breast cancer , the most common malignant tumor affecting women , with over 1 million new diagnosed cases each year , is ranked second in causing cancer deaths in women worldwide [ 13 ] . in 2013 , 232 340 us women were diagnosed with invasive breast cancer and there were 39 620 deaths due to this disease . breast cancer is a group of diseases with heterogeneous features encompassing numerous entities differing in biological behavior , morphology , response to therapy , and clinical outcome . according to the molecular profiling , it can be divided into 3 subtypes : luminal , basal - like , and human epidermal growth factor receptor-2 ( her2)-positive . the recognized molecular mechanisms for drug resistance include dna repair pathway alteration , resistance to the initiation of apoptotic pathway , cellular target mutations , and the development of signaling pathways constitutively activated [ 79 ] . recently , many micrornas ( mirnas ) such as mir-155 , mir-34 , and mir-195 have been characterized and identified as indispensable regulators and/or biomarkers in breast cancer development , including initiation , metastasis , and therapy resistance . mir-451 , which is mapped to chromosome 17 at 17q11.2 where the mir-144 gene is located 100 bp downstream , is dysregulated in many human malignant tumors , including breast cancer , playing a crucial role in oncogenesis . this mirna has been documented to be associated with the drug resistance mechanisms of multiple cancers , such as non - small - cell lung cancer ( nsclc ) and colon carcinoma   in 2008 , a study explored the role of mir-451 in the chemotherapeutic drug resistance of mcf-7 cells to doxorubicin , revealing that overexpressed mir-451 was capable of sensitizing mcf-7 cells to doxorubicin therapy through directly targeting the multi - drug resistance 1 ( mdr1 ) gene . however , whether and how the regulation of mir-451 influences the drug resistance of other agents , such as docetaxel and epirubicin , has not been fully determined . in the present study , we aimed to determine the influence of mir-451 on drug resistances of the paclitaxel - resistant breast cancer cell line via transfecting and regulating the levels of mir-451 and to explore the mechanisms of this influence . mcf-7 human breast adenocarcinoma cell line and its multidrug - resistant cell lines ( mcf-7/doc and mcf-7/epi ) were obtained from the affiliated shengjing hospital of china medical university . mcf-7/doc and mcf-7/epi , members of the mcf-7 cell line , were seeded in rpmi-1640 ( gibco , usa ) medium supplemented with 10% fetal bovine serum ( fbs , gibco , usa ) at 37c/5% co2 atmosphere , and then transferred into the culture flask . the adherent cells were detached by digesting in 0.25% trypsin/0.1% edta ( cellgro , va ) , centrifuged for 5 min at 1000 rpm , and then the original medium was replaced with a fresh complete medium , incubated at 37c/5% co2 . cells during logarithmic growth phase were collected , washed with pbs ( 3 times ) after the removal of the medium , digested with 0.25% trypsin for 1~2 min , and added to fresh rpmi-1640 medium . the adherent cells were released by repeated pipetting with a mouth - operated micropipette , and the cell suspension was put into a new centrifugal tube ( 1000 r / min ; 10 min ) . we discarded the supernatant , added rpmi 1640 medium with 10% fbs , pre - cooled it for 10 min at 4c , and added sterile glycerol . slow freezing and rapid thawing was performed for the cell recovery . for the establishment of drug - resistant cell lines , parental cells mcf-7 during logarithmic growth phase were collected , digested with 0.25% trypsin ( gibco , usa ) and centrifuged , and then a cell suspension ( 110 cells / l ) was obtained . then , 10 ml cell suspension was seeded to a culture bottle for 24 h. subsequently , medium containing epirubicin ( epi , 10 ng / ml , pfizer , inc . ) was added . after culturing for 48 h , the medium containing drugs was discarded . replacement and cell passage were performed and gradient concentration of epi ( 10 ng / ml  50 ng / ml  100 ng / ml  200 ng / ml  500 ng / ml ) was used for intermittent induction to obtain a 500 ng / ml epi - resistant cell line ( breast cancer drug - resistant cell strain mcf-7/epi ) . with the same method , 500 ng / ml docetaxel ( doc , qilu pharmaceutical factory , jinan , china ) was used to obtain breast cancer drug - resistant cell strain mcf-7/doc . mcf-7/epi and mcf-7/doc were cultured further in complete culture medium containing 10 ng / ml epi and doc . twenty - four hours before the transfection , the mcf-7 cell line , mcf-7/doc and mcf-7/epi were collected during logarithmic growth phase . after trypsinization and centrifugation , cell suspension was obtained , and seeded in a 24-well plate chamber ( medium without antibiotic ) at a density of 210 cells / well . cells were grown to 50% confluency and mir-415 mimics or inhibitors were transiently transfected using lipofectamine 2000 ( invitrogen , usa ) according to the specifications of the manufacturer . six hours after the transfection , the medium containing mirna - lipo2000 mixture was replaced with fresh complete medium , and the plate was placed in an incubator with 37c/5% co2 atmosphere . the cells were collected for mirna detection at 24 h after transfection and cells were harvested for protein analyses at 36 h after the transfection . the cell apoptosis assay was performed at 72 h after the transfection using flow cytometry . the annexin v - apc / pi apoptosis detection kit ( invitrogen , usa ) was used for the detection of cell apoptosis . for each group total rna was extracted from serum sample and cell line sample using the mirneasy mini kit ( qiagen , hilden , germany ) following the manufacturer s instructions . the concentration and purity of rna samples were determined using a uv spectrophotometer ( nanodrop 2000 ) . . then complementary dna ( cdna ) was synthesized utilizing 5 g of purified rna by using the miscript ii rt kit ( qiagen , hilden , germany ) based on the manufacturer s recommendations . for the detection of mir-451 expression in different cell lines , quantitative rt - pcr ( qrt - pcr ) was performed using miscript sybr green pcr kit ( qiagen , hilden , germany ) . the designed pcr primers were provided by shanghai sangon biotechnology co. , ltd . , china and are presented in table 1 . total volume of qrt - pcr reaction included : 5rt buffer ( 4.0 l ) , enzyme mix ( 2.0 l ) , template total rna ( 4.0 l ) , enzyme mix ( 2.0 l ) , template total rna ( 4.0 l ) , sybr green master mix ( 10.0 l ) , pcr primer mix ( 2.0 l ) , and diluter cdna template ( 8.0 l ) . qrt - pcr reaction conditions were : pre - denaturing at 95c for 10 min , denaturing at 95c for 10 s , and annealing at 60c for 1 min . total 40 cycles were conducted in this qrt - pcr reaction . relative expression of mir-451 was determined using the 2ct method : ct=ctstudy group mirnactcontrol group mirna , ct = ctmir-451ctu6 rna . for each group , this experiment was repeated 5 times . after being washed with pbs , total cells were added to ripa buffer provided by beijing puli lai gene technology co. , ltd . , beijing , china , digestion at 4c for 15 min , and the supernatant was obtained by centrifugation . the soluble proteins ( 30 g ) were electrophoresed using 10% sds - page , electro - transferred onto pvdf and blocked with pbs containing 5% dried skimmed milk for 2 h. the primary antibodies ( p - bcl-2 and p - caspase 3 , both 1:1000 dilution ) were added and incubated with the membrane overnight at 4c , then washed with tbs . then goat anti - rabbit igg labeled by horseradish peroxidase ( hrp ) was added as a second antibody and incubated at room temperature for 2 h. finally , the membranes were rinsed with pbs and the bands were visualized using the ecl detection kit ( amersham international plc ) and exposed to a kodak x - omat film . the same membrane was stripped and re - blotted with an antibody specific to -actin ( sigma , 1:5000 dilution ) . bands of bcl-2 and capase-3 were semi - quantified by densitometry using scanimage software and normalized by -actin levels . all data are expressed as mean  standard deviation ( sd ) and statistical analyses were performed using spss 18.0 software . mcf-7 human breast adenocarcinoma cell line and its multidrug - resistant cell lines ( mcf-7/doc and mcf-7/epi ) were obtained from the affiliated shengjing hospital of china medical university . mcf-7/doc and mcf-7/epi , members of the mcf-7 cell line , were seeded in rpmi-1640 ( gibco , usa ) medium supplemented with 10% fetal bovine serum ( fbs , gibco , usa ) at 37c/5% co2 atmosphere , and then transferred into the culture flask . the adherent cells were detached by digesting in 0.25% trypsin/0.1% edta ( cellgro , va ) , centrifuged for 5 min at 1000 rpm , and then the original medium was replaced with a fresh complete medium , incubated at 37c/5% co2 . cells during logarithmic growth phase were collected , washed with pbs ( 3 times ) after the removal of the medium , digested with 0.25% trypsin for 1~2 min , and added to fresh rpmi-1640 medium . the adherent cells were released by repeated pipetting with a mouth - operated micropipette , and the cell suspension was put into a new centrifugal tube ( 1000 r / min ; 10 min ) . we discarded the supernatant , added rpmi 1640 medium with 10% fbs , pre - cooled it for 10 min at 4c , and added sterile glycerol . for the establishment of drug - resistant cell lines , parental cells mcf-7 during logarithmic growth phase were collected , digested with 0.25% trypsin ( gibco , usa ) and centrifuged , and then a cell suspension ( 110 cells / l ) was obtained . then , 10 ml cell suspension was seeded to a culture bottle for 24 h. subsequently , medium containing epirubicin ( epi , 10 ng / ml , pfizer , inc . ) was added . after culturing for 48 h , the medium containing drugs was discarded . replacement and cell passage were performed and gradient concentration of epi ( 10 ng / ml  50 ng / ml  100 ng / ml  200 ng / ml  500 ng / ml ) was used for intermittent induction to obtain a 500 ng / ml epi - resistant cell line ( breast cancer drug - resistant cell strain mcf-7/epi ) . with the same method , 500 ng / ml docetaxel ( doc , qilu pharmaceutical factory , jinan , china ) was used to obtain breast cancer drug - resistant cell strain mcf-7/doc . mcf-7/epi and mcf-7/doc were cultured further in complete culture medium containing 10 ng / ml epi and doc . twenty - four hours before the transfection , the mcf-7 cell line , mcf-7/doc and mcf-7/epi were collected during logarithmic growth phase . after trypsinization and centrifugation , cell suspension was obtained , and seeded in a 24-well plate chamber ( medium without antibiotic ) at a density of 210 cells / well . cells were grown to 50% confluency and mir-415 mimics or inhibitors were transiently transfected using lipofectamine 2000 ( invitrogen , usa ) according to the specifications of the manufacturer . six hours after the transfection , the medium containing mirna - lipo2000 mixture was replaced with fresh complete medium , and the plate was placed in an incubator with 37c/5% co2 atmosphere . the cells were collected for mirna detection at 24 h after transfection and cells were harvested for protein analyses at 36 h after the transfection . the cell apoptosis assay was performed at 72 h after the transfection using flow cytometry . the annexin v - apc / pi apoptosis detection kit ( invitrogen , usa ) was used for the detection of cell apoptosis . for each group total rna was extracted from serum sample and cell line sample using the mirneasy mini kit ( qiagen , hilden , germany ) following the manufacturer s instructions . the concentration and purity of rna samples were determined using a uv spectrophotometer ( nanodrop 2000 ) . . then complementary dna ( cdna ) was synthesized utilizing 5 g of purified rna by using the miscript ii rt kit ( qiagen , hilden , germany ) based on the manufacturer s recommendations . for the detection of mir-451 expression in different cell lines , quantitative rt - pcr ( qrt - pcr ) was performed using miscript sybr green pcr kit ( qiagen , hilden , germany ) . the designed pcr primers were provided by shanghai sangon biotechnology co. , ltd . , china and are presented in table 1 . total volume of qrt - pcr reaction included : 5rt buffer ( 4.0 l ) , enzyme mix ( 2.0 l ) , template total rna ( 4.0 l ) , enzyme mix ( 2.0 l ) , template total rna ( 4.0 l ) , sybr green master mix ( 10.0 l ) , pcr primer mix ( 2.0 l ) , and diluter cdna template ( 8.0 l ) . qrt - pcr reaction conditions were : pre - denaturing at 95c for 10 min , denaturing at 95c for 10 s , and annealing at 60c for 1 min . relative expression of mir-451 was determined using the 2ct method : ct=ctstudy group mirnactcontrol group mirna , ct = ctmir-451ctu6 rna . for each group after being washed with pbs , total cells were added to ripa buffer provided by beijing puli lai gene technology co. , ltd . , beijing , china , digestion at 4c for 15 min , and the supernatant was obtained by centrifugation . the soluble proteins ( 30 g ) were electrophoresed using 10% sds - page , electro - transferred onto pvdf and blocked with pbs containing 5% dried skimmed milk for 2 h. the primary antibodies ( p - bcl-2 and p - caspase 3 , both 1:1000 dilution ) were added and incubated with the membrane overnight at 4c , then washed with tbs . then goat anti - rabbit igg labeled by horseradish peroxidase ( hrp ) was added as a second antibody and incubated at room temperature for 2 h. finally , the membranes were rinsed with pbs and the bands were visualized using the ecl detection kit ( amersham international plc ) and exposed to a kodak x - omat film . the same membrane was stripped and re - blotted with an antibody specific to -actin ( sigma , 1:5000 dilution ) . bands of bcl-2 and capase-3 were semi - quantified by densitometry using scanimage software and normalized by -actin levels . all data are expressed as mean  standard deviation ( sd ) and statistical analyses were performed using spss 18.0 software . the mirbase database was used to analyze the basic mir-451 information , suggesting that mir-451 was located near chromosome 17 centromere , and formed a gene cluster with mir-144 at l00 bp upstream of mir-451 . we also found that the 72 nt precursor mir-451 , perfectly conserved among vertebrates , became 23 nt mature mir-451 through the direct cleavage of ago2 , but not dicer cleavage , to exert its biological function . bioinformatics analysis based on targetscan target prediction software , pita target prediction software , and miranda database , demonstrated bcl-2 protein as a potential target gene for mir-451 . in order to explore the expression of mir-451 in mcf-7 , mcf-7/epi , and mcf-7/doc cells after transfection by mir-451 mimics and mir-451 inhibitors , the melting peaks of mir-451 and internal reference u6 were tm , which represents the temperature of 50% opening of base pairs of double - stranded dna . the unimodal melting curves of mir-451 and u6 revealed a good specificity of pcr amplification , and tm values of mirna between 30c and 35c at the single peak . results showed that mir-451 expression increased obviously in all 3 types of cells after transfection with mir-451 mimics , as compared with the control group ( p<0.01 ) . the mir-451 expression in all these 3 types of cells transfected with mir-451 inhibitors was lower than that in the control group ( figure 1 ) . figure 2 displays the apoptosis of mcf-7 , mcf-7/epi , and mcf-7/doc cells transfected with mir-451 mimics and mir-451 inhibitors by flow cytometry analysis . as compared with the control group , the apoptotic capability of all mcf-7 , mcf-7/epi , and mcf-7/doc cells evidently enhanced in varying degrees after being transfected with mir-451 mimics ( p<0.01 ) , as shown in figure 2 . however , no observable deceasing tendency of cell apoptosis was detected between the mir-451 inhibitors group and the control group ( figure 2 ) . after mcf-7 , mcf-7/epi , and mcf-7/doc cells were transfected with mirna-451 mimics and mirna-451 inhibitors , the protein expression of bcl-2 was detected by using western blotting . after transfecting mir-451 mimics into mcf-7 , mcf-7/epi , and mcf-7/doc cells , the expression levels of bcl-2 protein in the 3 types of cells all obviously decreased in comparison with the negative control group , which presented a statistical significance with p values less than 0.01 . however , no significant difference was found in the bcl-2 protein expression between the mir-451 inhibitors group and the control group ( figure 3 ) . after mcf-7 , mcf-7/epi , and mcf-7/doc cells were transfected with mirna-451 mimics and mirna-451 inhibitors , the protein expression of caspase 3 was detected by using western blotting . results showed that caspase 3 protein expression in the 3 kinds of cells increased obviously as compared with negative control groups after transfections ( all p<0.01 ) . nevertheless , transfection with mir-451 inhibitors into mcf-7 , mcf-7/epi , and mcf-7/doc cells made no obvious difference in caspase 3 protein expression from the control group ( figure 4 ) . the mirbase database was used to analyze the basic mir-451 information , suggesting that mir-451 was located near chromosome 17 centromere , and formed a gene cluster with mir-144 at l00 bp upstream of mir-451 . we also found that the 72 nt precursor mir-451 , perfectly conserved among vertebrates , became 23 nt mature mir-451 through the direct cleavage of ago2 , but not dicer cleavage , to exert its biological function . bioinformatics analysis based on targetscan target prediction software , pita target prediction software , and miranda database , demonstrated bcl-2 protein as a potential target gene for mir-451 . in order to explore the expression of mir-451 in mcf-7 , mcf-7/epi , and mcf-7/doc cells after transfection by mir-451 mimics and mir-451 inhibitors , qrt - pcr was used . the melting peaks of mir-451 and internal reference u6 were tm , which represents the temperature of 50% opening of base pairs of double - stranded dna . the unimodal melting curves of mir-451 and u6 revealed a good specificity of pcr amplification , and tm values of mirna between 30c and 35c at the single peak . results showed that mir-451 expression increased obviously in all 3 types of cells after transfection with mir-451 mimics , as compared with the control group ( p<0.01 ) . the mir-451 expression in all these 3 types of cells transfected with mir-451 inhibitors was lower than that in the control group ( figure 1 ) . figure 2 displays the apoptosis of mcf-7 , mcf-7/epi , and mcf-7/doc cells transfected with mir-451 mimics and mir-451 inhibitors by flow cytometry analysis . as compared with the control group , the apoptotic capability of all mcf-7 , mcf-7/epi , and mcf-7/doc cells evidently enhanced in varying degrees after being transfected with mir-451 mimics ( p<0.01 ) , as shown in figure 2 . however , no observable deceasing tendency of cell apoptosis was detected between the mir-451 inhibitors group and the control group ( figure 2 ) . after mcf-7 , mcf-7/epi , and mcf-7/doc cells were transfected with mirna-451 mimics and mirna-451 inhibitors , the protein expression of bcl-2 was detected by using western blotting . after transfecting mir-451 mimics into mcf-7 , mcf-7/epi , and mcf-7/doc cells , the expression levels of bcl-2 protein in the 3 types of cells all obviously decreased in comparison with the negative control group , which presented a statistical significance with p values less than 0.01 . however , no significant difference was found in the bcl-2 protein expression between the mir-451 inhibitors group and the control group ( figure 3 ) . after mcf-7 , mcf-7/epi , and mcf-7/doc cells were transfected with mirna-451 mimics and mirna-451 inhibitors , the protein expression of caspase 3 was detected by using western blotting . results showed that caspase 3 protein expression in the 3 kinds of cells increased obviously as compared with negative control groups after transfections ( all p<0.01 ) . nevertheless , transfection with mir-451 inhibitors into mcf-7 , mcf-7/epi , and mcf-7/doc cells made no obvious difference in caspase 3 protein expression from the control group ( figure 4 ) . previous studies indicated that mir-451 acts as a tumor suppressor oncogene in several cancers , including breast cancer [ 1618 ] . our study reveals that the expression of mir-451 may be regulated after the transfection of mir-451 mimics and mir-451 inhibitors , and the properties of these 2 synthetic oligonucleotides might contribute to the outcomes . the specific results of our study indicate that the expression of mir-451 was up - regulated relative to the control group after the transfecting mir-451 mimics , and was down - regulated to some extent after transfecting mir-451 inhibitors in mcf-7 , mcf-7/epi , and mcf-7/doc cells , but no without statistical significance , which may lead to the inherent low level of mir-451 expression in breast cancer cells . results of the present study also show that mir-451 might influence the sensitivity to neo - adjuvant chemotherapy through regulating cell apoptosis . after transfection of mir-451 mimics , the apoptosis of mcf-7 , mcf-7/epi , and mcf-7/doc cells increased by various extents , suggesting that the up - regulation of mir-451 expression may promote the apoptosis of tumor cells . some toxic drugs can induce changes in dna sequence , including mutations , deletions , and rearrangements , which may lead to the initiation of signaling that will result in cell death or the deactivation of several cancer suppressor genes . moreover , cell death in response to dna damage in most cases has been shown to result from cell apoptosis . consequently , we believe that regulation of apoptosis - associated proteins is an important approach to alter the drug - resistance in cancer cells . mir-451 may modulate cell apoptosis to make tumor cells have drug - resistances and then influence the sensitiveness to neo - adjuvant chemotherapy . according to our results , mir-451 could down - regulate the expression of bcl-2 proteins via negatively regulating the levels of bcl-2 mrna , which might impact the caspase 3 expression , thereby playing an effective role in apoptosis in human breast cancer cells . the bcl-2 family , proteins of which are key regulators in the process of apoptosis , comprises both prosurvival and proapoptotic proteins , and the balance shifting toward the first has been elucidated as a mechanism by which cancer cells evade apoptosis . evidence has shown that an elevated bcl-2 level could contribute to the docetaxel - involved chemotherapy efficacy in breast cancer , indicating that bcl-2 expression may be regarded as a predictive biomarker for the sensitivity to this chemotherapy . based on these conditions , we considered that serum mir-451 might impact the drug resistance of neoadjuvant chemotherapy in breast cancer by regulating bcl-2 expression . as has been noted by nan et al . , mir-451 can affect the invasion and survival of cells via the regulation of target proteins , including bcl-2 , suggesting that the mir-451 could lead to an inhibition of bcl-2 mrna translation through the binding of its 3 untranslated region , thus down - regulating the expression of bcl-2 protein . caspase-3 , a major protein that executes the caspase signaling pathway , is a downstream effector caspase playing a critical role in apoptosis induction . its activation has been considered as the terminal event before cell death , while its deficiency in tumor cells or stroma may result in significant sensitivity to radiotherapy in mouse or xenograft tumors . in general , caspase activation is strongly associated with the release of cytochrome c ( cyt - c ) , both of which could be impaired in tumors involving overexpression of bcl-2 [ 2931 ] . additionally , a heightened ratio of bax / bcl-2 may enhance the activity of caspase-3 , and the up - regulation of mir-451 may increase the caspase-3-dependent apoptosis . thus , we conclude that mir-451 could influence the apoptosis of breast cancer cells via regulating and impacting the expression of bcl-2 mrna and caspase 3 protein . the up - regulation of serum mir-451 could negatively regulate the expression of bcl-2 mrna and bcl-2 its protein to impact the expression of caspase 3 protein and consequently play an effective role in the apoptosis of human breast cancer cells , which might explain how serum mir-451 influenced the multidrug resistance in breast cancer . our research provides a basis for further studying the influence and mechanism of the regulation of mir-451 on the expression of bcl-2 and caspase 3 in multidrug resistance in breast cancer cells . however , our study still has some limitations , such as no bcl-2 transfection after mir-451 transfection to ensure the uniqueness of bcl-2 in mir-451-induced cell apoptosis , which may influence the reliability of our results ; therefore , further research with more rigorous study design is needed .
backgroundthis study aimed to investigate the potential influence of microrna-451 ( mir-451 ) in drug resistances of the paclitaxel - resistant breast cancer cell line by transfecting mir-451 mimics and mir-451 inhibitors to mce-7 , mcf-7/epi , and mcf-7/doc.material / methodsreal - time quantitative pcr ( qrt - pcr ) was performed for detecting whether transfected mir-451 mimics and mir-451 inhibitors could regulate the expression of mir-451 effectively . 
 the apoptosis of the 3 cell lines was measured by applying annexin v - apc / pi staining . 
 western blot was used for the detection of the protein expression of bcl-2 and caspase 3 after the transfection of mir-451 mimics /inhibitors . 
 bioinformatics analysis demonstrated that bcl-2 protein is a potential target gene for mir-451.resultsin comparison to the control group , after transfection with mir-451 mimics , there was a significant increase in mir-451 expression in mcf-7 , mcf-7/epi , and mcf-7/doc . 
 cells in the three cell lines had increased apoptosis , bcl-2 protein expression decreased significantly , and caspase protein expression increased obviously . 
 after the transfection with mir-451 inhibitors , mir-451 expression was significantly decreased and apoptosis in the 3 cell lines had no significant decrease compared with the control group.conclusionsincreased mir-451 expression may negatively regulate bcl-2 mrna and protein expression , followed by affecting the protein expression of caspase 3 , and accelerate the apoptosis in breast cancer , indicating that mir-451 might influence the drug resistances of the paclitaxel - resistant breast cancer cell line .