of both mir-141 and mir-200c resulted in the induction of epithelial differentiation of undifferentiated cancer cells ( fig 3a ; supplementary fig 2a , b online ) . in particular , the overall effect of mir-200c was comparable with the strong effect of zeb1 knockdown . notably , mir-200c , and to a lesser extent mir-141 , also led to increased e - cadherin expression and cell  cell adhesion , as well as reduced spreading of normal human fibroblasts ( supplementary fig 2c online ) . overexpression of mir-200c strongly reduced cancer cell migration and invasion into matrigel , indicating a potential inhibitory role in malignant tumour progression ( fig 3b ; supplementary fig 2d online ) . on the contrary , treatment of differentiated cancer cell lines hpaf2 and dld1 with inhibitors of mir-200c , and to a lesser extent of mir-141 , resulted in a mesenchymal transition as indicated by cell scattering , upregulation of vimentin and reduced expression of e - cadherin ( fig 3c ) . knowing that the breast cancer cell line mda - mb231 , which almost completely lost both mirnas , is derived from a basal type of breast cancer , we analysed various types of human breast cancer . as predicted from using cancer cell lines , we confirmed that basal types of breast cancers , characterized by an undifferentiated phenotype and poor clinical prognosis , show reduced expression of both mirnas compared with the common ductal invasive type of breast cancer ( supplementary fig 2e online ) . to explain further the mechanisms by which both mirnas induce epithelial differentiation , we searched for putative target genes on the basis of the predicted mrna recognition sequence of the conserved stem  loop sequences by using the targetscan search programme ( lewis et al , 2003 ) . notably , among the highest scored target genes for mir-200c were zeb1 and zeb2 , which confirms recent findings published during the course of our work ( hurteau et al , 2007 ; gregory et al , 2008 ; park et al , 2008 ) . in addition , a putative target of mir-141 is tgf2 , indicating that both mirnas are functionally linked by affecting different members of the same emt - inducing pathway . we further selected the putative target factors leptin receptor and cofilin 2 because they are known promoters of malignant tumour progression ( attoub et al , 2000 ; wang et al , 2007 ) . the predicted mirna binding sites in the 3 untranslated region ( utr ) were highly conserved during evolution ( fig 4a ; supplementary fig 3a online ) . we could show that overexpression of mirnas led to reduced expression of zeb1 , tgf2 and the other candidate genes in undifferentiated cancer cells ( fig 4b , c ; supplementary fig 3b online ) . as predicted , mir-141 had the strongest inhibitory effect on tgf2 and mir-200c on zeb1 expression , as shown by rna and protein levels . on the contrary , treatment of differentiated cancer cell lines hpaf2 and mcf7 with inhibitors of mir-200c or mir-141 resulted in a change in the expression of characteristic genes , including an increase in zeb1 and tgf2 expression ( supplementary fig 3c online ) . a negative regulation of zeb1 and tgf2 expression by mir-200c and mir-141 was indicated after cloning of their highly conserved , putative mrna 3utr target sequences in a luciferase reporter vector . after transfection into sw480 shzeb1 clones with reduced zeb1 and enhanced expression of both mirnas , both reporters showed reduced luciferase activity in two independent shzeb1 clones compared with shctl clones ( fig 4d ) . overexpression of mir-141 and mir-200c in sw480 cells led to a reduced activity of tgf2 - 3utr and zeb1 - 3utr constructs , respectively ( fig 4e ) . on the contrary , selective inhibition of mir-141 and mir-200c in sw480 shzeb1 cells by specific anti - mirnas resulted in a selective increase in the activity of tgf2 - 3utr and zeb1 - 3utr constructs , respectively ( supplementary fig 3d online ) . by applying a mirna expression array screen for various human cancer cells , we detected several mirnas suppressed by the emt inducer zeb1 . zeb1 directly suppressed transcription of two members closely linked on human chromosome 12 , mir-141 and 200c , by binding to at least two highly conserved sites in their putative promoter . in confirmation with the data published during the course of our work ( hurteau et al , 2007 ; gregory et al , 2008 ; park et al , 2008 ) , the detected mirnas induced a mesenchymal to epithelial transition ( met ) and inhibited emt , migration and invasion of undifferentiated cancer cells . we further identified putative target genes , which are known promoters of emt and malignant tumour progression . one target of mir-200c is zeb1 itself , indicating an emt - enhancing feedforward loop in invading cancer cells . this regulatory loop might be stabilized further by downregulation of mir141 , as one of its putative targets is tgf2 . there is increasing evidence that zeb1 has crucial effects on various processes of malignant tumour progression ( peinado et al , 2007 ) and promotes metastasis ( spaderna et al , 2008 ) . in the light of the important role of zeb1 and other emt inducers , such as snail ( olmeda et al , 2007 ) , twist ( yang et al , 2004 ) and of emt as a whole in tumour progression , our data , indicating that zeb1 promotes an emt - stabilizing feedforward loop by suppressing specific mirnas , add functional evidence for the molecular mechanisms underlying these processes . our work addressed the clinically relevant question of how putative tumour - suppressive mirnas can be inactivated in cancer progression . the fact that both the zeb1 binding sites and the overall structure of the mir-200c and mir-141 genes are highly conserved in vertebrates from zebrafish to human suggests that the tumour cells use a long - established regulatory mechanism of mirna expression . moreover , the second mirna cluster of the mir-200 family on human chromosome 1p36 also contains highly conserved putative zeb1 binding sites in the upstream sequence , indicating that the whole mir-200 family can be suppressed by zeb1 ( supplementary fig 3e online ) . in addition , both the strong transcriptional inhibition of the two mirnas by zeb1 and their putative tumour - suppressive effect were detected in tumour cell lines of various important cancer entities , namely pancreatic , colorectal and breast cancer . a clinical relevance is indicated by the fact that both mirnas are lost in the highly aggressive basal type of breast cancer , which , in contrast to the luminal and ductal invasive type , is poorly differentiated , shows no expression of oestrogen and progesterone receptors , and has a worse clinical prognosis ( sempere et al , 2007 ) . both mirnas affect the expression of different molecules , which all work in the same proinvasive manner , as is known for tgf2 , zeb1 , cofilin and leptin receptor . the differential function of the two mirnas can synergize , as they are coexpressed , possibly through coactivation by a common promoter . the intriguing fact is that both mirnas inhibit members of their own repressing pathway : mir-200c targets zeb1 and mir-141 targets tgf2 . thus , zeb1 becomes a crucial regulator , as its aberrant expression in cancer might start a self - enhancing feedforward loop by downregulating its own inhibitors mir-141 and mir-200c ( fig 4f ) . moreover , if the initial signal breaks down ( for example , tumour environmental tgf ) , such a loop might as well re - enforce expression of the mirnas , thereby re - inducing an epithelial phenotype . recently , liu et al ( 2008 ) showed that zeb1 is crucial for tgf-mediated emt in various steps of organ development . this important role of zeb1 points out that the predicted regulatory loop might also have a physiological role in separating mesenchymal from epithelial tissue in development and organogenesis . in conclusion , we suggest that zeb1 is a crucial promoter of tumour progression by reducing transcription of both mrnas and mirnas . thus , zeb1 is a central molecular regulator of a mirna - mediated feedforward loop , which can re - enforce emt . a 50 g portion of total rna including small rnas isolated from 4  10 cells using the trizol reagent ( invitrogen , carlsbad , ca , usa ) was shipped to capital bio ( beijing , china ) . an expression screening was carried out using capitalbio mammalian mirna array v2.0 containing 743 human , rat and mouse non - redundant mirna probes . the microarray data have been deposited on arrayexpress ( http://www.ebi.ac.uk/arrayexpress/ ) with the accession code e - tabm-461 . dna constructs . for the hsa - mir-200c promoter reporter plasmid nucleotides 683 to 67 , and for the spacer reporter nucleotides + 66 to + 403 ( relative to first nucleotide of mir-200c stem  loop ) were cloned into pgl3basic ( promega , mannheim , germany ) . for the 3utr reporter plasmids , nucleotides + 3,399 to + 3,953 of human zeb1 complementary dna and nucleotides + 1,427 to + 1,695 of human tgf2 cdna were amplified and cloned downstream of the luciferase gene in the pmir - report vector ( ambion , austin , tx , usa ) . standard cell culture , transient transfections , reporter assays , electromobility shift assays , immunoblots , transient short interfering rna ( sirna)-mediated knockdown and quantitative real - time reverse transcription  pcr were carried out as described previously ( brabletz et al , 1999 , 2004 ; hlubek et al , 2001 ) . for tgf/tnf stimulation 3  10 cells per well were seeded in a 12-well plate , transfected at day 1 as indicated , and stimulated with 2 ng / ml tgf and 10 ng / ml tnf for 5 days . mirna modulation : a total of 5  10 cells per well were seeded in a 12-well plate . after 24 h , cells were transfected with 30 pmol oligonucleotides for mir-141 , mir-200c or control mirna-16 ( ambion , austin , tx , usa ) using oligofectamine reagent ( invitrogen , carlsbad , ca , usa ) for overexpression , or with 420 nm of specific anti - mirs ( ambion ) for inhibition . cell invasion was evaluated using the chemicon cell invasion assay kit as described previously ( chemicon international , millipore , schwalbach , germany ) , using 20,000 transiently ( mirna or sirna ) transfected cells . cell migration assay : cells were transfected with mirnas and controls as described ( spaderna et al , 2006 ) . after reaching confluence , cells were scratched with a pipette tip and the migration potential was observed for up to 50 h. quantitative real - time pcr for mirnas : rna from cultured cells was extracted using the mirvana paris kit ( ambion , austin , tx , usa ) . total rna of formalin - fixed , paraffin - embedded samples of breast carcinomas retrieved from the archives of the department of pathology , university of erlangen was extracted after microdissection using the total nucleic acid isolation kit for ffpe ( ambion , austin , tx , usa ) . specific quantitative real - time pcr experiments were carried out using taqman microrna assays for mir-141 , mir-200c and control mirna-16 ( applied biosystems , foster city , ca , usa ) on a roche lightcycler 480 . chip analysis : the chip it kit ( active motif , carlsbad , ca , usa ) was applied according to the manufacturer 's instructions . a 3 g portion of control rabbit antiserum or antisera against zeb1 was used for immunoprecipitation . for oligonucleotide sequences , plasmids and antibodies used in this study ,
the embryonic programme  epithelial  mesenchymal transition ' ( emt ) is thought to promote malignant tumour progression . 
 the transcriptional repressor zinc - finger e - box binding homeobox 1 ( zeb1 ) is a crucial inducer of emt in various human tumours , and was recently shown to promote invasion and metastasis of tumour cells . here , we report that zeb1 directly suppresses transcription of microrna-200 family members mir-141 and mir-200c , which strongly activate epithelial differentiation in pancreatic , colorectal and breast cancer cells . 
 notably , the emt activators transforming growth factor 2 and zeb1 are the predominant targets downregulated by these micrornas . 
 these results indicate that zeb1 triggers an microrna - mediated feedforward loop that stabilizes emt and promotes invasion of cancer cells . alternatively , depending on the environmental trigger , this loop might switch and induce epithelial differentiation , and thus explain the strong intratumorous heterogeneity observed in many human cancers .