. differentiation of nave cd4 + t cells into th17 cells requires the coordinated activities of several microenvironmental cues .
the cytokines il-6 and tgf- induce th17 cell differentiation by activating retinoid - related orphan receptor ( ror)-t , a master transcription factor required for the generation of th17 cells .
accumulating evidence suggests that th17 cells are major drivers of inflammation and have been implicated in the pathogenesis of multiple autoimmune and immune - mediated inflammatory diseases , including rheumatoid arthritis , multiple sclerosis , asthma , and behet s disease [ 48 ] .
given the important role of il-17 cells in regulating autoimmunity and inflammation , elaborating the factors and processes that govern the differentiation of th17 cells are necessary for developing th17-targeted therapies .
almost every immune cell type has been found to express vitamin d receptor ( vdr ) and/or the enzyme cyp27b1 that is required for the generation of the bioactive form of vitamin d , 1,25(oh)2d3 ( also known as calcitriol ) .
the action of 1,25(oh)2d3 is based on its binding to vdr , which is a ligand - dependent transcription factor , belonging to the steroid receptor superfamily . upon ligand
binding , vdr undergoes heterodimerization with the retinoid x receptor ( rxr ) and binds to hormone response elements on dna , resulting in expression or transrepression of specific gene products . both in vitro and in vivo studies
have found that 1,25(oh)2d3 treatment inhibits t cell commitment to the th17 lineage as well as th17 production of il-17a .
1,25(oh)2d3 directly inhibits th17 cell differentiation through the vdr signal in cd4 + t cells , independent of the il-2 , il-10 , and stat1 signals .
in addition , 1,25(oh)2d3 suppresses cytokines in th17 cells by inducing the c / ebp homologous protein ( chop ) , a molecule involved in endoplasmic reticulum stress and translational inhibition .
furthermore , 1,25(oh)2d3 inhibits production of il-17a by blocking nuclear factor for activated t cells ( nfat ) , recruitment of histone deacetylase ( hdac ) , sequestration of runt - related transcription factor 1 ( runx1 ) , and induction of forkhead box p3 ( foxp3 ) , a transcription factor associating with nfat and runx1 for transcriptional repression .
although previous studies have obtained some encouraging evidence , our understanding of the mechanisms by which 1,25(oh)2d3 regulates th17 differentiation remains limited .
micrornas ( mirnas ) are endogenous , small , non - coding rnas approximately 22 nucleotides ( nt ) in length that are highly conserved across various species of eukaryotes .
mirnas posttranscriptionally repress gene expression by binding to complimentary sequences in the 3 untranslated region ( utr ) of target messenger rna ( mrna ) .
mirnas are vital for controlling many processes within the immune system including the development and differentiation of lymphocytes , secretion of cytokines and chemokines , antibody switching , and regulation of immune tolerance .
mir-155 promotes th17 cell differentiation and il-17a production by targeting suppressor of cytokine signaling 1 ( socs-1 ) , which is a molecule responsible for the negative regulation of the jak - stat pathway   and suppressing the inhibitory effects of the dna - binding protein jarid2 .
mir-155-deficient mice show a defect in th17 differentiation and strong resistance to experimental autoimmune encephalomyelitis ( eae ) , an experimental animal model for central nervous system demyelinating disease .
mir-20b expression is down - regulated in th17 cells during mouse eae , and over - expression of mir-20b inhibits th17 differentiation and ameliorates eae by targeting ror-t and stat3 .
in addition , mir-21 , mir-181a , mir-210 , and mir-301a are found to be involved in the development of th17 cells [ 2125 ] .
since both mirnas and 1,25(oh)2d3 are crucial regulators of th17 differentiation , it is of great interest to investigate their interaction in regulating th17 differentiation . in the present study
, we aim to evaluate the expression of several candidate mirnas during th17 differentiation and the effects of 1,25(oh)2d3 on th17 differentiation and mirna expression .
human peripheral blood was obtained by venipuncture from 3 young healthy subjects ( lzb , ky , and jfj ) and was collected into k2-ethylene diamine tetraacetic acid ( k2-edta ) vacutainer tubes ( shenzhen medrey medical technology , china ) .
whole blood ( 4 ml ) was mixed with 4 ml of phosphate - buffered saline and layered gently on 4 ml of lymphocyte separation medium ( dakewe biotech company , china ) in a 15-ml centrifugate tube .
pbmc were separated by using density centrifugation in a 25-min centrifugation step at 2500 rpm at room temperature .
the mononuclear layer was collected from the interface of the blood plasma and the separation medium .
after separation , pbmc were washed and cultivated at a density of 1.010 cells / ml in rpmi 1640 ( keygen biotech , china ) containing 10% fetal bovine serum , l - glutamine , nahco3 , penicillin , and streptomycin .
cells were incubated in a humidified atmosphere ( 37c and 5% co2 ) . to generate th17 cells in pbmcs , anti - cd3 mab ( 0.5 ng / l ; biolegend , usa ) , anti - cd28 mab ( 0.5 ng / l ; biolegend , usa ) , recombinant human il-1 ( rhil-1 ; 100 ng / ml ) , rhil-23 ( 100 ng / ml ) , rhil-6 ( 100 ng / ml ) , and rhtgf-1 ( 25 ng / ml ) ( all cytokines from peprotech , usa ) were added to the medium on day 0 . when 1,25(oh)2d3 ( selleck chemicals , usa ) was used to treat the cells , a final concentration of 100 nm was employed according to the results of our pre - experiments ( not shown ) .
after 72 hours of culture , supernatants were collected , and il-17a production in culture supernatants was analyzed by enzyme - linked immunosorbent assay ( elisa ) ( dakewe biotech company , china ) according to the manufacturer s instructions .
after 72 hours of culture , total rna containing small - size rna was isolated from pbmcs using the mircury rna isolation kit ( exiqon , denmark ) .
the concentration of rna was quantified using the nanodrop 2000 spectrophotometer ( thermo , usa ) .
first strand cdna synthesis of mirna was performed with the mir - x mirna first - strand synthesis kit ( clontech , japan ) . for generating cdna , 5 l of 2 mrq buffer
, 1.25 l of mrq enzyme , and 3.75 l of rna were used , for a total volume of 10 l .
the synthesis reaction was carried out at 37c for 60 min , followed by 85c for 5 min .
cdna was diluted with 90 l of ddh2o to bring the total volume to 100 l after reaction .
for mirna quantification , qpcr analysis was performed using the mir - x mirna qrt - pcr sybr kit ( clontech , japan ) and the cfx96 qpcr system ( biorad , usa ) .
the delta - delta ct method was used to determine the expression of hsa - mir-20b , hsa - mir-21 , hsa - mir-155 , hsa - mir-181a , hsa - mir-210 , and hsa - mir-301a normalized against that of u6 snrna .
the 3 primer for qpcr was the mrq 3 primer supplied with the kit , whereas the 5 primers for mirnas were designed and synthesized by takara biotechnology ( dalian ) ( table 1 ) .
reactions were incubated at 95c for 10 seconds , followed by 40 cycles at 95c for 5 seconds and 60c for 20 seconds .
each sample was run in duplicate for analysis . at the end of the pcr cycles ,
amplicons were also analyzed by agarose gel electrophoresis and visualized with ethidium bromide under uv transillumination to validate the specific generation of the expected pcr .
data are presented as median with interquartile range ( irq ) and were analyzed by nonparametric statistics with stata version 11.0 .
human peripheral blood was obtained by venipuncture from 3 young healthy subjects ( lzb , ky , and jfj ) and was collected into k2-ethylene diamine tetraacetic acid ( k2-edta ) vacutainer tubes ( shenzhen medrey medical technology , china ) .
whole blood ( 4 ml ) was mixed with 4 ml of phosphate - buffered saline and layered gently on 4 ml of lymphocyte separation medium ( dakewe biotech company , china ) in a 15-ml centrifugate tube .
pbmc were separated by using density centrifugation in a 25-min centrifugation step at 2500 rpm at room temperature .
the mononuclear layer was collected from the interface of the blood plasma and the separation medium .
after separation , pbmc were washed and cultivated at a density of 1.010 cells / ml in rpmi 1640 ( keygen biotech , china ) containing 10% fetal bovine serum , l - glutamine , nahco3 , penicillin , and streptomycin .
to generate th17 cells in pbmcs , anti - cd3 mab ( 0.5 ng / l ; biolegend , usa ) , anti - cd28 mab ( 0.5 ng / l ; biolegend , usa ) , recombinant human il-1 ( rhil-1 ; 100 ng / ml ) , rhil-23 ( 100 ng / ml ) , rhil-6 ( 100 ng / ml ) , and rhtgf-1 ( 25 ng / ml ) ( all cytokines from peprotech , usa ) were added to the medium on day 0 .
when 1,25(oh)2d3 ( selleck chemicals , usa ) was used to treat the cells , a final concentration of 100 nm was employed according to the results of our pre - experiments ( not shown ) .
after 72 hours of culture , supernatants were collected , and il-17a production in culture supernatants was analyzed by enzyme - linked immunosorbent assay ( elisa ) ( dakewe biotech company , china ) according to the manufacturer s instructions .
after 72 hours of culture , total rna containing small - size rna was isolated from pbmcs using the mircury rna isolation kit ( exiqon , denmark ) .
the concentration of rna was quantified using the nanodrop 2000 spectrophotometer ( thermo , usa ) .
first strand cdna synthesis of mirna was performed with the mir - x mirna first - strand synthesis kit ( clontech , japan ) . for generating cdna , 5 l of 2 mrq buffer ,
1.25 l of mrq enzyme , and 3.75 l of rna were used , for a total volume of 10 l .
the synthesis reaction was carried out at 37c for 60 min , followed by 85c for 5 min .
cdna was diluted with 90 l of ddh2o to bring the total volume to 100 l after reaction .
for mirna quantification , qpcr analysis was performed using the mir - x mirna qrt - pcr sybr kit ( clontech , japan ) and the cfx96 qpcr system ( biorad , usa ) .
the delta - delta ct method was used to determine the expression of hsa - mir-20b , hsa - mir-21 , hsa - mir-155 , hsa - mir-181a , hsa - mir-210 , and hsa - mir-301a normalized against that of u6 snrna .
the 3 primer for qpcr was the mrq 3 primer supplied with the kit , whereas the 5 primers for mirnas were designed and synthesized by takara biotechnology ( dalian ) ( table 1 ) .
reactions were incubated at 95c for 10 seconds , followed by 40 cycles at 95c for 5 seconds and 60c for 20 seconds .
each sample was run in duplicate for analysis . at the end of the pcr cycles ,
amplicons were also analyzed by agarose gel electrophoresis and visualized with ethidium bromide under uv transillumination to validate the specific generation of the expected pcr .
data are presented as median with interquartile range ( irq ) and were analyzed by nonparametric statistics with stata version 11.0 . a p value of < 0.05 was considered significant .
to generate th17 cells , we isolated pbmc from healthy donors and activated them in vitro with anti - cd3 and anti - cd28 antibodies in the presence of polarizing cytokines il-23 , il-1 , tgf-1 , and il-6 .
after 72 hours of culture , we collected the culture medium and assessed il-17a production by elisa .
as shown in figure 1 , il-17a was undetectable when cells received no stimulation , whereas a high level of il-17a was detected under th17-polarizing conditions ( median [ irq ] , 2535.4 [ 2153.3 ] pg / ml , p<0.05 ) ( figure 1 ) , indicating a substantial increase in the number of il-17a - expressing t cells .
we tested 1,25(oh)2d3 for its capacity to inhibit th17 differentiation by adding 1,25(oh)2d3 to the medium at a final concentration of 100 nm .
after 72 hours of culture , il-17a production was significantly decreased by 1,25(oh)2d3 under th17-polarizing conditions ( median [ irq ] , 745.7 [ 473.5 ] pg / ml vs. 2535.4 [ 2153.3 ] pg / ml , p<0.05 ) ( figure 1 ) .
the expression of mirnas hsa - mir-20b , hsa - mir-21 , hsa - mir-155 , hsa - mir-181a , hsa - mir-210 , and hsa - mir-301a was found to be changed under th17-polarizing conditions . of these mirnas ,
hsa - mir-155 was significantly up - regulated ( median fold change : 3.61 , p<0.05 ) , whereas hsa - mir-20b , hsa - mir-21 , hsa - mir-181a , hsa - mir-210 , and hsa - mir-301a were significantly down - regulated ( median fold change : 0.44 , 0.37 , 0.18 , 0.15 , and 0.26 , respectively ; p<0.05 ) ( table 2 , figure 2 ) .
although 1,25(oh)2d3 significantly suppressed human th17 differentiation , there was no change of mirna expression after 1,25(oh)2d3 treatment ( table 2 , figure 2 ) .
melting curve analysis and agarose gel electrophoresis of qpcr products confirmed that the amplicons were specific ( not shown ) .
to generate th17 cells , we isolated pbmc from healthy donors and activated them in vitro with anti - cd3 and anti - cd28 antibodies in the presence of polarizing cytokines il-23 , il-1 , tgf-1 , and il-6 .
after 72 hours of culture , we collected the culture medium and assessed il-17a production by elisa .
as shown in figure 1 , il-17a was undetectable when cells received no stimulation , whereas a high level of il-17a was detected under th17-polarizing conditions ( median [ irq ] , 2535.4 [ 2153.3 ] pg / ml , p<0.05 ) ( figure 1 ) , indicating a substantial increase in the number of il-17a - expressing t cells .
we tested 1,25(oh)2d3 for its capacity to inhibit th17 differentiation by adding 1,25(oh)2d3 to the medium at a final concentration of 100 nm .
after 72 hours of culture , il-17a production was significantly decreased by 1,25(oh)2d3 under th17-polarizing conditions ( median [ irq ] , 745.7 [ 473.5 ] pg / ml vs. 2535.4 [ 2153.3 ] pg / ml , p<0.05 ) ( figure 1 ) .
the expression of mirnas hsa - mir-20b , hsa - mir-21 , hsa - mir-155 , hsa - mir-181a , hsa - mir-210 , and hsa - mir-301a was found to be changed under th17-polarizing conditions . of these mirnas ,
hsa - mir-155 was significantly up - regulated ( median fold change : 3.61 , p<0.05 ) , whereas hsa - mir-20b , hsa - mir-21 , hsa - mir-181a , hsa - mir-210 , and hsa - mir-301a were significantly down - regulated ( median fold change : 0.44 , 0.37 , 0.18 , 0.15 , and 0.26 , respectively ; p<0.05 ) ( table 2 , figure 2 ) .
although 1,25(oh)2d3 significantly suppressed human th17 differentiation , there was no change of mirna expression after 1,25(oh)2d3 treatment ( table 2 , figure 2 ) .
melting curve analysis and agarose gel electrophoresis of qpcr products confirmed that the amplicons were specific ( not shown ) .
the main findings of the present study are as follows : ( 1 ) we observed increased expression of hsa - mir-155 and decreased expression of hsa - mir-20b , hsa - mir-21 , hsa - mir-181a , hsa - mir-210 , and hsa - mir-301a during human th17 differentiation ; and ( 2 ) 1,25(oh)2d3 significantly suppressed human th17 differentiation , but did not affect mirna expression during this process .
as far as we know , we provide the first evidence that 1,25(oh)2d3 suppresses human th17 differentiation without influencing mirna expression .
the classical function of 1,25(oh)2d3 is to maintain calcium - phosphate homeostasis and skeletal function .
the study by ikeda et al . demonstrated that 1,25(oh)2d3 significantly inhibited th17 differentiation by down - regulating the mrna expression levels of rorc and aryl hydrocarbon receptor .
in addition , the inhibitory effect of 1,25(oh)2d3 on th17 differentiation was found to be via the vdr signal in cd4 + t cells , but independent of il-2 , il-10 , and stat1 signals .
furthermore , it was shown that 1,25(oh)2d3 suppressed th17 differentiation by blocking nfat , recruitment of hdac , sequestration of runx1 , and inducing chop and foxp3 . recently ,
nanduri et al . reported that 1,25(oh)2d3 repressed th17-specific genes by the smad pathway and extracellular signal - regulated kinase activation . despite of these findings , our understanding of the mechanisms whereby 1,25(oh)2d3 regulates th17 differentiation remains limited .
mirnas represent a large family of about 22 nt long , noncoding , single - stranded rna molecules . in mammals ,
mirnas predominantly regulate gene expression by induction of mrna degradation , and they play an important role in a variety of physiological and pathological processes . an involvement in the regulation of th17 differentiation
has recently been proposed for several mirnas , including mir-20b , mir-21 , mir-155 , mir-181a , mir-210 , and mir-301a [ 2025 ] .
given that both 1,25(oh)2d3 and mirnas are important regulators of th17 differentiation , it is necessary to evaluate whether 1,25(oh)2d3 affects mirna expression when exerting its inhibitory effects on th17 differentiation .
in this study , we focused on six candidate mirnas , including mir-20b , mir-21 , mir-155 , mir-181a , mir-210,and mir-301a , based on existing evidence from previous studies showing their relation with th17 differentiation , rather than performing mirna microarray analysis to identify candidates , because the former method was more efficient and targeted .
in addition , we did not identify candidate mirnas using in silico analysis because this method was relatively unreliable .
the change of mirna expression during th17 differentiation was evaluated using qpcr . in agreement with the results from previous studies ,
all six mirnas were found to be significantly dysregulated in the development of th17 cells , with five down - regulated ( hsa - mir-20b , hsa - mir-21 , hsa - mir-181a , hsa - mir-210 , and hsa - mir-301a ) and one up - regulated ( hsa - mir-155 ) .
it was noteworthy that most evidence on expression of mirnas during th17 differentiation to date has been derived from results using animal cell cultures [ 2023,25 ] , whereas we evaluated these mirnas using human cell cultures , which could provide more valuable evidence .
in addition , most previous studies concentrated on a single mirna , but we evaluated several mirnas simultaneously in this study , which would provide more detailed information on change of mirna expression during th17 cell development .
1,25(oh)2d3 significantly suppressed th17 differentiation , but did not affect mirna expression during this process , suggesting that 1,25(oh)2d3-mediated th17 cell suppression was not dependent on regulation of mirna expression .
we noticed that our results were distinct from those on endothelial tissues and cancer cells , which showed that vitamin d regulated mirna expression and was involved in mirna - directed posttranscriptional mechanisms .
the study by zitman - gal et al . found that mir-20b expression was up - regulated by 1,25(oh)2d3 in endothelial cells exposed to a diabetic - like environment , and this change of expression played a role in the modulation of endothelial function .
demonstrated that vitamin d deficiency up - regulated mir-21 expression in the aorta , which contributed to the development of atherosclerosis .
in addition , cancer studies also showed that 1,25(oh)2d3 regulated the expression of a subset of mirnas with potential regulatory functions in cancer pathways [ 3032 ] .
however , we did not find mirna involvement in 1,25(oh)2d3-mediated suppression of th17 differentiation in this study .
our results indicate that 1,25(oh)2d3 exerts its functions via distinct mechanisms across different cell types , not through mirnas in th17 cells .
our study showed that six mirnas were dysregulated during human th17 differentiation , with one significantly up - regulated ( hsa - mir-155 ) and five significantly down - regulated ( hsa - mir-20b , hsa - mir-21 , hsa - mir-181a , hsa - mir-210 , and hsa - mir-301a ) .
 backgroundvitamin d is an import regulator of t helper 17 ( th17 ) differentiation , but our understanding of the underlying mechanisms remains limited . in the present study , we aimed to detect the expression levels of micrornas ( mirnas ) during human th17 differentiation and evaluate the effects of 1,25-dihydroxyvitamin d3 ( 1,25(oh)2d3 ) , the bioactive form of vitamin d , on th17 differentiation and mirna expression.material/methodswe cultured human peripheral blood mononuclear cells ( pbmc ) in vitro and activated them with anti - cd3 and anti - cd28 antibodies in the presence of th17-promoting cytokines interleukin ( il)-23 , il-1 , tgf-1 , and il-6 for 72 hours . 
 1,25(oh)2d3 was added to the medium at a final concentration of 100 nm on day 0 . 
 the production of il-17a in culture medium was detected by enzyme - linked immunosorbent assay ( elisa ) . 
 the expression levels of mirnas during th17 differentiation were determined by quantitative polymerase chain reaction ( qpcr).resultssix mirnas were found to be dysregulated during human th17 differentiation . of these mirnas , 
 hsa - mir-155 was significantly up - regulated ( median fold change : 3.61 , p<0.05 ) , whereas hsa - mir-20b , hsa - mir-21 , hsa - mir-181a , hsa - mir-210 , and hsa - mir-301a were significantly down - regulated ( median fold change : 0.44 , 0.37 , 0.18 , 0.15 , and 0.26 , respectively , p<0.05 ) . 
 1,25(oh)2d3 treatment significantly decreased il-17a production ( median [ interquartile range ] , 745.7 [ 473.5 ] pg / ml vs. 2535.4 [ 2153.3 ] pg / ml , p<0.05 ) . 
 however , expression of these mirnas was not changed after 1,25(oh)2d3 treatment.conclusions1,25(oh)2d3 suppressed human th17 differentiation without affecting mirna expression . 
Background
Material and Methods
Peripheral blood mononuclear cells (PBMC) isolation
Th17 cell generation
Quantitative PCR (qPCR)
Statistical analysis
Results
1,25(OH)
Detection of miRNA expression by qPCR
Discussion
Conclusions