Polyubiquitination inhibition of estrogen receptor alpha and its implications in breast cancer

Estrogen receptor alpha(ERα) is detected in more than 70% of the cases of breast cancer. Nuclear activity of ERα, a transcriptional regulator, is linked to the development of mammary tumors, whereas the extranuclear activity of ERα is related to endocrine therapy resistance. ERα polyubiquitination is induced by the estradiol hormone, and also by selective estrogen receptor degraders, resulting in ERα degradation via the ubiquitin proteasome system. Moreover, polyubiquitination is related to the ERα transcription cycle, and some E3-ubiquitin ligases also function as coactivators for ERα. Several studies have demonstrated that ERα polyubiquitination is inhibited by multiple mechanisms that include posttranslational modifica-tions, intera-ctions with coregula-tors, a-nd forma-tion of specific protein complexes with ERα. These events are responsible for an increase in ERα protein levels and deregulation of its signaling in breast cancers. Thus, ERα polyubiquitination inhibition may be a key factor in the progression of breast cancer and resistance to endocrine therapy.

Estrogen receptor alpha gene amplification in breast cancer:25 years of debate

Twenty-five years ago,Nembrot and colleagues reported amplification of the estrogen receptor alpha gene(ESR1) in breast cancer,initiating a broad and still ongoing scientific debate on the prevalence and clinical significance of this genetic aberration,which affects one of the most important genes in breast cancer.Since then,a multitude of studies on this topic has been published,covering a wide range of divergent results and arguments.The reported prevalence of this alteration in breast cancer ranges from 0% to 75%,suggesting that ESR1 copy number analysis is hampered by technical and interpreter issues.To date,two major issues related to ESR1 amplification remain to be conclusively addressed:(1) The extent to which abundant amounts of messenger RNA can mimic amplification in standard fluorescence in situ hybridization assays in the analysis of strongly expressed genes like ESR1,and(2) the clinical relevance of ESR1 amplification:Such relevance is strongly disputed,with data showing predictive value for response as well as for resistance of the cancer to anti-estrogen therapies,or for subsequent development of cancers in the case of precursor lesions that display amplification of ESR1.This review provides a comprehensive summary of the various views on ESR1 amplification,and highlights explanations for the contradictions and conflicting data that could inform future ESR1 research.

T29C genotype polymorphism of estrogen receptor alpha is associated with initial response to interferon-alpha therapy in chronic hepatitis B patients

BACKGROUND: Virological clearance, delayed progression to cirrhosis or liver cancer, and increased survival are the long-term goals of antiviral therapy in chronic hepatitis B patients. Identification of host factors correlated with therapeutic response may contribute greatly to individual treatment. This study aimed at investigating whether T29C genotype polymorphism of estrogen receptor alpha (ESR1) is associated with the initial response to interferon-alpha (IFN-alpha) therapy in chronic hepatitis B patients. METHODS: The initial responses of 100 patients to IFN-alpha therapy were evaluated and compared by classifying them into three groups according to T29C genotype polymorphism of ESR1: T/T, TIC, and C/C genotype groups. Polymerase chain reaction-restriction fragment length polymorphism was used to analyze the genotype polymorphism in T29C. RESULTS: The frequency of initially combined response was markedly higher in both the T/T and TIC groups than in the C/C group (Z=10.326, P=0.006 and Z=26.247, P=0.000, respectively). In addition, the initial virological response was higher in the T/T and T/C groups than the C/C group (chi(2)=5.674, P=0.017 and chi(2)=4.980, P=0.026, respectively). In 78 initially HBeAg-positive patients, however, the frequency of initial e-antigen disappearance or seroconversion among the T/T, T/C, and C/C genotype groups was 34.15%, 27.78% and 15.79%, respectively, which were not significantly different. CONCLUSION. The T29C genotype polymorphism of ESR1 is associated with the initial response to IFN-alpha in patients with chronic hepatitis B, and might be a significant marker for predicting the initial response to IFN-alpha, at least in this study population. (Hepatobiliary Pancreat Dis Int 2010; 9: 275-279)

Effects of Estrogen-related Receptor alpha (ERRα) on Proliferation and Metastasis of Human Lung Cancer A549 Cells

Estrogen-related receptor alpha （ERRα） plays an important role in the development of hor- monezdependent cancers, but its roles in lung cancer remain elusive. The present study was aimed to investigate the effects of ERRα on the proliferation and metastasis of lung cancer A549 cells. The mRNA and protein levels of ERRor were detected in lung cancer A549 and MCF-7 cells and bronchial epithelial BEAS-2B cells by qRT-PCR and Western blotting, respectively. ERRor plasmid transfection and XCT-790 （an inverse agonist of ERRc0 were used to up-regulate or down-regulate ERRα expression in A549 cells, respectively. The viability of A549 cells was measured by cell counting kit-8 （CCK-8） and the motility of A549 cells by wound healing assay and Transwell migration/invasion assay. The epithelial markers E-cadherin （E-Cad） and zona occludin-1 （ZO-1）, the mesenchymal markers fi- bronectin （FN） and vimentin （Vim） and the transcription factors （Snail, Zebl Twist and Slug） were fur- ther detected at mRNA and protein levels by qRT-PCR and Western blotting, respectively. The results showed that ERRor promoted the growth of lung cancer A549 cells in vitro. XCT-790 significantly in- hibited the migration and invasion of A549 cells. Over-expression of ERRα promoted the epithe- lial-to-mesenchymal transition （EMT） of A549 ceils, down-regulated the epithelial makers E-Cad and ZO-1, and up-regulated the mesenchymal makers FN and Vim. Silencing of Slug, but not other tran- scription factors, significantly abolished the ERRs-induced EMT of A549 cells. It was suggested that ERRor promoted the migration and invasion of A549 cells by inducing EMT, and Slug was involved in the process. Targeting ERRor might be an efficient approach for lung cancer treatment.

Expression of estrogen receptor alpha,nerve growth factor,interleukin-2,and androgen receptor in the cerebellum of ovariectomized rats following soybean isoflavone treatment

BACKGROUND： Studies have shown that estrogen receptor alpha （ERα）, nerve growth factor （NGF）, interleukin-2 （IL-2）, and androgen receptor （AR） expression in the cerebellum decreases when estrogen levels decrease in vivo. Soybean isoflavone, a type of non-steroid estrogen with similar molecular structure and function to estradiol, exhibits estrogen-like characteristics. OBJECTIVE： To investigate the effects of various doses of soybean isoflavone on expression of ERa, NGF, IL-2, and AR in the cerebellum of ovariectomized rat, and to determine whether there is a dose-dependent effect.DESIGN, TIME AND SETTING： Controlled trial at the cellular and molecular level. The study was performed at the Experimental Animal Engineering Center, College of Veterinary Medicine, Sichuan Agricultural University from July 2006 to May 2008. MATERIALS： Soybean isoflavone, comprised of daidzin, genistein and isoflavone, was provided by Taiyuan Yuantai Biochemical Industry, China. The ERα, NGF, IL-2, and AR in situ hybridization kit, rabbit anti-rat ERa, NGF, IL-2, and AR monoclonal antibodies, and SABC kit were purchased from Wuhan Boster Biological Technology, China. METHODS： A total of 50 female, Sprague Dawley rats, aged 3 months, were randomly assigned to 5 groups, with 10 animals in each group. With the exception of the sham-operation group （abdominal cavity opening alone）, all rats underwent bilateral ovariectomy. At 14 days after surgery, rats in the high-, middle-, and low-dose soybean isoflavone groups were subcutaneously injected with 1.5, 1.0, and 0.5 mg/kg soybean isoflavone, respectively, every 2 days for 6 consecutive weeks. Rats in the sham-operation and ovariectomized groups were subcutaneously injected with absolute alcohol （0.5 mL/kg）. MAIN OUTCOME MEASURES： Expression levels and distribution of ERα, NGF, IL-2, and AR in the cerebellum were detected by immunohistochemistry and in situ hybridization. RESULTS： Compared with the sham-operation group, immunoreactive products and hybridization signals of ERa, NGF, IL-2, and AR were significantly decreased in the cerebellar cortex and nuclei of ovariectomized rats （P 〈 0.05 or P 〈 0.01）, but increased following soybean isoflavone treatment. In particular, levels of the high-dose soybean isoflavone group were almost restored to levels of the sham-operation group （P 〉 0.05）. The immunoreactive products were primarily located in the cytoplasm and neurites, and rarely in the cell membrane and nuclei. However, the hybridization signals were predominantly located in the nuclei, but rarely in the cytoplasm, cell membrane, or neurites. CONCLUSION： Soybean isoflavone upregulated ERα, NGF, IL-2, and AR protein and gene expression in a dose-dependent manner, and played an important role in sustaining and protecting structure and function of cerebellar neurons. Moreover, the similarity of expression patterns of these molecules indicated that they were mutually interactive during the regulation of soybean isoflavone to the cerebellum.

The Effect of GnRHa Induced Superovulation on Endometrial Morphology and Estrogen Receptor and Progesterone Receptor in Mouse

To evaluate the effect of GnRHa induced superovulation protocol on endometrial morphology and function. Material & Methods Forty ICR mice were randomly allocated into 4 groups, among them, 2 experimental groups were injected with GnRHa+HMG+hCG, another 2 groups were given saline of same volume as control group. The uterine tissues were investigated at 24 h and 48 h after administration (experimental group) or ovulation (control group).The endometrial thickness, the size of gland and glandular lumen, the total area of glandular cells, the average height of glandular epithelium were measured from routine histological slides using computerized image analysis. The SP immunohistochemistry techniques with monoclonal antibodies were employed to semi quantitatively analize the estrogen receptor (ER) and progesterone receptor (PR) in glandular cells. Results The endometrial thickness was not significantly different between experimental groups and control groups at 24 h and 48 h (P>0.05).The average area, perimeter, maximal diameter of single gland and glandular lumen, the total area, average height of glandular epithelium in experimental groups were significantly smaller than those of in control groups at equivalent time stages (all P<0.01). The asynchronous development of gland epithelium and stroma cells, namely, pesudostratified glandular epithelium and predecidual changes of stroma cells were seen at same time in experimental groups. The positive percentage (%) and expression intense of ER and PR in glandular epithelium cells were significantly lower in experimental groups than in control groups (P<0.05). Conclusion The protocol with GnRHa had a negative effect on endometrial histological structure and down regulated the express of ER and PR, suggesting that this protocol effect on the endometrial morphology and function and could not facilitate the formation of a physiologic endometrium completely, which may be one of the causes of low pregnancy rates.

Expression of estrogen receptor alpha in preimplantation mice embryos

Objective:To study the expression of estrogen receptor alpha(ERα) in preimplantation mice embryos.Methods:Mice zygotes were collected from superovulated Kunming mice and cultured in vitro.Embryos at different developmental stages were collected at 0,24,36,48,72 and 96hours after cultivation.The expression of ERα in early mice embryos was detected by reverse transcription-PCR(RT-PCR) and immunocytochemistry.Results:The expression of ERα mRNA was detected in all of the examined embryonic stages.The relative amount of ERα mRNA showed no significant difference between 1-cell stage embryos and 4-cell stage embryos(P>0.05).However,the relative level of ERα mRNA significantly decreased(P<0.05) at 2-cell stage and was the lowest at this stage.Over 2-cell stage,the ERα mRNA relative level would increase and achieve the peak level at blastocyst stage.The location of immunocytochemistry showed that ERα immunopositive cells could be firstly detected at 8-cell stage,after which they are consistently detected until blastocyst stage.In addition,the intensity of ERα positive staining was higher at blastocyst stage compared with that at 8-cell stage and morula stage.Conclusion:ERα is expressed in preimplantation mice embryos in a temporal and spatial pattern and may be involved in regulating the development of early mice embryos,which probably plays crucial roles in early embryonic development.

The expression of estrogen receptors in thyroid cancer and its significance

Objective The study aimed to detect the expression of estrogen receptors(ERs) in thyroid cancer and investigate the correlation between their expression and clinical features and different pathological types.Methods The expression of ERs in 56 samples of thyroid cancer tissues was detected by an immunochemical approach. The expression of ERs in thyroid cancer tissues and different pathological types were analyzed using the χ~2 test. Results The number of cases with positive expression of ER in thyroid cancer tissues was 36. The number of papillary thyroid cancers(PTCs) was 48, with positive expression of ERs in 32 cases. The number of follicular thyroid cancers was 4, with positive expression of ERs in 2 cases. The number of medullary thyroid cancers was 4, with negative expression of ERs in all cases. The difference between the expression and different pathological types showed statistical significance. The expression of ERs showed no correlation with sex, age, or TNM stage, with no statistical significance. However, the expression of ERs was correlated with metastasis of lymph nodes, which had statistical significance. The expression of ERs was negatively correlated with pathological types and metastasis of lymph nodes. The correlated coefficient index was –0.313 and –0.334, respectively. Conclusion The expression of ERs showed no correlation with sex, age, or TNM stage, but was negatively correlated with pathological types and metastasis of lymph nodes.

Expression of estrogen receptor (ER) -α and -βtranscripts in the neonatal and adult rat cerebral cortex, cerebellum, and olfactory bulb

In the present study expression of estrogen receptor subtype -α(ERα) and -β (ERβ) in the cerebral cortex, cerebellum, and olfactory bulb was investigated and compared between neonatal (1～3-days-old) and adult (250～350g) rats, using reverse transcription-polymerase chain reaction (RT- PCR). No ERα transcripts were detectable in the adult cerebellum and olfactory bulb, whereas very weak expression of ERα was present in the adult cerebral cortex. No significant difference in ERβ transcripts was detectable between the neonatal and adult rats. While transcripts for both ER subtypes were co-expressed in these brain areas of neonatal rats, although ERα expression was significantly weaker than ERβ. Even in the cerebral cortex known to contain both ER subtypes in adult rats, ERα transcripts in neonatal rats were much higher than in adult. These observations provide evidence for the existence of different expression patterns of ERcα/ERβ transcripts in these three brain areas between the neonatal and adult rats, suggesting that each ER subtype may play a distinct role in the regulation of differentiation, development, and functions of the brain by estrogen.

Correlation and Significance of Midkine and Estrogen Receptor Beta Protein Expression in Non-Small Cell Lung Cancer

OBJECTIVE Midkine (MK),a new member of the heparin-binding growth factor family,was found recently to have a highexpression level in many carcinoma specimens,including thoseof the esophagus,gall bladder,pancreas,colorectum,breast andlung.Estrogen receptor beta (ER-β),a recently cloned estrogenreceptor subtype,was also found to be highly expressed in lungtumor tissue,in contrast to a lower level of expression in normallung tissue.However,few relevant studies on these proteins havebeen published.The aims of our study were to investigate theexpression of midkine and ER-β proteins in non-small cell lungcancer (NSCLC) and to examine the relationship between theirexpression and the clinicopathologic data as well as to analyse thecorrelation of their expression in NSCLC.METHODS By immunohistochemistry,MK and ER-β were ex-amined in 24 surgically resected cases of NSCLC with their corre-sponding paraneoplastic and normal lung tissues.RESULTS MK and ER-β were overexpressed in NSCLC.Thelevels of MK and ER-β expression in NSCLC were found to be sig-nificantly negatively correlated with the pathological classification(P=0.042 and 0.021,respectively),and their expression decreasedwith a raise in the classification.Spearman's correlation analysisshowed that the correlation of their expression in NSCLC wasstrong (correlation coefficient[r_s]= 0.535,P=0.007<0.01).CONCLUSION The expression levels of MK and ER-β to someextent reflect the malignant degree of NSCLC,and their combineddetection may be of great value in early diagnosis,treatments ofpatients with NSCLC and can predict the prognoses.

Increased Midkine and Estrogen Receptor-β Expression in Human Non-Small Cell Lung Cancer

Objective： Midkine （MK）, a new member of the heparin-binding growth factor family, has been found recently to have a high expression level in many tumor specimens including lung carcinoma. Estrogens may be involved in lung carcinogenesis, and estrogen receptors, mainly estrogen receptor-β （ER-β）, are present and functional in normal lung and tumor cell lines and tissues. In addition, estrogens and growth factors may promote the progression of human non-small cell lung cancer （NSCLC）. Previously, we have immunohistochemically demonstrated that MK and ER-β proteins were overexpressed in NSCLC and their expression levels were both significantly negatively correlated with the pathological classification. The purpose of this study was to further verify their expression and its correlation with NSCLC. Methods： Taking NSCLC tissues and their corresponding paraneoplastic and normal lung as research objects, we further examined the expression of MK and ER-β by meas of RT-PCR, in situ hybridization and Western blot analyses at the levels of messenger RNA （mRNA） and protein, respectively. Results： The increased MK and ER-β mRNA expression was found in NSCLC by RT-PCR and in situ hybridization analyses. Furthermore, Western blot analysis also displayed increased expression of MK and ER-β proteins in NSCLC. Finally, their correlation analysis at the levels of mRNA and protein expression in NSCLC demonstrated that MK protein level was significantly correlated to estrogen receptor-β （P〈0.01, rs=0.535）; in spite of their correlation at the mRNA level, there was no remarkable difference between MK and ER-β （P〉0.05, rs=0.178）. Conclusion： All these results in the present study confirmed that MK and ER-β were overexpressed in human NSCLC.

Correlation of Hormonal Receptors Estrogen Receptor, Progesterone Receptor and Her-2/Neu with Tumor Characteristics in Breast Carcinoma: Study of 100 Consecutive Cases

Introduction-Breast cancer is the most common cancer and leading cause of death in women. In India, its incidence is rapidly rising over last few decades. Present study is aimed to study the pattern of expression of hormonal receptors and Her-2/neu in invasive breast carcinoma and to correlate estrogen receptor (ER), progesterone receptor (PR) and Her-2/neu expressions with various clinicopathological parameters. Material and methods: The present study was carried out in Department of Pathology, S.C.B. Medical College, Cuttack in the year 2013 taking consecutive 100 cases. Routine H&E staining for histological diagnosis and IHC analysis for ER, PR and Her 2/neu was carried out in all 100 cases of breast malignancies. Results: 99% of cases are invasive breast carcinoma, not otherwise specify (IDC-NOS). The age ranges from 23 years to 72 years. Majority of tumors are of grade 2 (70%) followed by grade 3 (30%). ER PR and Her-2/neu expression are seen in 45%, 35% and 30% respectively. Triple negative cases comprise 35%. Higher number of grade 2 tumor shows ER, PR positivity as compared to grade 3 tumors. Her-2/neu expression does not show any significant correlation with age or lymph node status of the patient. Conclusion: ER and PR expression in breast cancers in the current study are found to be comparable to the findings of other authors, but the frequency of HER-2/neu expression is slightly higher. Significant correlation is observed between hormonal receptor status and the grade of the tumor. Inverse relationship is found between Her-2/neu expression and ER, PR receptor status. Her-2/neu expression is increased with size and high grade of tumor.

Studies of the Expression of Estrogen Receptor Gene in the Rat Uterus during the Estrous Cycle and Periimplantational Period

The correlation or serum estradiol concentrstion and uterine estrogen receptor (ER) gene expression (ERn and ERc quantitated by Dextrsn Coat Charcoal assay and ER mRNA by Northern blotting) was studied during the rat estrous cycle and early Pregnant stage (dl-d10). The ER gone expression was up - regulated by estrogen and the levels of ER mRNA synchronized with the changes of ER protein, suggesting that estrogen influenced the trsnscriPtional step of the ER gene. Post-coitum ER expression increased with the serum estrsdiol progressively, reached a peak on d4-ds (Just before implantation), but drastically dropped to the nadir on d6-d7 (during implantation) and then recovered. It was of interest to discover that ER mRNA level in the nonimplantstion sites (NIS) of uterus was much higher than that in the implantstion sites (IS).

Estrogen Receptor Alpha 36 Gene Knockdown Promote the Expression of NF-κB in PC12 Cells

The nuclear transcription factors κB (NF-κB) is widely existing in various kinds of cell types in the nervous system and plays an important role in neuron apoptosis and neurodegenerative diseases. Estrogen receptor alpha 36 (ER-α36), is a novel variant of ERα (as known ER-α66) which can transduce both estrogenand antiestrogen-dependent activation of MAPK signal pathway and stimulate cell growth. Here, we aimed to detect the effect of ER-α36 gene silencing on the expression of NF-κB in normal cultured PC12 cells and to provide an experimental foundation for understanding the function of ER-α36 innerve cells. PC12 cells with ER-α36 expression knocked down by the shRNA method. Then Western blot and immunocytochemical staining were performed to detect the expression and translocation of NF-κB after transfection. The results showed that NF-κB expression was significantly higher comparing with the control group after transfection (P 0.01). Also, NF-κB subunit entered nuclear after transfection;Immunofluorescence staining and immunocytochemical staining of PC12 cells demonstrated that ER-α36 was expressed mainly on the plasma membrane and on the cell nucleus membrane. These data indicate that ER-α36 gene silencing can increase the expression of NF-κB and promote its nuclear translocation in PC12 cells.

ER_α通路代偿性激活在乳腺癌细胞的拉帕替尼获得性耐药中的作用

目的了解雌激素受体α(estrogen receptorα,ERα)通路代偿性激活在乳腺癌细胞BT474的拉帕替尼获得性耐药发生过程中的作用和可能机制。方法利用real-time PCR和蛋白质印迹检测BT474被拉帕替尼作用后人表皮生长因子受体2(HER2)通路和ERα通路活性的改变;利用拉帕替尼浓度递增、持续培养的方法建立乳腺癌细胞BT474的拉帕替尼获得性耐药细胞(rBT474)模型;采用流式细胞术检测拉帕替尼对rBT474细胞凋亡的影响,进一步以蛋白质印迹法分析BT474和rBT474在HER2通路和ER通路上的差别;应用MTT法检测rBT474在拉帕替尼和氟维司群作用下的生长情况;利用克隆形成实验观察双靶点治疗对预防拉帕替尼获得性耐药的可能性。结果 Real-time PCR和蛋白质印迹检测显示拉帕替尼抑制BT474细胞HER2磷酸化,同时诱导叉头蛋白3A(FOXO3a)和孕激素受体(PR)的表达升高;成功获得的耐药细胞株rBT474在含有5μmol/L拉帕替尼的培养液中仍可持续生长;蛋白质印迹结果显示,rBT474细胞与BT474细胞相比,其磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/AKT)通路受抑制,而促分裂素原活化蛋白激酶(MAPK)通路激活,ER通路的活化更加明显;MTT法检测结果显示,与单用拉帕替尼相比,联合使用拉帕替尼和氟维司群可抑制rBT474细胞活力(P<0.01);克隆形成实验结果表明,与二甲亚砜组、拉帕替尼组和氟维司群组相比,拉帕替尼和氟维司群联合用药组可抑制BT474细胞克隆形成(P<0.01),具有预防获得性耐药发生的可能。结论 ERα通路的代偿性激活可能是导致HER2(+)/ERα(+)的乳腺癌细胞对拉帕替尼产生获得性耐药的机制之一,而PI3K/AKT抑制和MAPK激活可能是ERα代偿激活的主要原因。

Effects of Estrogen on ER, NGF, and ChAT Expression in Cerebellum of Aging Female Sprague-Dawley Rat

This article discusses the effects of estrogen on the expression of estrogen receptor （ER）, nerve growth factor （NGF）, and choline acetyltransferase （CHAT） in the cerebellum of rats. The model of aging female rat was established to study the expression and distribution of ER, NGF, and ChAT in the cerebellum following 17β-estradiol treatment using the technique of immunohistochemical ultrasensitive SP in sprague-dawley rat. The immunoreactive productions were distributed in stratum Purkinje cell, nucleus dentatus, nucleus interpositus, and nucleus fastigii of cerebellum, and the ER positive production was mainly located in the plasma, cytoplasmic membrane, and neurite, and also existed in nucleus. The general tendency of the expression of ER, NGF, and ChAT positive production in the cerebellum cortex and nuclei of aging rat significantly decreases, while the intensity and quantity of the immunoreactive production ascends predominantly after 17β-estradiol treatment. Simultaneously, the positive neurite of Purkinje cell shows a similar tendency. The above- mentioned results suggest that the estrogen upregulates the expression of NGF and CHAT, and plays a vital role in sustaining and protecting the structure and function of cerebellum neurons. Furthermore, the similarity of their changing tendency implies that they were correlated and cooperated during the course in effect of estrogen on cerebellum. It also showed that the action of estrogen in cerebellum could be via genomic and nongenomic mechanism.

METTL14介导ERα的m6A修饰调控子宫内膜癌转移的机制研究

背景与目的:甲基转移酶样因子14(methyltransferase-like factor 14,METTL14)失调引起的异常N6-甲基腺苷(N6-methyladenosine,m6A)修饰在多种癌症的进展中发挥重要作用,目前尚不清楚其是否参与子宫内膜癌(endometrial cancer,EC)的进展。本研究旨在探讨METTL14失调引起的异常m6A修饰在EC侵袭和转移中的作用。方法:收集96例2017—2021年在青海省人民医院接受治疗性手术的EC患者。从冷冻组织中分离RNA(70对)或蛋白质(10对),用于实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR)或免疫印迹分析,以评估METTL14在EC中的表达。评估METTL14的表达及其与EC临床病理学特征的相关性。在体外和体内测定METTL14在EC中的生物学效应。将甲基化RNA免疫沉淀测序(methylated RNA immunoprecipitation sequencing,MeRIP-seq)与RNA测序(RNA sequencing,RNA-seq)相结合,并在m6A斑点印迹后,采用MeRIP-RTFQ-PCR、RIP-RTFQ-PCR或双荧光素酶报告基因分析来筛选和验证METTL14的候选靶标。结果:与匹配的相邻组织相比,EC中METTL14的mRNA表达和蛋白质水平显著下调。与METTL14高表达组相比,METTL14低表达组国际妇产科联盟(International Federation of Gynecology and Obstetrics,FIGO)分期、浸入深度、淋巴管血管侵犯、淋巴结转移及肿瘤转移例数显著增加(P<0.05)。在功能上,METTL14在体外和体内抑制EC细胞的增殖和侵袭能力。从机制上讲,METTL14介导的m6A去甲基化导致雌激素受体α(estrogen receptor alpha,ERα)转录后抑制。此外,与METTL14相比,ERα诱导肿瘤的致癌行为。结论:METTL14通过m6A依赖性方式在EC细胞中减弱ERα的表达,进而抑制肿瘤转移和侵袭。

乳腺癌组织中COL11A1表达与ER、PR、HER-2和Ki-67的相关性分析

目的探讨乳腺癌组织中Ⅺ型胶原α1(COL11A1)表达与雌激素受体(ER)、孕激素受体(PR)、人表皮生长因子受体-2(HER-2)和Ki-67的关系。方法采用免疫组化EnVision两步法检测18例乳腺纤维腺瘤组织和120例乳腺癌组织(36例原位癌组织、84例浸润性癌组织)中COL11A1的表达,同时对乳腺癌组织进行ER、PR、HER-2和Ki-67免疫组化检测,对HER-2(2+)进行荧光原位杂交检测;分析COL11A1表达与乳腺癌临床病理特征和多种免疫标记(ER、PR、HER-2和Ki-67)的关系。结果免疫组化结果显示COL11A1主要表达于乳腺癌细胞中。在纤维腺瘤组织中COL11A1表达呈弱阳性。COL11A1在原位癌中表达高于浸润性乳腺癌(P<0.05)。COL11A1表达与乳腺癌患者的年龄、部位、肿块大小和淋巴结转移无关(P>0.05),而中、高分化组织的COL11A1表达水平高于低分化组织,差异有统计学意义(P<0.05)。进一步分析COL11A1与乳腺癌其他免疫标记间的相关性发现,在Ki-67高表达组织中COL11A1低表达的比例更高,差异有统计学意义(P<0.05),而其他3种标记与COL11A1的表达无关(P>0.05)。结论COL11A1在浸润性乳腺癌中表达较原位癌低,在低分化乳腺癌中表达水平较中高分化更低,且与Ki-67表达有关,提示COL11Al表达在乳腺癌发生发展过程中是动态变化的,为乳腺癌的诊断治疗提供潜在标记物。

Effects of delayed estrogen treatment and 20-HETE synthesis inhibition on postischemic pial artery response to acetylcholine in rats

Relatively little is known about the effects of estrogen on postischemic cerebral vasomotor dynamics after ischemic injury. Emerging hypotheses suggest that the timing after menopause at which hormone replacement is initiated might be important and might modulate the potential benefits of estrogen on brain rescue once a cerebral ischemic event occurs. Therefore, we sought to determine if protracted hypoestrogenicity modifies estrogen’s protective effects on postischemic pial artery dilatory dysfunction and if the arachidonic acid metabolite 20-hydroxyeicosatetraeonic (20-HETE) contributes to the dysfunction. Pial artery dilation to acetylcholine was examined before and 1 hour after 15 minutes forebrain ischemia. The rat study groups included: sexually mature males (M), naive (N), OVX (OV), estrogen-treated OVX females (E1;estrogen started 1 week post ovariectomy) and delayed estrogen-treated (E10;started 10 weeks post ovariectomy) females. Postischemic responses were assessed before and after superfusion of the 20-HETE synthesis inhibitor N-hydroxy-N’-(4-butyl-2-methylphenyl)-formamidine (HET0016). Postischemic acetylcholine dilation was depressed in M, OV and E10 compared to N and E1 rats. Compared to E1, delayed estrogen replacement worsened acetylcholine-induced dilation. Postischemic microvascular estrogen receptor alpha (ERα) density was similar in the OV, E1 and E10 rats. Postischemic application of HET0016 failed to improve acetylcholine dilation. Continuous infusion of HET0016 during and after ischemia did not reverse postischemic pial vasodilatory dysfunction. Timing of estrogen replacement may be critical for vascular health after cerebral ischemic injury. Postischemic loss of acetylcholine reactivity does not appear to involve mechanisms related to 20-HETE synthesis or microvascular ERα expression.