17-estradiol ( e2 ) is a member of the family of steroid hormones which controls many aspects of mammalian physiology .
although its ability to stimulate breast cell proliferation is one of e2 normal roles , it increases the risk of breast cancer [ 1 , 2 ] .
classically , e2 actions are mediated upon binding on two intracellular nuclear receptors , estrogen receptor ( er ) alfa and er beta , which bind to dna and control gene expression . like other steroid hormones
it is well known that e2 has direct and indirect effects on the thyroid , and this subject has been recently reviewed .
although many studies have shown the expression of er in thyroid cancer , there is no study associating aggressiveness of thyroid cancer and absence of er , as it is seen in breast cancer .
a novel 7-transmembrane g - protein - coupled receptor , which responds to e2 stimulation with rapid cellular signaling , including erk activation , pi3k activation , calcium mobilization , and camp production , has been extensively studied .
this receptor was named g - protein estradiol receptor 30 ( gpr30 ) , g - protein estradiol receptor ( gper ) , and , more recently , g - protein estradiol receptor 1 ( gper1 ) .
it has been shown to be expressed in many estradiol - responsive tissues , and some studies have proposed a potential use of selective antagonists of gper1 as a new targeted therapy for cancer [ 8 , 9 ] .
gper1 expression was described in thyroid carcinoma cell lines [ 10 , 11 ] but has not been studied yet in normal or benign thyroid tissues or cells .
the knowledge of the expression of this receptor in normal and nonmalignant thyroid could be a potential tool to better understand the nongenomic effects of e2 , contributing to developing targets to treatment of diseases .
so , the aim of this study was to evaluate the expression of gper1 in human normal thyroid and goiter .
the project was submitted to , and approved by , the research ethics committee of the hospital de clnicas de porto alegre ( hcpa ) , porto alegre , rs , brazil .
a written informed consent from the participants was considered unnecessary by the institutional review board of the hcpa . in accordance with the resolution
hcpa 02/97 , based on the resolution cns 196/96 of the national health council , brazil , and guideline 9 of the international ethical guidelines for biomedical research involving human subjects ( cioms , who , geneva , 1993 ) , there was no need to obtain informed consent of the patients , because only after the surgical procedure the researchers would know if a tissue sample would be available , they would not know the identity nor have access to the files of the patients , and the tissue samples would be discharged by the pathologists , so there was no interference with these exams .
all tissue samples were considered medical waste and all data was anonymized ( permit number : 12 - 0272 ) .
thyroid samples were obtained from 16 normal thyroid and 19 goiter patients who underwent total thyroidectomy as part of treatment for differentiated thyroid cancer in hcpa .
after macroscopic and frozen sections evaluation of surgical specimens by two pathologists , normal thyroid or goiter tissue , which would be discharged , was frozen in liquid nitrogen and stored at 80c until further processing . the pattern used to confirm the presence of goiter was a well - defined fibrous capsule with a mixture of macrofollicles and microfollicles and , in some cases , some degenerative changes such as fibrosis and hemorrhage .
total rna was isolated from thyroid tissues using trizol reagent ( sigma - aldrich , st .
louis , mo , usa ) and quantified using a nanodrop nd-1000 ( thermo fisher scientific , wilmington , de ) via absorbance measurements at 260 and 280  nm . only samples that presented a260/a280 ratios between 1.80 and 2.04 were used in this study .
cdna was synthesized from 2  g of total rna in a 20  l reaction using oligo - dt primers and the superscript iii reverse transcriptase ( invitrogen ) according to the manufacturer 's guidelines .
gper1 and -actin genes were amplified in parallel using applied biosystems stepone real - time pcr system ( invitrogen life technologies ) and kit platinum sybr green qpcr supermix - udg ( invitrogen life technologies ) .
the reaction for both genes was initiated by preheating at 95c for 10  s , followed by 40 cycles of denaturation for 15  s at 95c , annealing for 30  s at 58c min , and extension for 30  s at 72c .
primer pairs used were described previously by maggiolini et al . for gper1   and by souza et al .
the specificity of the products was verified by melting curve analysis and 2.0% agarose gel electrophoresis containing 0.025  g / ml ethidium bromide .
the gper1 and -actin quantification in samples was performed based on amplification of a standard curve with five successive tenfold dilution points of a pool of cdna samples .
thyroid tissues were homogenized in ripa buffer containing 20  mm ( ph 8.0 ) tris - hcl , 150  mm nacl , 5  mm edta , 10% glycerol , plus 1  ug / ml aprotinin , 1  ug / ml leupeptin , and 0.1  mm phenylmethylsulfonyl fluoride ( pmsf ) .
lysates were centrifuged at 12.000  g for 30  min at 4c , and supernatants were used for the assay .
protein content was measured by the bradford assay , and 100 micrograms of protein was separated in a 12% sds - polyacrylamide gel with a standard molecular weight marker ( spectra multicolor broad , thermo fisher scientific inc , rockford , il , usa ) .
proteins were transferred to immobilon - p polyvinylidene difluoride ( 0.45  m , pvdf ) blotting membrane with a semidry transfer cell ( bio - rad trans - blot sd , hercules , ca , usa ) .
afterwards , membranes were blocked by incubation with tris - buffered saline containing 0.1% tween 20 and 3% nonfat dry milk for 2  h at room temperature .
thereafter , membranes were incubated with rabbit anti - gpr30 antibodies ( 1  :  500 , santa cruz biotechnology ) , or with mouse anti--tubulin antibody ( 1  :  1.000 , invitrogen ) overnight at 4c .
membranes were then incubated in horseradish peroxidase - conjugated anti - rabbit or anti - mouse immunoglobulin ( 1  :  7.500 , santa cruz biotechnology ) .
antigen - antibody complexes were detected by chemiluminescence and exposed to kodak medical x - ray processor 102 ( eastman kodak , rochester , ny , usa ) .
films were scanned and the optical density of each specific band was analyzed using the image station 4000 mm pro ( rochester , ny , usa ) .
comparisons between the two groups were performed by the mann - whitney test ; using spss statistical significance was established at p values of < 0.05 .
gper1 mrna was present in all samples studied ( figure 1 ) ; normal and goiter samples were obtained from the same patient in 9 cases . results from rt - qpcr are shown as the ratio of gper1 expression versus -actin expression in arbitrary units ( au ) , and data are shown as median [ percentile 25/percentile 75 ( p25/p75 ) ] .
gper1 mrna was less expressed in goiter than in normal thyroid with median ( p25/p75 ) , respectively , 0.8620 ( 0.09/4.68 ) and 9.6085 ( 0.44/16.12 ) .
gper1 protein levels were evaluated in 15 samples of normal thyroid and 13 samples of goiter ; normal and goiter samples were obtained from the same patient in 7 cases .
the presence of a band with a molecular weight of ~38  kda ( figures 2(a ) and 2(b ) , upper panel ) indicated the presence of gper1 .
all normal thyroid samples expressed gper1 protein , while it was not expressed in four of the goiter samples ( 28% ) .
densitometric scanning of the 38  kda band showed lower expression of gper1 protein levels in goiter when compared with normal thyroid ( p = 0.002 ) , with median ( p25/p75 ) , respectively , 0.3332 ( 0.09/0.88 ) and 1.5804 ( 0.81/2.43 ) .
as female sexual hormones could be directly involved in the pathogenesis of multinodular goiter , we studied gper1 gene and protein expression in 35 samples of normal thyroid and goiter .
likewise , gper1 protein levels were higher in normal thyroid than goiter , but the presence of gper1 protein was not observed in all samples of goiter .
this was the first time gper1 gene expression was studied in goiter ; nevertheless , gombos et al .
observed previously a lower expression of this gene , as measured by high - density oligonucleotide array , confirmed by rt - qpcr , in benign and malignant thyroid tumors , when compared to normal thyroid .
similarly , kumar et al . , studying papillary ( n = 2 ) and follicular ( n = 1 ) carcinoma cell lines , identified very low or absent levels of gper1 gene expression . on the other hand , vivacqua et al . were able to prevent estradiol - induced transduction pathways using specific inhibitors for gper1 in follicular ( n = 1 ) and anaplastic ( n = 2 ) thyroid carcinoma cell lines . in other tissues , gper1 mrna has also been evaluated .
reported that gper1 mrna levels were significantly downregulated in breast cancer tissues in comparison with their matched normal tissues .
interestingly , the receptor expression levels were lower in tumor tissues from patients who had lymph node metastasis , when with tumors without this condition .
equally , gper1 gene expression was observed to be lower in infiltrating ductal carcinoma than in nontumor mammary tissues   and also lower in the polycystic ovary syndrome group than in normal group . on the other hand ,
gper1 mrna levels were higher in malignant than benign ovarian endometriotic cysts ( eaoc ) and correlated with matrix metallopeptidase 9 ( mmp-9 ) expression , suggesting that the abnormal expression of this receptor may be involved in malignant transformation , invasion , and metastasis of eaoc .
although there are no studies concerning the functional activity of gper1 neither in normal thyroid cells nor in goiter , the lower gene and protein expression in goiter suggests a role of this gene in its pathogenesis .
demonstrated by immunohistochemistry that gper1 was positive in all samples of normal breast , while in primary breast cancer only 42% were gper1 positive .
however , in endometrial carcinoma , lung tumors , epithelial ovarian cancer , and uterine leiomyomas , the expression of this protein was higher when compared with their matched normal or benign tissues [ 2326 ] .
although the effect of e2 in activating the growth of thyroid cells has been shown to be an action directly mediated by er , manole et al . described nongenomic mechanisms mediating estradiol effect in thyroid growth .
the pathophysiological implications of the lower gper1 gene and protein expressions in goiter are unknown . nevertheless our data , although preliminary , suggest that gper1 abnormal gene and protein expressions could be involved in the pathogenesis of goiter as either a cause or a consequence of it .
 goiter is more common in women , suggesting that estrogen could be involved in its physiopathology . 
 the presence of classical estrogen receptors ( er and er ) has been described in thyroid tissue , suggesting a direct effect of estrogen on the gland . 
 a nonclassic estrogen receptor , the g - protein - coupled estrogen receptor ( gper1 ) , has been described recently in several tissues . however , in goiter , the presence of this receptor has not been studied yet . 
 we investigated gper1 gene and protein expressions in normal thyroid and goiter using reverse transcription quantitative polymerase chain reaction ( rt - qpcr ) and western blot , respectively . in normal thyroid 
 ( n = 16 ) and goiter ( n = 19 ) , gper1 gene was expressed in all samples , while gper1 protein was expressed in all samples of normal thyroid ( n = 15 ) but in only 72% of goiter samples 
 ( n = 13 ) . when comparing gper1 gene and protein levels in both conditions , gene expression and protein levels were higher in normal thyroid than in goiter , suggesting a role of this receptor in this condition . 
 further studies are needed to elucidate the role of gper1 in normal thyroid and goiter . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion