more than 1.3 million new cases are reported each year , accounting for approximately 23% of all new cancer cases in either sex worldwide . several strong risk factors , ranging from genetic to environmental , have been identified for breast cancer and incorporated into public awareness campaigns and clinical monitoring strategies of the general at - risk population . in addition , convenient and accurate early detection techniques have been developed and are in routine use . while these strategies have been able to reduce the rates of mortality associated with breast cancer , nearly half a million women still die of the disease each year ; among those , more than 90% are attributed to cancer metastasis . therefore , substantial research and clinical efforts have been devoted to understanding the underlying molecular mechanisms that control the metastatic spread of tumor cells . it is expected that molecularly targeting these mechanisms , by pharmaceutical or genetic - based approaches , may slow down or stop metastasis and improve the survival of cancer patients . microrna ( mirna ) is a family of endogenous , highly conserved , small non - coding rna molecules . the in vivo mature mirna molecules are between 18 to 22 nucleotides ( nt ) in length , and are generated from the genome by a 2-step process . first , the gene for mirna is transcribed in the nucleus by rna polymerase ii to produce primary mirna ( pri - mirna ) , which is then targeted by the ribonuclease drosha for processing into a stem - loop structure , known as the precursor mirna ( pre - mirna ) . second , with the help of exportin 5 and ran - gtp , pre - mirna is transported to the cytoplasm , where it is further processed by the ribonuclease dicer to an approximately 22-nt double - stranded rna ( dsrna ) molecule , which is the mature mirna . the 2 strands of the mature mirna are thermodynamically asymmetric , and the strand with less 5-end base - pairing stability , also called the guide strand , is preferentially selected by argonaute 2 and other proteins for subsequent assembly into the rna - induced silencing complex ( risc ) . by hybridizing , often imperfectly , to a homologous mrna sequence , the guide strand of mirna directs risc to the target mrna sequence . the mirna is then cleaved by the endonuclease component of the complex , leading to mrna degradation and gene silencing . most mirna target sites are located within the 3-untranslated regions ( 3-utr ) of genes . through this post - transcriptional regulatory mechanism of gene expression , mirna actively modulates many biological processes , including cell proliferation , apoptosis , differentiation , and motility ; likewise , mirna actions can influence the onset and progression of a number of human diseases , as has been demonstrated for heart disease , hematopoietic disease , neurological disease , immune disease and cancer . to date , more than 900 mature mirnas have been identified in the human genome ( mirbase database , http://www.mirbase.org/ ) . although the number of mirna - coding genes only accounts for approximately 1% of total genes , they are believed to regulate the expression of more than 30% of the protein - coding genes in humans . for example , mir-25 and mir-1792 are involved in the proliferation of normal mammary ducts and acini , let-7 regulates terminal differentiation of mammary cells , mir-29 mediates breast tissue remodeling , and mir-205 is considered as a marker for normal mammary stem cells . abnormal expression levels of mirnas have been clinically observed in cancerous breast tissues , where they are believed to function as oncogenes or tumor suppressors . recently , ma et al . identified the mir-10b gene as a target of the transcription factor twist , which is highly expressed in metastatic breast cancer cells and stimulates in vitro and in vivo tumor invasion . up - regulated expression of mir-10b was shown to result in suppression of the mirna-10b target homeobox gene d10 ( hoxd10 ) , which in turn led to induced expression of the pro - metastatic gene rhoc . in the study presented herein , we identified another target of mir-10b silencing activity , e - cadherin ( e - cad ) , an essential component for maintaining epithelial cell polarity and a potent suppressor of breast cancer invasion / metastasis . moreover , we showed that mir-10b is necessary and sufficient for driving the expression of e - cad in breast cancer cells in vitro . a high negative correlation was found between the expressions of these 2 molecules in human breast cancer tissues , and a higher level of mir-10b significantly correlated with advanced disease and higher lymph node metastasis . the human metastatic breast adenocarcinoma cell line mda - mb-231 was obtained from the center of molecular medicine , tongji hospital ( tongji medical college , wuhan , china ) and cultured in rpmi-1640 medium containing 10% fetal bovine serum ( fbs ; gibco , carlsbad , ca , usa ) . the 2-o - methyl mir-10b inhibitor ( in-10b , an antisense oligonucleotide specifically targeting mature mir-10b ; sequence 5-cacaaauucgguucuacagggua-3 ) , a control scrambled oligonucleotide ( in - ctrl , 5-caguacuuuuguguaguacaa-3 ) , the 2-o - methyl mir-10b mimic ( mi-10b , dsrna molecule mimicking the endogenous mature mir-10b molecules ; sense 5-uacccuguagaaccgaauuugug-3 , antisense 3-caaauucgguucuacaggguauu-5 ) , and its corresponding control scrambled rna duplex ( mi - ctrl , sense 5-uucuccgaacgugucacgutt-3 , antisense 5-acgugacacguucggagaatt-3 ) were designed and synthesized by genepharma ( shanghai , china ) . to observe the transfection efficiency , an fam - labeled mir-10b inhibitor or mimic for delivery of the inhibitor or mimic into the cells , mda - mb-231 were seeded at 1.510 cells / well in 6-well plates . twenty - four hours later , the mir-10 inhibitor or mimic was transfected into the cells using lipofectamine 2000 ( invitrogen , carlsbad , ca , usa ) by following the manufacturer s protocol . this study was approved by the ethics committee of tongji medical college , huazhong university of science and technology ( wuhan , china ) , and written consent was obtained from all subjects . a cohort of 44 breast cancer tissues were freshly isolated from female patients ( age range : 35 to 76 years ; average : 50.27 years ) in either tongji hospital ( huazhong university of science and technology , wuhan , china ) or the affiliated xuzhou hospital , medical college of southeast university ( xuzhou , china ) . according to the world health organization ( who ) guidelines for histological grading of tumors of the breast ( 2003 ) , the breast cancer tissue samples included 21 grade i , 14 grade ii , and 9 grade iii tumors . pathologically , these tissues included 35 invasive ductal carcinoma , 4 ductal carcinoma in situ , 2 mucinous carcinoma , 1 medullary carcinoma , 1 paget s disease , and 1 neuroendocrine carcinoma . based on the 2002 tnm classification of breast cancers developed by the international union against cancer ( uicc ) and the american joint committee on cancer ( ajcc ) , the sample cohort was composed of 21 stage i , 9 stage ii , and 14 stage iii cases . other available clinicopathological data associated with each breast cancer patient were collected , including tumor size , positivity for her2 , estrogen receptor ( er ) and progesterone receptor ( pr ) , and mitotic index ( as indicated by ki-67 staining ) . as controls , 16 samples of benign breast lesions , including 12 fibroadenomas , 2 adenosis , 1 mammary hamartoma , and 1 mammary duct ectasia , were freshly obtained from 16 female patients ( age range : 20 to 63 years ; average : 34.06 years ) in the second affiliated hospital of henan university of science and technology . all breast tissues were immediately transferred to liquid nitrogen and stored at 86c until future use . total rna was extracted from breast tissues or breast cancer cells using the trizol reagent ( invitrogen ) and following the manufacturer s instructions . to quantify the mir-10b amount in each of the total rna samples , the hairpin - it mir-10b qpcr quantitation kit ( genepharma ) this procedure involves exclusive stem - loop rt primer hybridization to mature , mirna , and not to pri - mirna or pre - mirna , followed by amplification of the mature mir-10b using a set of gene - specific primers . as an internal control , the small nuclear rna ( snrna ) u6 was amplified by using the u6 snrna qpcr normalization kit ( genepharma ) . to examine the expression of e - cad in various samples , total rna was reverse transcribed into cdna using mmlv reverse transcriptase ( promega , madison , wi ) and following the manufacturers protocol . qpcr was performed using the following primers provided in the custom rt - qpcr gene expression kit ( genepharma ) : human e - cad ( amplicon size of 176 base pairs ( bps ) ) : forward 5-atgccatcgttgttcactgga-3 , reverse 5-catgagaagtatgacaacagcct-3 ; human gapdh ( internal control ; amplicon size of 113 bps ) : 5-agtccttccacgataccaaagt-3 , reverse 5-attggaacgatacagagaagatt-3. the expression of the target gene was calculated using the 2 method , as previously described . tissue homogenate was prepared in an ice cold buffer containing 50 mm tris - hcl ( ph 6.7 ) , 2% sodium dodecyl sulfate ( sds ) , 4% glycerol , and 1 mm phenylmethanesulphonylfluoride ( pmsf ) . cell lysate was prepared in an ice - cold buffer containing 50 mm tris - hcl ( ph 8.0 ) , 0.5% triton x-100 , 10% glycerol , and 0.1 mm ethylenediaminetetraacetic acid ( edta ) . after centrifugation at 12,000g for 10 min , the supernatant containing the total protein sample was collected and used in subsequent analysis . following resolution by 10% sds - polyacrylamide gel electrophoresis ( page ) , the separated proteins were electro - transferred onto a polyvinylidene fluoride ( pvdf ) membrane . the membrane was then blocked against non - specific binding by room temperature incubation with tbst buffer ( 10 mm tris - hcl ( ph 7.4 ) , 100 mm nacl , and 0.2% tween-20 ) containing 5% skim milk for 2 h. after which , overnight incubation with rocking at 4c was carried out with the following primary antibodies : anti - e - cad antibody ( santa cruz biotechnology , santa cruz , ca , usa ) , anti--actin antibody ( sigma , st . the next day , incubation was carried out with hrp - conjugated secondary antibody ( jackson immunoresearch , west grove , pa , usa ) , and the signal was developed with supersignal west pico chemiluminescent substrates ( pierce , rockford , il , usa ) . immunoreactive signal intensities were analyzed by the gel - pro analyzer ( media cybernetics , bethesda , md , usa ) . transwell inserts in 24-well format ( 8.0-m pores ; corning , lowell , ma , usa ) were coated on the upper side with diluted matrigel ( 50 mg / l matrigel : dmem with 0.5% fbs ) and incubated at 37c for 4 h. after coating , 510 cells in 100 l dmem medium containing 5% fbs were seeded on top of the insert . to the lower chamber , 700 l of dmem medium containing 20% fbs was added . the invasion assay was allowed to proceed at 37c for 24 h. then , the transwell insert was taken out , washed once with phosphate buffered saline ( pbs ) , and fixed in methanol at 20c for 10 min . after another wash with pbs , the non - invasive cells and matrigel on the top of the insert were cleared with a cotton swab , and the insert was washed 3 times with pbs . after air drying , the insert was immersed in 200 l of 0.1% crystal violet and incubated at 37c for 30 min ; after a final 3 washes with ddh2o , the inserts were evaluated and digitally imaged under a light microscope . for each sample , 6 random fields were imaged under high magnification ( 200 ) and used to calculate the average number of invaded cells . statistical analysis was performed using spss 13.0 software ( chicago , il , usa ) . all quantitative data are presented as mean  standard deviation ( sd ) for 3 independent experiments or multiple samples , where indicated . comparison between 2 groups was first performed by levene s test , with those showing equal variance being applied to further testing by one - way analysis of variance ( anova ) . if the anova test was positive ( p<0.05 ) , a student - newman - keuls test was performed for pairwise comparisons . the variation of mir-10b expression according to distinct clinicopathological features was analyzed using the student s t - test with bonferroni correction , and the correlation between these parameters was examined by a general linear regression model . the correlation between the expression levels of mir-10b and e - cad was analyzed using spearman s rank correlation test , with the coefficient r calculated the human metastatic breast adenocarcinoma cell line mda - mb-231 was obtained from the center of molecular medicine , tongji hospital ( tongji medical college , wuhan , china ) and cultured in rpmi-1640 medium containing 10% fetal bovine serum ( fbs ; gibco , carlsbad , ca , usa ) . the 2-o - methyl mir-10b inhibitor ( in-10b , an antisense oligonucleotide specifically targeting mature mir-10b ; sequence 5-cacaaauucgguucuacagggua-3 ) , a control scrambled oligonucleotide ( in - ctrl , 5-caguacuuuuguguaguacaa-3 ) , the 2-o - methyl mir-10b mimic ( mi-10b , dsrna molecule mimicking the endogenous mature mir-10b molecules ; sense 5-uacccuguagaaccgaauuugug-3 , antisense 3-caaauucgguucuacaggguauu-5 ) , and its corresponding control scrambled rna duplex ( mi - ctrl , sense 5-uucuccgaacgugucacgutt-3 , antisense 5-acgugacacguucggagaatt-3 ) were designed and synthesized by genepharma ( shanghai , china ) . to observe the transfection efficiency , an fam - labeled mir-10b inhibitor or mimic for delivery of the inhibitor or mimic into the cells , mda - mb-231 were seeded at 1.510 cells / well in 6-well plates . twenty - four hours later , the mir-10 inhibitor or mimic was transfected into the cells using lipofectamine 2000 ( invitrogen , carlsbad , ca , usa ) by following the manufacturer s protocol . this study was approved by the ethics committee of tongji medical college , huazhong university of science and technology ( wuhan , china ) , and written consent was obtained from all subjects . a cohort of 44 breast cancer tissues were freshly isolated from female patients ( age range : 35 to 76 years ; average : 50.27 years ) in either tongji hospital ( huazhong university of science and technology , wuhan , china ) or the affiliated xuzhou hospital , medical college of southeast university ( xuzhou , china ) . according to the world health organization ( who ) guidelines for histological grading of tumors of the breast ( 2003 ) , the breast cancer tissue samples included 21 grade i , 14 grade ii , and 9 grade iii tumors . pathologically , these tissues included 35 invasive ductal carcinoma , 4 ductal carcinoma in situ , 2 mucinous carcinoma , 1 medullary carcinoma , 1 paget s disease , and 1 neuroendocrine carcinoma . based on the 2002 tnm classification of breast cancers developed by the international union against cancer ( uicc ) and the american joint committee on cancer ( ajcc ) , the sample cohort was composed of 21 stage i , 9 stage ii , and 14 stage iii cases . other available clinicopathological data associated with each breast cancer patient were collected , including tumor size , positivity for her2 , estrogen receptor ( er ) and progesterone receptor ( pr ) , and mitotic index ( as indicated by ki-67 staining ) . as controls , 16 samples of benign breast lesions , including 12 fibroadenomas , 2 adenosis , 1 mammary hamartoma , and 1 mammary duct ectasia , were freshly obtained from 16 female patients ( age range : 20 to 63 years ; average : 34.06 years ) in the second affiliated hospital of henan university of science and technology . all breast tissues were immediately transferred to liquid nitrogen and stored at 86c until future use . total rna was extracted from breast tissues or breast cancer cells using the trizol reagent ( invitrogen ) and following the manufacturer s instructions . to quantify the mir-10b amount in each of the total rna samples , the hairpin - it mir-10b qpcr quantitation kit ( genepharma ) this procedure involves exclusive stem - loop rt primer hybridization to mature , mirna , and not to pri - mirna or pre - mirna , followed by amplification of the mature mir-10b using a set of gene - specific primers . as an internal control , the small nuclear rna ( snrna ) u6 was amplified by using the u6 snrna qpcr normalization kit ( genepharma ) . to examine the expression of e - cad in various samples , total rna was reverse transcribed into cdna using mmlv reverse transcriptase ( promega , madison , wi ) and following the manufacturers protocol . qpcr was performed using the following primers provided in the custom rt - qpcr gene expression kit ( genepharma ) : human e - cad ( amplicon size of 176 base pairs ( bps ) ) : forward 5-atgccatcgttgttcactgga-3 , reverse 5-catgagaagtatgacaacagcct-3 ; human gapdh ( internal control ; amplicon size of 113 bps ) : 5-agtccttccacgataccaaagt-3 , reverse 5-attggaacgatacagagaagatt-3. the expression of the target gene was calculated using the 2 method , as previously described . tissue homogenate was prepared in an ice cold buffer containing 50 mm tris - hcl ( ph 6.7 ) , 2% sodium dodecyl sulfate ( sds ) , 4% glycerol , and 1 mm phenylmethanesulphonylfluoride ( pmsf ) . cell lysate was prepared in an ice - cold buffer containing 50 mm tris - hcl ( ph 8.0 ) , 0.5% triton x-100 , 10% glycerol , and 0.1 mm ethylenediaminetetraacetic acid ( edta ) . after centrifugation at 12,000g for 10 min , the supernatant containing the total protein sample was collected and used in subsequent analysis . following resolution by 10% sds - polyacrylamide gel electrophoresis ( page ) , the separated proteins were electro - transferred onto a polyvinylidene fluoride ( pvdf ) membrane . the membrane was then blocked against non - specific binding by room temperature incubation with tbst buffer ( 10 mm tris - hcl ( ph 7.4 ) , 100 mm nacl , and 0.2% tween-20 ) containing 5% skim milk for 2 h. after which , overnight incubation with rocking at 4c was carried out with the following primary antibodies : anti - e - cad antibody ( santa cruz biotechnology , santa cruz , ca , usa ) , anti--actin antibody ( sigma , st . the next day , incubation was carried out with hrp - conjugated secondary antibody ( jackson immunoresearch , west grove , pa , usa ) , and the signal was developed with supersignal west pico chemiluminescent substrates ( pierce , rockford , il , usa ) . immunoreactive signal intensities were analyzed by the gel - pro analyzer ( media cybernetics , bethesda , md , usa ) . transwell inserts in 24-well format ( 8.0-m pores ; corning , lowell , ma , usa ) were coated on the upper side with diluted matrigel ( 50 mg / l matrigel : dmem with 0.5% fbs ) and incubated at 37c for 4 h. after coating , 510 cells in 100 l dmem medium containing 5% fbs were seeded on top of the insert . to the lower chamber , 700 l of dmem medium containing 20% fbs was added . the invasion assay was allowed to proceed at 37c for 24 h. then , the transwell insert was taken out , washed once with phosphate buffered saline ( pbs ) , and fixed in methanol at 20c for 10 min . after another wash with pbs , the non - invasive cells and matrigel on the top of the insert were cleared with a cotton swab , and the insert was washed 3 times with pbs . after air drying , the insert was immersed in 200 l of 0.1% crystal violet and incubated at 37c for 30 min ; after a final 3 washes with ddh2o , the inserts were evaluated and digitally imaged under a light microscope . for each sample , 6 random fields were imaged under high magnification ( 200 ) and used to calculate the average number of invaded cells . statistical analysis was performed using spss 13.0 software ( chicago , il , usa ) . all quantitative data are presented as mean  standard deviation ( sd ) for 3 independent experiments or multiple samples , where indicated . comparison between 2 groups was first performed by levene s test , with those showing equal variance being applied to further testing by one - way analysis of variance ( anova ) . if the anova test was positive ( p<0.05 ) , a student - newman - keuls test was performed for pairwise comparisons . the variation of mir-10b expression according to distinct clinicopathological features was analyzed using the student s t - test with bonferroni correction , and the correlation between these parameters was examined by a general linear regression model . the correlation between the expression levels of mir-10b and e - cad was analyzed using spearman s rank correlation test , with the coefficient r calculated to gain a mechanistic understanding of mir-10b - regulated breast cancer metastasis , we sought potential human mir-10b targets by querying bioinformatics databases using the miranda ( http://www.microrna.org/microrna/home.do ) and pita ( http://genie.weizmann.ac.il/pubs/mir07/mir07_data.html ) algorithms . both analyses identified the human cdh1 gene that encodes the transmembrane glycoprotein e - cad as a potential target for mir-10b . the potential target site in the e - cad mrna was located between nucleotides 461 and 481 within the 3 utr ( figure 1a ) . to test whether mir-10b is required for the down - regulated e - cad expression commonly observed in breast cancer cells , we transiently transfected mda - mb-231 cells with the 2-o - methyl mir-10b inhibitor ( in-10b ) . mock - transfected cells ( mock ) , cells transfected with a scrambled oligonucleotide ( in - ctrl ) , or non - transfected cells ( blank ) were used as controls to evaluate changes in gene expressions associated with mir-10b silencing . by using fam - labeled mir-10b , we were able to visually assess transfection efficiency ; at 48 h after transfection , the transfection efficiency reached approximately 80% ( figure 1b ) . meanwhile , rt - qpcr analysis showed that the level of mir-10b was dramatically reduced in in-10b cells , by approximately 53% vs. mock cells , 50% vs. blank cells , and 58% vs. in - ctrl cells ( p<0.01 ; figure 1c ) . once reduced levels of mir-10b had been established , we examined the concomitant expression of e - cad at both steady - state mrna and protein levels . as shown in figure 2a and 2b , although we detected a slight up - regulation of both e - cad mrna and protein in in - ctrl cells , as compared to mock and blank cells , the e - cad expression in in-10b cells was significantly up - regulated as compared to the other 3 negative control cells ( p<0.05 ) . since e - cad is a crucial regulator of cell migration / invasion , we examined whether inhibition of mir-10b led to any alterations in cell invasive properties , presumably through the dysregulated e - cad . compared with blank cells , the invasion capability of in-10b cells next , we investigated whether mir-10b is sufficient to down - regulate e - cad in breast cancer cells . to achieve this , we transfected mda - mb-231 cells with a mir-10b mimic that functions as a mature mir-10b in cells ( mi-10b ) and would create an environment of mir-10b overexpression . mock - transfected cells ( mock ) , cells transfected with a control mimic ( mi - ctrl ) , or non - transfected cells ( blank ) were used as controls to evaluate the change in e - cad expression due to mir-10b overexpression . again , the transfection efficiency reached approximately 80% at 48 h post - transfection ( figure 3a ) . as expected , the mi-10b cells showed a dramatically up - regulated mir-10b level , by an approximately 2500-fold increase vs. mock or blank cells and 55-fold vs. mi - ctrl cells ( figure 3b ) . the up - regulation of mir-10b level was accompanied by significant reduction of e - cad mrna and protein expressions , by more than 90% and 80% vs. mock cells , respectively ( p<0.01 ; figure 4a and 4b ) . in functional analysis , the mi-10b cells also exhibited significantly higher invasion capability than the blank cells ( p<0.05 ; figure 4c ) . to analyze the clinical significance of the potential cross - talk between mir-10b and e - cad during breast cancer development , we examined the expressions of mir-10b and e - cad in benign breast lesions ( benign , n=16 ) , primary breast cancer that was limited in breast tissue without lymph node metastasis ( pri , n=21 ) , and metastatic breast cancer that was positive for lymph node metastasis ( met , n=23 ) . compared to cancers from benign breast lesions , primary breast cancers showed no obvious differences in detectable mir-10b level ( p>0.05 ; figure 5a ) . metastatic breast cancers , however , contained significantly higher levels of mir-10b , approximately 5- and 6-fold increases vs. benign tissue and primary breast cancers , respectively ( p<0.05 ) . corresponding to the up - regulated level of mir-10b in metastatic breast cancers , we also found that e - cad was significantly down - regulated at both the mrna and protein levels in metastatic breast cancers , as compared to that in benign breast lesions and primary breast cancers ( p<0.05 ) . the e - cad mrna and protein levels were not dramatically different among the benign breast lesions and primary breast cancers ( p>0.05 ; figure 5b and 5c ) . spearman s rank correlation test showed that there was a high negative correlation between mir-10b level and e - cad mrna or protein level in breast cancer ( r<0.5 , p<0.001 ; table 1 ) . the t - test statistical analysis showed that mir-10b level varied significantly in conjunction with the following clinicopathological parameters : tumor diameter ( p=0.008 ) , histological grading ( p=0.007 ) , clinical staging ( p=0.004 ) , status of lymph node metastasis ( p=0.003 ) , presence of her2 ( p=0.012 ) , er and pr expression ( p=0.039 and 0.041 , respectively ) , and the level of mitosis ( p=0.022 ; table 2 ) . multivariate linear regression analysis revealed that mir-10b level in breast cancer independently and positively correlated with tumor diameter ( p=0.0076 ) , histological grading ( p=0.0120 ) , clinical staging ( p=0.0006 ) , presence of lymph node metastasis ( p<0.0001 ) , positivity for her2 ( p=0.0098 ) , and higher mitotic level ( p=0.0230 ) , while negatively correlating with positivity for er and pr ( p=0.0184 and 0.0319 , respectively ; table 3 ) . to gain a mechanistic understanding of mir-10b - regulated breast cancer metastasis , we sought potential human mir-10b targets by querying bioinformatics databases using the miranda ( http://www.microrna.org/microrna/home.do ) and pita ( http://genie.weizmann.ac.il/pubs/mir07/mir07_data.html ) algorithms . both analyses identified the human cdh1 gene that encodes the transmembrane glycoprotein e - cad as a potential target for mir-10b . the potential target site in the e - cad mrna was located between nucleotides 461 and 481 within the 3 utr ( figure 1a ) . to test whether mir-10b is required for the down - regulated e - cad expression commonly observed in breast cancer cells , we transiently transfected mda - mb-231 cells with the 2-o - methyl mir-10b inhibitor ( in-10b ) . mock - transfected cells ( mock ) , cells transfected with a scrambled oligonucleotide ( in - ctrl ) , or non - transfected cells ( blank ) were used as controls to evaluate changes in gene expressions associated with mir-10b silencing . by using fam - labeled mir-10b , we were able to visually assess transfection efficiency ; at 48 h after transfection , the transfection efficiency reached approximately 80% ( figure 1b ) . meanwhile , rt - qpcr analysis showed that the level of mir-10b was dramatically reduced in in-10b cells , by approximately 53% vs. mock cells , 50% vs. blank cells , and 58% vs. in - ctrl cells ( p<0.01 ; figure 1c ) . once reduced levels of mir-10b had been established , we examined the concomitant expression of e - cad at both steady - state mrna and protein levels . as shown in figure 2a and 2b , although we detected a slight up - regulation of both e - cad mrna and protein in in - ctrl cells , as compared to mock and blank cells , the e - cad expression in in-10b cells was significantly up - regulated as compared to the other 3 negative control cells ( p<0.05 ) . since e - cad is a crucial regulator of cell migration / invasion , we examined whether inhibition of mir-10b led to any alterations in cell invasive properties , presumably through the dysregulated e - cad . compared with blank cells , the invasion capability of in-10b cells next , we investigated whether mir-10b is sufficient to down - regulate e - cad in breast cancer cells . to achieve this , we transfected mda - mb-231 cells with a mir-10b mimic that functions as a mature mir-10b in cells ( mi-10b ) and would create an environment of mir-10b overexpression . mock - transfected cells ( mock ) , cells transfected with a control mimic ( mi - ctrl ) , or non - transfected cells ( blank ) were used as controls to evaluate the change in e - cad expression due to mir-10b overexpression . again , the transfection efficiency reached approximately 80% at 48 h post - transfection ( figure 3a ) . as expected , the mi-10b cells showed a dramatically up - regulated mir-10b level , by an approximately 2500-fold increase vs. mock or blank cells and 55-fold vs. mi - ctrl cells ( figure 3b ) . the up - regulation of mir-10b level was accompanied by significant reduction of e - cad mrna and protein expressions , by more than 90% and 80% vs. mock cells , respectively ( p<0.01 ; figure 4a and 4b ) . in functional analysis , the mi-10b cells also exhibited significantly higher invasion capability than the blank cells ( p<0.05 ; figure 4c ) . to analyze the clinical significance of the potential cross - talk between mir-10b and e - cad during breast cancer development , we examined the expressions of mir-10b and e - cad in benign breast lesions ( benign , n=16 ) , primary breast cancer that was limited in breast tissue without lymph node metastasis ( pri , n=21 ) , and metastatic breast cancer that was positive for lymph node metastasis ( met , n=23 ) . compared to cancers from benign breast lesions , primary breast cancers showed no obvious differences in detectable mir-10b level ( p>0.05 ; figure 5a ) . metastatic breast cancers , however , contained significantly higher levels of mir-10b , approximately 5- and 6-fold increases vs. benign tissue and primary breast cancers , respectively ( p<0.05 ) . corresponding to the up - regulated level of mir-10b in metastatic breast cancers , we also found that e - cad was significantly down - regulated at both the mrna and protein levels in metastatic breast cancers , as compared to that in benign breast lesions and primary breast cancers ( p<0.05 ) . the e - cad mrna and protein levels were not dramatically different among the benign breast lesions and primary breast cancers ( p>0.05 ; figure 5b and 5c ) . spearman s rank correlation test showed that there was a high negative correlation between mir-10b level and e - cad mrna or protein level in breast cancer ( r<0.5 , p<0.001 ; table 1 ) . the t - test statistical analysis showed that mir-10b level varied significantly in conjunction with the following clinicopathological parameters : tumor diameter ( p=0.008 ) , histological grading ( p=0.007 ) , clinical staging ( p=0.004 ) , status of lymph node metastasis ( p=0.003 ) , presence of her2 ( p=0.012 ) , er and pr expression ( p=0.039 and 0.041 , respectively ) , and the level of mitosis ( p=0.022 ; table 2 ) . multivariate linear regression analysis revealed that mir-10b level in breast cancer independently and positively correlated with tumor diameter ( p=0.0076 ) , histological grading ( p=0.0120 ) , clinical staging ( p=0.0006 ) , presence of lymph node metastasis ( p<0.0001 ) , positivity for her2 ( p=0.0098 ) , and higher mitotic level ( p=0.0230 ) , while negatively correlating with positivity for er and pr ( p=0.0184 and 0.0319 , respectively ; table 3 ) . e - cad , the prototype member of the classical cadherin family , is of paramount importance in maintaining cell polarity and epithelial integrity . extensive studies have revealed that a reduction / loss of e - cad expression occurs concomitantly with the progression of breast cancer , from early - stage carcinoma to late - stage invasive carcinoma , and the reduced expression is significantly correlated with higher metastatic risk and worse prognosis . induced re - expression of e - cad , on the other hand , led to reduced motility and reversed the invasive phenotype of breast cancer cells , illustrating the tumor - suppressor potential of this gene . several mechanisms at different levels of gene expression have been demonstrated for the loss of e - cad in neoplastic cells . on the genetic level , loss of heterozygosity in the chromosome 16q region , where the human e - cad gene is located , in addition , polymorphic mutations in the e - cad gene were associated with development of the lobular subtype of sporadic breast cancer . on the epigenetic level , dna hypermethylation   and up - regulation of the snail / slug zinc - finger transcriptional repressors of the e - cad gene   have both been shown to contribute to loss of e - cad expression in breast cancer cells . other transcriptional repressors for e - cad have been identified , and include the basic helix - loop - helix transcription factors twist and e12/e47   and the zinc - finger e - box binding homeobox 1 and 2 ( zeb1 and zeb2 ) , the latter of which is controlled by microrna ( mir)-200 family members and mir-205 , which have both been characterized as down - regulated in clinical breast cancer specimens lacking e - cad . post - translationally , cell - surface e - cad level is controlled by matrix metalloproteinase ( mmp)-mediated proteolytic degradation , as well as by tyrosine phosphorylation - induced e3 ligase - targeted proteosomal degradation . upon cleavage by mmps , the extracellular domain of e - cad this event not only reduces cell adhesion by depleting intact e - cad available to participate in adhesion complex formation , but also promotes cell proliferation , invasion and migration in a paracrine manner by activating erbb receptor or up - regulating mmps . receptor tyrosine kinases ( rtks ) , such as epidermal growth factor receptor ( egfr ) , hepatocyte growth factor receptor ( hgfr ) and fibroblast growth factor receptor ( fgfr ) , are frequently found to be activated in breast cancer cells . these rtks phosphorylate e - cad , as well as catenins , targeting these molecules for e3 ligase - mediated proteosomal degradation . collectively , tumor cells appear to exploit every possible way to silence e - cad , supporting the theory that loss of e - cad expression is a prerequisite for tumor cell invasion and further metastasis . in this study , we identified a novel regulator for e - cad expression in tumor cells . the mir-10b controls the level of e - cad at the post - transcriptional level . by using the metastatic breast cancer cell line mda - mb-231 as a model system , we were able to demonstrate that liposomal - mediated intracellular delivery of the mir-10b inhibitor reduces its endogenous level by approximately 50% . moreover , this reduction is accompanied by a significant up - regulation of e - cad , at both the mrna and protein levels . meanwhile , ectopically expressing mir-10b produced dramatic reductions in e - cad mrna ( by > 90% ) and protein ( by ~80% ) . in addition , the changes of e - cad expression in response to mir-10b inhibitor or mimics inducing overexpression were accompanied by functional alterations in cell invasion . these data suggest that mir-10b is capable of actively regulating e - cad expression in metastatic breast cancer cells , and that targeted silencing of mir-10b may serve to recover e - cad expression in breast tumors and suppress or eliminate the potential for tumor invasion / metastasis . in addition to the above - mentioned in vitro findings , we found that in clinical samples of breast cancer tissues there is a significant negative correlation between the levels of these 2 molecules . thus , mir-10b appears to be a biologically important molecule for controlling e - cad expression in human breast cancer and may be amenable to pharmacologic or genetic intervention . mir-10b , together with mir-10a , constitutes the entire mir-10 family . mir-10a and mir-10b genes are localized within the homeobox ( hox ) clusters of genes on human chromosomes 17 ( 17p21 ) and 2 ( 2p31.1 ) , respectively . as such , they are co - regulated with the hox genes , and functionally target many hox mrnas . both genes have been detected as aberrantly expressed in a number of human tumors , including those of the brain , liver , colon and breast . . showed that mir-10b is specifically and highly expressed in metastatic breast cancer cells , as compared to its levels in normal mammary epithelial cells or non - metastatic breast cancer cells . ectopic expression of mir-10b in non - metastatic breast tumors was demonstrated as sufficient to drive potent invasion in vitro and metastasis in vivo . in our study , we compared the expression of mir-10b in 16 samples of benign breast lesions , 21 of primary breast cancers and 23 of metastatic breast cancers . , we found that mir-10b was approximately 4-fold higher in metastatic breast cancers than in benign lesions or primary breast cancers ( p<0.01 ) , while the levels in the latter 2 types of sample were not dramatically different from each other ( p>0.05 ) . more importantly , the higher level of mir-10b independently and significantly correlated with multiple clinicopathological features of advanced progression and metastatic diseases , including larger tumors , grade iii tumors , stage iii tumors , tumors with lymph node metastasis , tumors with positive her-2 expression , and tumors showing higher mitotic activity . in contrast , the mir-10b level in breast cancer negatively correlated with er and pr positivity , 2 key indicators of better prognosis , supporting the notion that mir-10b overexpression can be an effective indicator for breast cancers of advanced and metastatic status . it has been demonstrated that the basic helix - loop - helix transcription factor twist controls the expression of mir-10b in breast cancer cells . twist is also known to directly suppress the expression of e - cad in breast cancer . therefore , twist may control e - cad expression , either directly by binding to the promoter region for transcriptional regulation , or indirectly by up - regulating mir-10b for post - transcriptional regulation . it is also possible that breast cancer increases mir-10 expression through genomic amplification , which in turn targets e - cad mrna for degradation . these potential mechanisms further demonstrate the importance of down - regulated e - cad in maintaining the transformed and metastatic phenotypes of breast cancer . in summary , we present here the first evidence that e - cad is a potential target for mir-10b and that mir-10b may modulate cancer metastasis through targeting e - cad . mir-10b is not only necessary but also sufficient for regulating e - cad expression in breast cancer cells , and is biologically significant for suppressing e - cad in metastatic breast cancer . as an independent indicator for advanced and metastatic breast cancer ,
summarybackgroundrecent studies have suggested that microrna-10b ( mir-10b ) acts as a promoter of metastasis in breast cancer , although the underlying mechanism remains largely unknown . in this study 
 , we provide the first evidence that e - cadherin ( e - cad ) is a potential target of mir-10b.material/methodby applying gain - of - function and loss - of - function approaches in the metastatic breast cancer cell line mda - mb-231 , we demonstrated that mir-10b is necessary and sufficient to regulate the cellular expression of e - cad and in vitro tumor cell invasion.resultscomparative expression analysis of mir-10b in benign breast lesions ( n=16 ) , primary breast cancers ( n=21 ) , and metastatic breast carcinomas ( n=23 ) revealed that mir-10b transcription was uniquely up - regulated in metastatic cancers . 
 the expression level of mir-10b positively correlated with tumor size , pathological grading , clinical staging , lymph node metastasis , her2-positivity and tumor proliferation , but was negatively associated with estrogen receptor - positivity , progesterone receptor - positivity and e - cad mrna and protein levels.conclusionsthese findings indicate the existence of a novel e - cadherin - related mechanism by which mir-10b modulates breast cancer metastasis . in addition , mir-10b may be a useful biomarker of advanced progression and metastasis of breast cancer .