as one of the most common malignant tumors derived from the gastrointestinal tract , esophagus carcinoma has a high incidence and mortality , and more than 300 000 people died from this cancer , making it the sixth leading cause of cancer death worldwide . due to various factors , including environment , diet , and modern life - styles , china has one of the world s highest incidence rates of esophagus cancer .
esophagus carcinoma has been reported as one of top 10 cancers in chinese [ 35 ] .
the etiology of esophagus carcinoma has revealed the involvement of multiple factors , including genetics , diet , and environmental influences . in its early stage
, esophagus cancer normally manifests as only minor discomfort when chewing solid food and lacks typical features , causing a high rate of misdiagnosis .
it is thus usually diagnosed in late stage , when opportunity for optimal treatment has passed , leading to difficulty of treatment and unfavorable prognosis , and severely compromising patient life quality and survival rate .
current studies agree on the genetic component underlying risk of malignant tumors , which are the results of accumulation of minor genetic mutations across different genes in a cascade manner .
micro rna , which is a type of small molecule , also named as mirna or small molecule rna , is widely expressed in animal and plant cells for gene expression regulation . via its base paring with a target gene or to inhibit its downstream protein expression , mirna mediates mrna degradation and protein expression . a close relationship between mirnas and tumor pathogenesis
has been suggested , as certain mirnas facilitate tumor proliferation and metastasis , while the silencing of certain mirna may cause it to lose its tumor - suppressor activity .
recent finding indicated the role of mir-503 in various tumor cells , including tumor cell growth , differentiation , and invasion , as well as prognosis . the detailed function of mir-503 and its effects on the immune system , however , remain unclear .
this study thus investigated the role and effects of mir-503 in cell growth , proliferation , and invasion of esophagus cells and measured its influences on immune cytokine secretion , in an attempt to study the related mechanism and to develop novel drug targets against esophagus cancer .
resuscitated ec9706 and heec cells ( atcc cell bank , usa ) were thawed at 37c , followed by 1 000 rpm centrifugation for 3 min and re - suspension in 1 ml dmem medium ( hyclone , us ) .
cells were incubated in a humidified 37c chamber with 5% co2 perfusion for 24~48 h. both cell lines were then inoculated into a culture dish at 110/cm using high - glucose dmem medium containing 10% fetal bovine serum ( fbs , evergreen , china ) , 100 u / ml penicillin , and 100 g / ml streptomycin .
log - phased heec cells were used as the control group , while ec9706 cells were randomly divided into a tumor cell control group and a mir503 inhibitor group .
mir-503 inhibitor oligonucleotide ( 5-gagcauuucggu cuggaa-3 , gimma , shanghai ) was mixed with 0.2 ml serum - free dmem medium and lipo2000 transfection reagent ( invitrogen , usa ) , and was added into cultured ec9706 cells ( confluence : 70~80% ) for 30-min incubation . after the removal of serum and rinsing by pbs , 1.6 ml of serum - free medium was added for further 6-h incubation .
trizol reagent ( invitrogen , usa ) was used to extract mrna from all groups . after measuring concentration and purity by ultraviolet spectrometer ,
total rna was used as the template for in vitro reverse - transcription using a test kit ( invitrogen , us ) to synthesize cdna .
real - time pcr was then used to amplify the target gene using specific primers ( table 1 , sangon , china ) under the following condition : 55c for 1 min , followed by 35 cycles each with 92c denaturing for 30 s , 58c annealing for 45 s , and 72c elongation for 35 s. using gapdh as the internal reference , fluorescent signals were quantified to determine ct values of all samples and standards .
log - phased ec9706 cells were seeded into 96-well plates at 5 x 10 density in dmem medium containing 10% fbs .
after 24-h incubation , 20 l of sterile mtt reagents was added into each well .
four hours later , supernatants were removed , followed by the addition of 150 l dmso .
the plate was shaken until complete resolving of the crystal violet . a microplate reader ( bio - rad , usa )
was used to measure the absorbance value at 570 nm . a transwell chamber ( hyclone , usa )
after 48-h transfection , cells were incubated using serum - free medium for 24 h. the transwell chamber was put into a 24-well plate which containing 0.5 ml dmem medium ( with 10% fbs ) .
we added a 100-l suspension of tumor cells inside the chamber with serum - free medium .
after 48-h incubation , the chamber was rinsed in pbs and fixed in cold ethanol .
the number of cells translocated to the lower side of the membrane was counted using an inverted microscope .
cytokine levels including il-2 , ifn- , il-4 and il-10 were quantified by elisa using test kits ( ebioscience , usa ) following the manual instructions . in brief , serially diluted standard samples were added into 96-well plates , along with tested samples in triplicates .
after washing and rinsing 5 times , enzyme - linked reagent was applied to each well , followed by 37c incubation for 30 min .
chromogenic substrate a and b were sequentially added into each well for development in the dark ( 10 min ) .
the reaction was stopped by adding quenching buffer to each well . optical density ( od )
the spss16.0 software package was used to analyze all collected data , of which measurement data are presented as mean  standard deviation ( sd ) .
resuscitated ec9706 and heec cells ( atcc cell bank , usa ) were thawed at 37c , followed by 1 000 rpm centrifugation for 3 min and re - suspension in 1 ml dmem medium ( hyclone , us ) .
cells were incubated in a humidified 37c chamber with 5% co2 perfusion for 24~48 h. both cell lines were then inoculated into a culture dish at 110/cm using high - glucose dmem medium containing 10% fetal bovine serum ( fbs , evergreen , china ) , 100 u / ml penicillin , and 100 g / ml streptomycin .
log - phased heec cells were used as the control group , while ec9706 cells were randomly divided into a tumor cell control group and a mir503 inhibitor group .
mir-503 inhibitor oligonucleotide ( 5-gagcauuucggu cuggaa-3 , gimma , shanghai ) was mixed with 0.2 ml serum - free dmem medium and lipo2000 transfection reagent ( invitrogen , usa ) , and was added into cultured ec9706 cells ( confluence : 70~80% ) for 30-min incubation .
after the removal of serum and rinsing by pbs , 1.6 ml of serum - free medium was added for further 6-h incubation .
trizol reagent ( invitrogen , usa ) was used to extract mrna from all groups . after measuring concentration and purity by ultraviolet spectrometer ,
total rna was used as the template for in vitro reverse - transcription using a test kit ( invitrogen , us ) to synthesize cdna .
real - time pcr was then used to amplify the target gene using specific primers ( table 1 , sangon , china ) under the following condition : 55c for 1 min , followed by 35 cycles each with 92c denaturing for 30 s , 58c annealing for 45 s , and 72c elongation for 35 s. using gapdh as the internal reference , fluorescent signals were quantified to determine ct values of all samples and standards .
log - phased ec9706 cells were seeded into 96-well plates at 5 x 10 density in dmem medium containing 10% fbs .
after 24-h incubation , 20 l of sterile mtt reagents was added into each well .
four hours later , supernatants were removed , followed by the addition of 150 l dmso .
the plate was shaken until complete resolving of the crystal violet . a microplate reader ( bio - rad , usa )
a transwell chamber ( hyclone , usa ) was coated with 50 mg / l matrigel dilution .
after 48-h transfection , cells were incubated using serum - free medium for 24 h. the transwell chamber was put into a 24-well plate which containing 0.5 ml dmem medium ( with 10% fbs ) .
we added a 100-l suspension of tumor cells inside the chamber with serum - free medium .
after 48-h incubation , the chamber was rinsed in pbs and fixed in cold ethanol .
the number of cells translocated to the lower side of the membrane was counted using an inverted microscope .
cytokine levels including il-2 , ifn- , il-4 and il-10 were quantified by elisa using test kits ( ebioscience , usa ) following the manual instructions . in brief , serially diluted standard samples were added into 96-well plates , along with tested samples in triplicates .
after washing and rinsing 5 times , enzyme - linked reagent was applied to each well , followed by 37c incubation for 30 min .
chromogenic substrate a and b were sequentially added into each well for development in the dark ( 10 min ) .
optical density ( od ) value at 450 nm was measured by the microplate reader within 15 min .
the spss16.0 software package was used to analyze all collected data , of which measurement data are presented as mean  standard deviation ( sd ) .
using real - time pcr , we found significant elevation of mir-503 expression in ec9706 esophagus carcinoma cells compared to normal heec cells ( p<0.05 , figure 1 ) .
mtt assay was used to describe the effect of mir-503 on the proliferation of esophagus carcinoma cells .
results showed significantly depressed proliferation rate after the transfection of mir-503 inhibitor compared to the control group ( p<0.05 , figure 2 ) , suggesting the participation of mir-503 in cell proliferation of ec9706 .
after the transfection of mir-503 , the percentage of invasive cells was significantly decreased compared to controlled ec9706 cells ( p<0.05 , figures 3 , 4 ) .
the transfection of mir-503 inhibitor significantly decreased the secretion of th1 cytokines il-2 and inf- as compared to control group ( p<0.05 , figure 5a , 5b ) , suggesting the potentiation of mir-503 in th1 cytokine secretion from esophagus carcinoma cells .
opposite effects occurred when checking the level of th2 cytokines including il-4 and il-10 , both of which had elevated secretion after mir-503-specific silencing ( p<0.05 , figure 5c , 5d ) .
using real - time pcr , we found significant elevation of mir-503 expression in ec9706 esophagus carcinoma cells compared to normal heec cells ( p<0.05 , figure 1 ) .
mtt assay was used to describe the effect of mir-503 on the proliferation of esophagus carcinoma cells .
results showed significantly depressed proliferation rate after the transfection of mir-503 inhibitor compared to the control group ( p<0.05 , figure 2 ) , suggesting the participation of mir-503 in cell proliferation of ec9706 .
after the transfection of mir-503 , the percentage of invasive cells was significantly decreased compared to controlled ec9706 cells ( p<0.05 , figures 3 , 4 ) .
the transfection of mir-503 inhibitor significantly decreased the secretion of th1 cytokines il-2 and inf- as compared to control group ( p<0.05 , figure 5a , 5b ) , suggesting the potentiation of mir-503 in th1 cytokine secretion from esophagus carcinoma cells .
opposite effects occurred when checking the level of th2 cytokines including il-4 and il-10 , both of which had elevated secretion after mir-503-specific silencing ( p<0.05 , figure 5c , 5d ) .
as one of the most common malignant tumors in china , esophagus carcinoma has relatively high invasion and recurrence rates , both of which cause unfavorable prognosis . currently available treatment against esophagus cancer , including surgical resection and chemo- and radio - therapy , can not significantly improve its 5-year recurrence rate .
such difficulty , plus the insidious onset of esophagus cancer , make it an important focus in cancer research .
therefore , the illustration of the biological mechanism underlying occurrence and progression of esophagus cancer may help early diagnosis and treatment of cancer , thus improving the prognosis and patient life quality .
mirna is a kind of pluripotent small molecule involved in multiple cellular processes , including cell proliferation , apoptosis , signal transduction , differentiation , hormone secretion , lipid metabolism , and maintaining potency of embryonic stem cells , all of which can regulate body growth and environmental acclimation .
recent studies have revealed the participation of mirna in tumor occurrence , progression , invasion , and metastasis , as well as other biological features . as a newly discovered mirna ,
mir-503 has been suggested to be abnormally expressed in various tumors and can be the target for regulating tumor biology .
its expressional profiles , however , vary across different tumors . in hepatocellular carcinoma , mir-503 can inhibit tumor growth and proliferation via inducing g1 phase arrest . in other tumors , such as osteosarcoma and brain glioma ,
therefore , it is generally believed that mir-503 acts like a tumor - suppressor gene . however , in a study of esophagus carcinoma tissue , mir-503 expression was found to facilitate tumor progression , although its detailed role and mechanism in esophagus cancer has not been defined .
this study demonstrated the elevation of mir-503 in esophagus carcinoma , in agreement with a previous observation .
further in vivo gene silencing assay by transfecting mir-503 inhibitor into esophagus cancer cell revealed significantly inhibited cell proliferation and lowed invasion ability , suggesting the participation of mir-503 in facilitating tumor proliferation and metastasis .
we also analyzed the effect of mir-503 on cytokine release from cancer cells and found decreased th1 cytokines ( il-2 and ifn- ) but elevated th2 cytokines ( il-4 and il-10 ) in mir-503 inhibitor transfected cells .
it has been shown that th1 sub - population of t cells mainly induce pathogenic immune response via secreting il-2 and tumor necrotic factor- ( tnf- ) to facilitate cell apoptosis , increased expression of adhesion molecules , and degrading normal tissues .
th2 sub - type of lymphocytes , however , can inhibit th1-induced immune damage and stimulate the differentiation of b cells into plasma cells for secreting antibody and production of memory b cells , thus potentiating humoral immunity .
the inhibition of mir-503 can depress proliferation and invasion ability of tumors , in addition to improving body immune function .
this study suggests the potency of mir-503 as a novel molecular target for diagnosis and treatment of esophagus cancer .
 backgroundmicrorna ( mir ) has been proved to be an important biomarker for tumors because it can regulate occurrence , progression , invasion , and metastasis of cancer . 
 a previous study has shown the involvement of mir-503 in multiple gastrointestinal tumors . 
 its detailed role and immune regulatory function in esophagus carcinoma , however , remains unknown . 
 this study thus investigated the effect of mir-503 in regulating growth , proliferation , and invasion of esophagus cancer and its influence on cytokine secretion.material/methodsesophagus carcinoma cell line ec9706 and normal esophageal epithelial cell line heec were transfected with mir-503 inhibitor . 
 mtt assay was used to quantify the cell proliferation , and a transwell chamber was used to evaluate cell invasion . 
 release of cytokines , including interleukin-2 ( il-2 ) , il-4 , il-10 , and interferon- ( ifn- ) , was measured by enzyme - linked immunosorbent assay ( elisa).resultsmir-503 expression was significantly elevated in esophagus carcinoma cells ( p<0.05 ) . 
 the specific inhibition of mir-503 expression remarkably suppressed proliferation and invasion of tumor cells . 
 it can also down - regulated il-2 and ifn- expression and facilitate secretion of il-4 and il-10 when compared to the control group ( p<0.05 in all ceases).conclusionsthe inhibition of mir-503 can effectively inhibit tumor progression and improve immune function , suggesting its potency as a novel drug target for esophagus cancer treatment . 
Background
Material and Methods
Cell culture
Transfection of miR-503 inhibitor
Real-time PCR
MTT assay
Cell invasion assay
Enzyme-linked immunosorbent assay (ELISA)
Statistical analysis
Results
Expression of miR-503
Effect of miR-503 on EC9706 cell proliferation
Cell invasion ability
Cytokine secretion from EC9706 cells
Discussion
Conclusions