thyroid cancer is the most prevalent endocrine malignancy ( accounting for > 92% endocrine malignancy ) , which can be classified histologically into follicular epithelial cell - derived papillary thyroid cancer ( ptc ) , follicular thyroid cancer ( ftc ) , anaplastic thyroid cancer ( atc ) , and parafollicular c - cell - derived medullary thyroid cancer ( mtc ) [ 1 , 2 ] . in the past several decades
, the incidence of thyroid cancer worldwide has been steadily increasing , and this rising is mainly attributed to the increased diagnosis of ptc [ 29 ] .
ptc is the most common histological type of thyroid cancers and accounts for approximately 80% of reported cases .
however , the mortality of ptc with aggressive clinicopathological features was much higher when compared with ptc without these clinicopathological features .
therefore , it is of great importance to identify biomarkers that are associated with aggressive clinicopathological features of ptc , and these biomarkers might also serve as potential pharmacological targets for ptc . 

braf mutation is the most common genetic alteration in thyroid malignances , exclusively existing in ptc and ptc - derived atc , but not in ftc and mtc .
numerous studies have consistently shown that braf mutation , which upregulates thyroid cell division and proliferation by activating mapk pathway , is mutually exclusive with other common genetic alterations such as the rearrangement in transformation / papillary thyroid carcinoma ( ret / ptc ) signal and ras mutation [ 1113 ] . importantly , many studies have proved that braf mutation is correlated with high - risk clinicopathological characteristics , such as larger tumor size , extrathyroidal invasion , local lymph node metastasis , distant metastasis , and advanced disease stages , suggesting that it is not only an independent oncogenic event for ptc tumorigenesis , but also involved in progression of ptc [ 1416 ] .
micrornas ( mirnas ) are small noncoding rna molecules , about 2125 nucleotides in length , which negatively regulate gene expression at the posttranscriptional level .
more than 1000 mirnas are found in humans and up to one - third of the total human mrnas are predicted to be mirna targets .
aberrant mirna expression has been described in a variety of tumors , including ptc [ 1721 ] .
reported that 17 mirnas were upregulated in ptc compared to adjacent normal tissue and defined five mirnas ( mirna-221 , mirna-222 , mirna-146b , mirna-181 , and mirna-21 ) being able to predict cancer status .
revealed the upregulation of mirna-21 , mirna-31 , mirna-221 , and mirna-222 by real - time rt - pcr in ptc compared to multinodular goiter . among the various studies
reported , the expression of 5 mirnas , including mirna-221 , mirna-222 , mirna-146b , mirna-181 , and mirna-21 , has been shown to be dysregulated . in our study
, we sought to further validate the expression of these 5 previously reported mirnas in differentiating ptc from benign thyroid nodules ( btns ) in chinese patients .
moreover , the associations between mirnas and braf mutation , as well as other clinicopathological features , will be addressed in the present study .
data of patients ( shown in table 1 ) , archival ffpe surgical samples previously diagnosed as ptc ( 52 samples ) and btn ( 52 samples ) , were obtained from the first affiliated hospital and cancer center of sun yat - sen university ( guangzhou , china ) .
informed consents were provided by all subjects and the study protocol was approved by the ethics committee of sun yat - sen university . for rna isolation
, eight 10  m sections per sample were obtained from the selected block for extraction of mirna with the mirneasy ffpe kit ( qiagen , hilden , germany ) .
genomic dna was extracted from ffpe samples using the qiaamp dna ffpe tissue kit ( qiagen ) .
the rna and dna yields were determined using the nanodrop nd-1000 spectrophotometer ( nanodrop , usa ) .
a panel of 5 mirnas ( mirna-221 , mirna-222 , mirna-146b , mirna-181 , and mirna-21 ) was selected based on previous reports [ 1722 ] .
quantification of these five mirnas was performed by using miscript pcr system ( qiagen ) as previously described . in brief , each 5  l sample of dissolved rna was polyadenylated and reverse - transcribed to cdna in a final volume of 20  l using the miscript reverse transcription kit ( qiagen ) .
real - time pcr was performed in duplicate using the miscript sybr green pcr kit ( qiagen ) with the abi prism 7500 sequence detection system ( applied biosystems , foster city , ca , usa ) .
the miscript primer assays are part of the miscript pcr system for mirna detection and quantification .
exon 15 , which contains the locus of the t1799a mutation , was amplified using primer pairs as previously reported .
all statistical analyses were performed using statistical package for the social sciences ( spss ) software ( version 16.0 , chicago , il , usa ) .
the mann - whitney u test was used to determine the significance of different levels of mirna expression .
receiver operating characteristic ( roc ) curves were used to analyze the diagnostic utility of different markers .
chi square test was used to identify possible associations between braf mutation and clinicopathological features of ptc patients .
all p values were two - sided and a p value less than 0.05 was considered statistically significant .
expression of five mirnas ( mirna-221 , mirna-222 , mirna-146b , mirna-181 , and mirna-21 ) in ffpe tissues from patients with ptc or btn was measured by qrt - pcr .
all these five mirnas were significantly higher in ptcs than those in btns ( 20.98  46.52 versus 2.51  4.66 , 15.82  26.07 versus 2.03  3.24 , 195.67  426.14 versus 2.87  5.74 , 5.98  11.20 versus 2.82  11.16 , and 14.78  23.98 versus 3.44  12.03 , resp .
, all p values < 0.001 ) ( figure 1 ) .   to evaluate the diagnostic value of these five mirnas for ptc ,
individually , the aucs for mirna-221 , mirna-222 , mirna-146b , mirna-181 , and mirna-21 were 0.872 ( cutoff value 0.022 , sensitivity 82.7% , specificity 78.8% ) , 0.868 ( 0.069 , 71.2% , 88.5% ) , 0.952 ( 0.021 , 90.4% , 88.5% ) , 0.837 ( 0.079 , 71.2% , 84.6% ) , and 0.877 ( 0.071 , 76.9% , 88.5% ) , respectively ( figures 2(a)2(e ) ) . the 5-mirna set had an area under the roc curve ( auc ) of 0.937 ( 95% confidence interval ( ci ) = 0.8910.983 ) , with a sensitivity of 90.4% and a specificity of 88.5% at the cutoff value of 0.471 ( figure 2(f ) ) . when assessing the relationship between braf mutation and clinicopathological features , we found that braf mutation occurred more frequently in ptc patients ( 19/52 ) with advanced tnm stage ( p = 0.014 ) .
however , other clinicopathological features , including tumor size and cervical lymph node metastasis , were not significantly different between patients with or without braf mutation .  
ptc patients were further divided into braf mutation group and non - braf mutation group and the expression of mirna was compared .
the mirna-221 , mirna-222 , mirna-146b , and mirna-181 expression levels were significantly higher in braf mutation group than in non - braf mutation group ( mirna-221 , 15.29 ( 2.7062.02 ) versus 6.36 ( 0.68269.35 ) , p = 0.001 ; mirna-222 , 11.82 ( 2.7343.93 ) versus 4.73 ( 0.04168.94 ) , p = 0.019 ; mirna-146b , 182.19 ( 10.701123.18 ) versus 55.11 ( 0.722917.96 ) , p = 0.003 ; and mirna-181 , 5.58 ( 2.0818.86 ) versus 2.46 ( 0.0980.72 ) , p = 0.005 ) .
however , there was no significant difference in mirna-21 expression level between the two groups ( p = 0.104 ) ( figure 3(a ) ) .  
we next accessed the differences in the expression levels of these 5 mirnas in ptc patients with different clinicopathological characteristics ( table 2 ) .
the levels of mirna-221 and mirna-181 were significantly higher in patients with tumor diameter 2  cm than < 2  cm ( p = 0.004 and p = 0.008 , resp . ) ( figure 3(b ) ) .
the expression levels of mirna-221 and mirna-222 were significantly higher in ptcs with advanced tumor - node - metastasis ( tnm ) stage ( p = 0.004 and p = 0.041 , resp . )
in addition , patients with lymph node metastasis had higher expression levels of mirna-221 and mirna-222 ( p = 0.033 and p = 0.014 , resp . )
there were no significant differences in these mirna levels in ptc patients with other clinicopathological parameters , including age , gender , tumor multicentricity , heteromorphic nucleus , thyroid globulin quantification , and ames ( risk definition including age , metastases , extent , and size ) score ( table 2 ) .
in the present study , we have reported the association of 5 previously reported mirnas and braf mutation and the clinicopathological features in chinese patients with ptc .
we showed that the expression levels of these five mirnas ( mirna-221 , mirna-222 , mirna-146b , mirna-181 , and mirna-21 ) were significantly higher in ffpe tissues from ptcs than btns .
mirna-221 , mirna-222 , mirna-146b , and mirna-181 expression levels were significantly higher in ptc patients with braf mutation .
enhanced expression of mirna-221 and mirna-222 was found in patients with cervical lymph node metastasis and advanced tnm stage .
although most of the thyroid nodules are benign , 5% to 30% of nodules are still malignant and require surgical intervention . at present , pathological study is the golden criteria of diagnosis of thyroid nodules .
however , there are several limitations in the pathological diagnosis of thyroid nodules , including inadequate sampling , indeterminate result , and possible misdiagnosis . during the past decades ,
gene detection techniques have developed considerably , which make it possible to further explore tumorigenesis and progression of ptc at molecular level and to discover potential diagnostic and prognostic biomarkers . among these techniques ,
detection of some specific mirna expression levels is a promising method to provide information for molecular diagnosis of ptc [ 1820 ] . in our study on utility of mirna
profiling , we have correlated this data with pathology of the surgical specimen , a gold standard of diagnosis , and we believe that our findings are certainly helpful in providing potentially diagnostic biomarker for ptc before operation .
our results demonstrated that each of these 5 mirnas or the 5-mirna set ( mirna-221 , mirna-222 , mirna-146b , mirna-181 , and mirna-21 ) could be used as a potential biomarker to differentiate ptc from btn , with highest diagnostic value for mirna-146b ( sensitivity of 90.4% and specificity of 88.5% at an auc of 0.952 ) and 5-mirna set ( sensitivity of 90.4% and specificity of 88.5% at an auc of 0.937 ) .
therefore , we suggest that use of the 5-mirna set as a potential diagnostic biomarker for ptc might be more helpful . in order to further validate the potential diagnostic value of mirna in the preoperative evaluation of thyroid nodules
, we will analyze the proposed mirnas data in  intermediate  or  suspicious 
braf mutation is the most common mutation in ptc , which leads to a constitutive activation of the mapk pathway .
activation of this pathway is a common and important mechanism in genesis and progression of human cancers through upregulating cell division and proliferation [ 1113 ] .
a number of studies have reported that braf mutation was associated with aggressive ptc characteristics , such as extrathyroidal extension , lymph node metastasis , tumor recurrence , decreased survival , and need for cervical reoperation [ 1416 ] . in our present study , we found that braf mutation occurred more frequently in the ptc patients with advanced tnm stage , which is consistent with previous studies [ 14 , 27 , 28 ] .
chou et al . demonstrated that mirna-146b was overexpressed in ptc with braf mutation . yip
et al . also documented that overexpression of mirna-146b was associated with aggressive behavior in braf mutant ptc
. however , sheu et al . reported that there was no correlation between braf mutation and the expression profile of a set of mirnas ( mirna-221 , mirna-222 , mirna-146b , mirna-181 , and mirna-21 ) in ptc . in our study , mirna-221 , mirna-222 , mirna-146b and mirna-181 expression levels were significantly higher in ptc patients with braf mutation .
the discrepancy between these studies could be due to different sample origins and the fundamental methodological differences used in the profiling studies .
therefore , it is of interest to further elucidate how these mirnas and braf mutation interact with each other in the pathogenesis and progression of ptcs . mirna-221 and mirna-222 , which are encoded in tandem on the x chromosome , have been shown to be overexpressed in numerous human tumors including ptc [ 3235 ] .
over - expression of mirna-221 and mirna-222 in cancer cell lines facilitates cell proliferation via downregulation of the expression of p27/kip1 and/or p57/kip2 , both of which are cell cycle inhibitors and tumor suppressors .
have demonstrated the upregulation of mirna-221 in uninvolved thyroid tissue from individuals with ptc compared to normal thyroid tissue from individuals without thyroid disease .
confirmed that not only mirna-221 but also mirna-222 was overexpressed in some cases of multinodular goiter in comparison with ptc , suggesting that upregulation of mirna-221 and mirna-221 is perhaps early events in thyroid carcinogenesis .
nevertheless , our current study showed that expression of mirna-221 and mirna-222 is significantly upregulated in ptc compared to btn and in patients with advanced tnm stage and lymph node metastasis .
similar to our findings , a report from taiwan showed that mirna-221 and mirna-222 were significantly associated with extrathyroidal invasion of ptc .
taken together , upregulation of mirna-221 and mirna-222 may not only be involved in the development of ptc , but also in invasion and metastasis of ptc .
mir-181 has been demonstrated to function both as oncogene and tumor suppressor depending on the origin of tissues and cells .
the downregulation of this mirna has also been noticed in gliomas   and aggressive cll . in our study ,
mir-181 expression was significantly higher in ptc versus btn , suggesting that mir-181 might serve as an oncogene .
moreover , the expression level of mirna-181 was higher in ptc with larger tumor size .
reported that mir-181 , by directly targeting on rassf1a , timp3 , and nlk , might be involved in hepatocellular cancer stem cells differentiation and invasion .
considering the roles of mir-181 in carcinogenesis , the target genes of mirna-181 in ptc are required to be identified in the future .  
mirna-146b over - expression has been shown in the majority of ptc studies [ 19 , 20 , 41 ] . in a series composing of seven pathologically distinct thyroid nodules , chen et al . demonstrated that mirna-146b , but not mirna-221 and mirna-222 , was most consistently overexpressed in ptc .
chou et al . suggested that mir-146b is a novel prognostic factor of ptc , and overexpression of mir-146b significantly increased cell migration and invasiveness . in our study , we showed that mirna-146b might be more helpful for distinguishing patients with ptc from btn subjects , while there was no significant difference in mirna-146b expression in ptc patients with different clinicopathological features .
as to the expression levels of mirna-21 , although it was significantly higher in ptc versus btn , there was substantial expression overlap between the two lesions and there was also no significant difference in mirna-21 level in ptc patients with any tested clinicopathological features .
these mirnas might act as a tumor - related mirna , but their roles in the pathogenesis of ptc still need further investigation . in conclusion
, we have confirmed that 5 previously reported mirnas ( mirna-221 , mirna-222 , mirna-146b , mirna-181 , and mirna-21 ) were up - regulated in chinese patients with ptc .
enhanced expression of these mirnas occurred more frequently in ptc patients with braf mutation and some clinicopathological features .
our findings showed a role of selected mirnas as potential biomarkers in differentiating ptc from btn and as a prognostic indicator of ptc in chinese population .
 micrornas ( mirnas ) dysregulation has been shown to play a critical regulatory role in papillary thyroid carcinomas ( ptcs ) . 
 braf mutation is associated with poor clinicopathological outcomes in ptc . in order to identify a possible association between dysregulated mirna expression and braf mutation as well as clinicopathological features in chinese patients with ptc 
 , we examined the expression levels of five reported dysregulated mirnas ( mirna-221 , mirna-222 , mirna-146b , mirna-181 , and mirna-21 ) and determined braf mutation status in 52 patients with ptc and 52 patients with benign thyroid nodules ( btns ) . 
 the expression levels of all five mirnas were significantly increased in ptc when compared to btn . 
 the braf mutation occurred more frequently in ptc cases with advanced tnm stage . 
 importantly , mirna-221 , mirna-222 , mirna-146b , and mirna-181 expression levels were significantly higher in ptc patients with braf mutation . 
 in addition , enhanced expression of mirna-221 and mirna-222 was found in patients with cervical lymph node metastasis and advanced tnm stage . 
 increased expression of mirna-221 and mir-181 was evidenced in patients with larger tumors . 
 these findings showed a potential role of this distinct profile of mirnas in differentiating ptc from btn . 
 braf mutation might regulate or interact with mirna in the pathogenesis and progression of ptc . 
1. Introduction
2. Patients and Methods
3. Results
4. Discussion