Estrogen Level And Expression Of Estrogen Metabolism Related Genes In Postmenopausal Thyroid Nodule

Objective: Thyroid nodule is the most common endocrine-relatedproliferative disease with increasing incidence in recent years. Epidemicalstudies have shown that thyroid nodule occurs3-4times in women than inmen. Thus the formation of thyroid nodule may be related to the estrogenlevel.Estrogen is known to have direct and indirect effects through genetic andnon-genetic pathways on thyroid, including increasing the thyroxine bindingglobulin, and regulating cell cycle, etc.The effects of estrogen depend different estrogen receptors. Expression ofestrogen receptor α(ER-α) in thyroid promotes cell proliferation whileestrogen receptor β (ER-β) promoting cell apotosis. The membrane receptorG-protein-coupled receptor30(GPR30) promotes cell growth. It is reportedthat the relative ratio of ER-α to ER-β in thyroid tissue plays an important rolein the genesis of thyroid cancer.17β-Hydroxysteroid dehydrogenases, are series of enzymes that regulatethe estradiol and estrone level in human body. But there is few reports on theexpression of them in thyroid tissue.In this study, to explore how estrogen(especially estradiol) affects thyroidproliferative diseases, we analyzed the estrone, estradiol and2-Methoxyestradiol level in serum and tissue of thyroid nodule patients, aswell as the expression of estrogen receptor genes(ERα,ERβ and GPR30) andestrogen metabolism relative enzyme genes(HSD17B1,HSD17B2,HSD17B4,HSD17B7,HSD17B8and HSD17B14) in thyroid tissue of patients withthyroid nodule.Methods:1Materials and grouping The thyroid tissues were obtained from the operation of patients withnodular goiter in the2ndHospital of Hebei Medical University (Shijiazhuang,China) and stored at-80℃. Informed consent was obtained from these patientsbefore operation. The nodules and normal tissue were identified according topathology results. Serum samples were collected for determination of thyroidfunction, E1, E2and2MTE2concentration.2Determination of thyroid functionConcentration of serum FT3and FT4were detected by enzymeimmunoassay (RIA). Concentration of serum TSH was detected byimmunoradiometric assay (IRMA).3Determination of E1、E2、2ME2in serum and thyroid tissue3.1Determination of protein level in thyroid tissueThe content of protein in thyroid tissue was tested by Coomassie BrilliantBlue-stained Protein Quantitative Determination Kit （Nanjing JianchengBioengineering Institute）.3.2Determination of E1level in serum and thyroid tissueE1was determined by the method of enzyme-linked immunosorbentassay(ELISA). The content of E1in serum and in throid tissue was expressedas picogram per milliliter（pg/ml）and picogram per milligram protein sample(pg/mg), respectively.3.3Determination of E2level in serum and thyroid tissueThe content of E2was detected by the method of Radioimmunoassy(RIA),using Iodine[125I]-E2Radioimmunoassay Kit(Tianjin Nine Tripods Medical&Bioengineering Co. Ltd). The content of E2in serum and in thyroid tissue wasexpressed as picogram per milliliter（pg/ml）and picogram per milligramprotein sample (pg/mg), respectively.3.4Determination of2ME2in serum and thyroid tissueThe content of2ME2was detected by the method of enzymeimmunoassay(EIA). The content of2ME2in serum and in thyroid tissue wasexpressed as picogram per milliliter（pg/ml）and picogram per milligramprotein sample (pg/mg), respectively. 4Realtime-PCR AnalysisTotal RNA was extracted by E.Z.N.A.TMTotal RNA KitⅡ R6934-01(OMEGA bio-tek）and stored at-80℃. cDNAs were synthesized from5000ngof total RNA using M-MLV First Strand Kit（invitrogen, cat no.C28025-032）and stored at-20℃. An Realtime-PCR kit(Bestar SybrGreen qPCRmastermix, DBI Bioscience) was employed in the amplification of cDNA.After an initial2-minute denaturation step at95°C,40cycles of PCR werecarried out on a Light Cycler PCR machine(CFX96Touch Real-Time PCRDetection System#185-5196, BIO-RAD) under the following condition:10seconds of denaturation at95°C,1minute and30seconds of annealing andextension at60℃. Expression of genes was showed as2（－△C t）.Results:1The serum E2level in patients with thyroid nodule was higher than that inhealthy people without thyroid disease.The median of E2level in serum of patients with thyroid nodule is58.87（40.21，114.62）pg/ml, higher than the maximum value of normalpostmenopausal women without thyroid disease (maximum:31pg/ml，P＜0.01).2E1, E2,2ME2level in thyroid tissue2.1The E2level of nodule groupwas higher than that in control groupThe E2level in nodule group was3.17（1.30，10.21）pg/mg，significantlyhigher than in control group---2.16（0.51，7.30）pg/mg (Z=2.052，P＜0.05）.While, the level in thyroid tissue was not correlated to serum E2level, either innodule group or control group(nodule group: r=0.182，P=0.593；control group:r=0.110，P=0.721). These results showed the increased E2level in nodulegroup was not correlated to the increased E2level in serum. And the high levelof E2might contribute to the formation of thyroid nodule.2.2No difference of E1level between nodule group and control groupE1level in nodule group and control group were25.05（11.76，104.56）pg/mg and18.62（6.37，86.89）pg/mg（Z=1.127，P=0.260）, respectively.While, the E1level in thyroid tissue was not correlated to the E1level in serum either in nodule group or control group(r=0.865，P=0.067；controlgroup: r=0.637，P=0.183). The result showed E1level in nodule group is notcorrelated with the E1level in serum.2.3No difference of2ME2level between nodule group and control groupThe2ME2level in nodule group and control group were3.52（1.42，13.82）pg/mg and2.87（0.78，9.11）pg/mg，respectively, without significantdifference（Z=1.851，P=0.064）。The2ME2level in thyroid tissue was notcorrelated to the serum2ME2level, either in nodule group or control group(nodule group: r=0.533，P=0.139；control group: r=0.450，P=0.224）.3Expression of estrogen receptor and estrogen metabolism related genes3.1Genes of estradiol synthesis were expressed in both nodule group andcontrol group. The expression of HSD17B1mRNA in nodule group was0.16（0.01，7.10）, higher than that in control group---0.09（0.01，4.52）(Z=2.068，P＜0.05）.The expression of HSD17B7mRNA in nodule group and controlgroup were9.11（1.08，60.63）and6.22（1.96,57.59），respectively, withoutstatistical difference (Z=1.125，P＞0.05). These results showed that theincreased level of E2in nodule group might be up-regulated by the increasedHSD17B1mRNA, while might not be related with the expression ofHSD17B7mRNA.3.2Genes of estradiol degration were expressed in both nodule group andcontrol group. The expression of HSD17B2mRNA in nodule group andcontrol group were0.03（0.0004，1.09）and0.01（0.0003，1.64），respectively,without statistical difference （Z=1.703， P＞0.05）. The expression ofHSD17B4mRNA in nodule group and control group were7.68（1.74，62.34）and6.84（0.81，19.44），respectively, without statistical difference（Z=1.399，P＞0.05）. The expression of HSD17B8mRNA in nodule group and controlgroup were57.29（5.29，149.82） and47.84（5.19，151.56），respectively,without statistical difference （Z=0.274， P＞0.05）. The expression ofHSD17B14mRNA in nodule group and control group were11.05（2.38，90.56）and12.52（0.72，140.63），respectively, without statistical difference（Z=0.213，P＞0.05）. These results showed that the increased E2level in nodule was not contributed by the decreased expression of estradiol degradinggenes.3.3Genes of estrogen receptors were expressed in both nodule group andcontrol group. The expression of ERα mRNA in nodule group was0.43（0.01，49.89），higher than that in control group---0.07（0.01,7.34）（Z=2.062，P＜0.05）. The expression of ERα mRNAin thyroid tissue was not correlated toE2level in thyroid tissue, either in nodule group or control group（nodulegroup r=0.081，P=0.650；control group r=-0.239，P=0.220）. The expressionof ERβ mRNA in nodule group and control group were0.0284（0.0006，0.61）and0.02（0.003，0.27），respectively, without statistical difference（Z=1.616，P＞0.05）. The expression of GPR30mRNA in nodule group and controlgroup were12.01（1.71,150.59）and13.70（0.47，33.61），respectively, withoutstatistical difference（Z=0.152，P＞0.05）. These results showed the increasedE2level in nodule group might be contributed by the enhancement of ERαpathway.Conclusion:1The serum E2level of postmenopausal thyroid nodule patients washigher than normal level.2The high level of E2in nodule group of postmenopausal thyroid nodulepatient may be related to the high expression of HSD17B1in nodule group3The high level of E2in nodule group may contribute to the formation ofthyroid nodule through ERα pathway.