Expression Of Estrogen Receptor-related Receptor α, γ And Estrogen Receptors In The Endometrioid Adenocarcinoma

The Purpose and SignificanceEndometrial cancer as one of the three most common gynecological cancer, accounting for about 20%-30% in gynecological cancer, in which endometrioid adenocarcinoma could account for about 75%.In recent years, its incidence is on the rise. The cause of endometrioid adenocarcinoma has been a hot research and now is mainly due to the sustained, long-term, abnormal estrogen stimulation, and/or lack of progesterone hormone antagonist. There is a widely distributed sources of estrogen in human body, mainly from the ovary, placenta, and mainly from the adrenal gland in post-menopausal women. It is considered that estrogen plays an important role in the occurrence and development of endometrioid adenocarcinoma. Therefore, to study the action mechanism of estrogen will contribute to understanding the specific pathogenesis of endometrioid adenocarcinoma. The classic mechanism of estrogen is induced by the estrogen receptor (ER).In the beginning, estrogens bind to the ER and induced its conformational changes.ER dissociates heat shock protein (HSP) and form E-ER complex. This nuclear estrogen-ER complex binds to estrogen response element sequences directly or indirectly through protein-protein interactions in the promoter, increased or decreased mRNA levels and associated protein production,and a physiological response.Nuclear receptor superfamily (NRs) are a large class of transcription factors, including the classic ligand-dependent receptors and a large number of orphan nuclear receptors. Estrogen receptor-related receptor (ERR) and ERs both belong to the larger NR3 class of nuclear receptors. At present, there are two subtypes of ER have been found, namely ERαand ERβ.ERRs include three nuclear receptors referred to as ERRα, ERRβand ERRγ, respectively. In the eighties of the last century ERαwas found and considered to be the only ER, until ten years later ERβwas not discovered. ERRα, ERRβwere screened from the kidney cDNA library under low stringency hybridization conditions using by DBD region of ERαas a probe.ERRγwere screened useing GRIP1 protein as bait under the yeast two-hybrid conditions in 1999. ERRs shared NRs family structural characteristics with ERs. There are high homology between the two family in amino acid sequences and gene sequences. As for conventional NRs, ERRs and ERs are organized into several modular regions, which have distinct biological functions. There mainly are five modular regions of biological functions, which are more research is A/B, C, E regions:The A/B regions are located at the N-terminus and include the activation function 1 (AF-1). The C region includes the DBD, which is the most evolutionary conserved region and contributes to the special DNA-binding by the two zing-finger motif. The homologies between ERαand ERβwere 95%. ERR a and ER a reached to 66%. ERRγand ERRαreached to 93%. The E region contains the LBD, which contains the ligand-binding hydrophobic pocket and contributes to the receptors' dimerization. The homologies between ERR a and ER a were 33%.In estrogen signaling, ERs and ERRs responsive element (ERRE) also has a high homology. ERE is a 5'-TGACCTnnnAGGTCA-3 'reverse palindromic sequence (n is any nucleotide), which core sequence is 5'-AGGTCA-3'. ERRE has an extended ERE-half site of sequence 5'-TnA-AGGTCA-3',which has the same core sequence. Therefore, ERs and ERRs can identified the same hormone response element of the core sequence. Furtherly, there was not find the corresponding natural ligand. The ERRs and ERs have similar activity to the ERE transcription. the HRE core sequence of the variant structure exist widely in the human body, such as: complete or incomplete ERE element, ERRE element, palindromic response elements of thyroxine (TRE) as well as the steroidal growth factor-1 (SF-1 factor) response element (SFRE). These were the basis of interaction between the ERRs and ERs for broad participation in metabolism. Consequently, the two families may has played an important role in the occurrence of endometrioid adenocarcinoma.There was some studies shown that ERαand ERβcan form ERαhomodimer, ERβhomodimer, or ERα/ERβheterodimers which can bind to ERE to induce the different biological effects. ERRαalso can form monomer, homodimer and identify ERRE to induce estrogen effects. It also can interfere with ERαthrough forming the ERα/ERRαheterodimer. The activity of ERRαdepends on the combination of ERαto ERE to some extent. ERRγis also the closely family members of ERRα. A studies have shown that ERRγhave a moderating role in the expression of ERRα. The studies in mice shows that lack of ERRαcan result in the increase of ERRγ. Therefore, the study of expression of ERRα, ERRy, ERαand ERβhas important significance in the occurrence and development of endometrioid adenocarcinoma. Ariazi etal. measured the expression of ERRs and ERs by real-time PCR and evaluate the role of ERRα, ERRγas a tumor marker for breast cancer. Kraus etal.studied the cells of breast cancer with ERα(+) and cervical cancer with ERα(-).he found that ERRαand ERαhave a closely relation. Sun Peng Ming investigated the positive rates of the ERRs and ERs in ovarian cancer tissue mRNA by real-time PCR, and presume that the cross-talking of ERRs and ERs may be the molecules foundation of complex biological behavior in ovarian cancer. Summing up, we presume that the cross-talking among ERRs and ERs may be related to the molecular mechanism of the development of endometrial carcinoma,as well as other estrogen-dependented carcinoma.The present study is the first collection of endometrial tissue samples tested and incorporated into the adjacent tissues and cancer tissues, normal tissue comparison. Taqman fluorescent quantitative PCR method is used to detect the mRNA expression levels of ERRα, ERRγ, ERαand ERβin endometrial cancer tissues, normal tissue,and paracancer tissue. Incorporating the biological behavior of tumors, we considered the paracancer as lesion precancerous.The clinical and pathological data of endometrial cancer patients will be analysized to investigate the roles and relevance of ERRs and ERs in endometrioid adenocarcinoma occurrence and development. ERR subtype-mediated signaling pathway and its relationship with estrogen-ER pathway between the relationships will be furtherly studied to reveal the occurrence mechanisms of endometrioid adenocarcinoma and to provide a new clinical outcome index for endometrioid adenocarcinoma.Methods and Contents1. Research Objects44 cases of endometrial carcinoma,30 cases of normal tissues and 30 cases of paracancer tissues were collected, all specimens were stained by HE pathological diagnosis, surgical pathologic staging according to the 2000 International League of Obstetrics and Gynecology (FIGO) criteria for carried out in phases, all patients were not receiving preoperative radiotherapy and chemotherapy and hormone treatment.2. Research MethodsThe total RNA in all samples were extracted by one-step TRIzol method and detected for the quality and integrity, then the total RNA were reverse transcripted into cDNA, real-time quantitative PCR with fluorescent Taqman probe method was adopted to measure the expression levels of ERα, ERβ, ERRαand ERRy mRNA in all of the specimens, the amount of target gene expression was calculated by standard curve method.3. Statistical MethodsSPSS 13.0 software was used for statistical analysis. ERα, ERβ, ERRα, ERRγmRNA differences and expression levels of ERα/ERβ, ERα/ERRα, ERRα/ ERRy change in the ratio were analyzed by non-parametric Kruskal-Wallis H test, Nemenyi method was used for comparison between groups. Spearman rank correlation analysis was used to analyze the relationship of ERα, ERβ, ERRα, ERRγwith stage and grade of carcinoma.Results1.44 in-patients with endometrial adenocarcinoma at the General Hospital of Nanjing Military Region, were selected from January 1,2008 to September 30,2009. The age of all patients are among 33 to 79 years old and pathologically diagnosed as endometrial adenocarcinoma, in which 25 patients with stageⅠ,Ⅱstage of 13 cases,Ⅲ6 cases,Ⅳstage 0 cases; Pathological grading:G1 grade in 7 patients, G2 in 24, G3 in 8; menopause:postmenopause in 24, premenopause in 20. At the same time the samples away from the foci of endometrial tissue were selected as an paracancer group, totally 30 cases; 30 samples were taken from patients operated hysterectomy but not due to endometrial diseases as normal control group, pathologically diagnosed as normal endometrial tissue,in which 16 cases of postmenopause, premenopause in 14 cases. It is between 35 and 77 of age. Comparison of age and menopause among groups showed no significant difference (P>0. 10).2. The quality and integrity of total RNA in all samples were in good condition, the standard curve showed that the experimental amplification were efficient, the experimental data was credible.3. The expression of ERα, ERβ, ERRα, ERRγmRNA in each groups:the expression of ERα, ERRγin the three tissues were the highest in paracancer tissues and the lowest in cancer tissues; while the expression of ERβ, ERRαwere the highest in paracancer tissues and the lowest in normal tissues. Comparisons of the four indicators between the three groups are statistically significant. Further pairwise comparison, the expression of ERα, ERβ, ERRαgene in the paracancer tissues were statistically significantly higher than other two groups, but only the expression of ERαshowed statistical significance compared between the normal tissues and the cancer tissues. The expression of ERRγshowed significant difference only between the cancer tissues and paracancer tissues.4.The ratios of ERα/ERβ, ERα/ERRα, ERRα/ERRγin each groups:The ratios of ERα/ERRαand ERα/ERβwere highest in the normal tissues, then the paracancer tissues and cancer tissues; the ratios of ERRα/ERRγshowed highest in the cancer tissues,then the paracancer tissues and normal tissues. Furthermore pairwise comparison showed statistical significances in the three ratios between the normal tissues group and the other two groups, but not between the paracancer tissues and the cancer tissues.5. The expression of ERα, ERβ, ERRα, ERRγmRNA and the ratio of ERα/ERβ, ERα/ERRα, ERRα/ERRγin cancer tissues of different stages:Only the expression of ERRαin Carcinoma showed statistically significant moderate positive correlation with stage, Namely, the higher expression of ERRαin cancer tissues, the higher stages in pathology; Only ERα/ERRαand stages had slightly negative correlation, showing statistical significance. The higher ratio of ERα/ERRα, the lower in phases. The other indicators had no statistical correlation with stages.6. The expression of ERα, ERβ, ERRα, ERRγmRNA and the ratio of ERα/ERβ, ERα/ERRα, ERRα/ERRγin cancer tissues of different grades:The expression of ERα, ERβwas moderately positively related to the grade in cancer tissues, while the expression of ERRαhad mild positive correlation with grade, both showing statistical significance. Namely, the higher expression of ERα, ERβand ERRαin carcinoma, the higher the grade and the lower the degree of differentiation; No statistical significance showed in the correlation between expression of ERRγand the grade.ERα/ERβ, ERα/ERRα, ERRα/ERRγand the grade had no statistically significant correlation in cancer tissues.Conclusions1. The four indicators in the paracancer tissues were significantly higher than in normal tissues and cancer tissues, in that, the high expression of ERs and ERRs in lesion precancerous may be the basis for further canceration of endometrial cells and the reason of abnormal estrogen action, only the expression of ERαshowed statistical significance compared between the normal tissues and the cancer tissues.2. The partly inactivation of the ERα,ERRγand the relatively high expression of ERβ, ERRαplay an important role in the progression of endometrial cells to cancer cells, especially the change of the ratios of ERα/ERβ,ERα/ERRα,ERRα/ERRγ. The changes of the ratios maybe contribute to imply the development of endometrioid adenocarcinoma.