【作者】 曲学玲；

【导师】 贾玉杰；

【作者基本信息】 大连医科大学 ， 病理学与病理生理学， 2014， 博士

【摘要】 宫内膜癌（endometrial carcinoma）别名为子宫体部癌，该肿瘤具备较强的侵犯肌层组织和周围扩散的能力，是源发自子宫内膜上皮组织的恶性肿瘤，是妇女生殖系统最为多见的恶性肿瘤之一，近年来发病率有上升趋势。常规治疗方法除了手术、放化疗外，缺少新的治疗手段，生存率和治疗有效率在很长时间内均无明显提高。目前，新型的肿瘤靶向治疗技术有望解决这一难题。近年来，肿瘤靶向治疗成为国际癌症研究的热点与难点，该技术代表着未来癌症治疗领域新的发展方向。肿瘤分子靶向治疗是选择具有恶性肿瘤细胞特异性的分子靶标，也就是肿瘤细胞本身特有的受体、激酶和分子结构等等特异性作用的靶向位点，利用作用于靶标的各种药物直接或间接阻断肿瘤细胞的信号通路，从而有效抑制细胞的生长增殖或者促进肿瘤细胞的凋亡，该方法不会破坏和影响正常组织细胞的功能与结构，对人体更为安全、有效，副作用与毒性反应均不明显，有利于在肿瘤治疗中的实际应用。新型靶向治疗证实均对一些恶性肿瘤有明显的疗效，但是目前针对子宫内膜癌还没有有效的靶向治疗目标和方向。芳香烃受体（Aryl hydrocarbon receptor，AhR）是一种配体激活性的转录因子，属于PER-ARNT-SIM (PAS)家族。没有与配体结合的芳香烃受体AhR在细胞质中以多聚体的形式存在，与配体结合以后，AhR会转移到细胞核内，随即与芳香烃受体的核转运蛋白（Aryl hydrocarbon receptor nuclear translocator，ARNT）结合，形成具有功能活性的转录因子复合物。特异性增强子序列和复合物结合，特异性增强子临近二恶英反应元件（Dioxin responsive elements，DREs）的靶向启动子，可以调控细胞色素P4501A1（Cytochrome P4501A1，CYP1A1）以及其他的异型性靶基因的表达，从而化学污染物的不良影响。本课题通过组织芯片技术检测AhR在子宫内膜癌中的表达与分布规律，掌握AhR表达与病人病理信息的关系。在此基础上建立子宫内膜癌细胞系（Ishikawa细胞和EEC-1细胞）离体模型，深入研究并阐释AhR受体在子宫内膜癌细胞增殖与侵袭中的作用机制，为子宫内膜癌的新型靶向治疗提供新的思路和理论支持，具有重要的科研价值和社会意义。本课题研究分三个部分：（1）AhR在子宫内膜癌组织中表达分布及相关临床意义；（2）AhR对子宫内膜癌细胞增殖和侵袭能力的影响研究；（3）AhR配体3-MC对子宫内膜癌细胞增殖和侵袭能力的影响。1. AhR在子宫内膜癌组织中表达分布及相关临床意义本部分收集2008年5月至2011年10月在大连市多家医院行子宫切除或诊断性刮宫的子宫内膜组织标本200例。应用组织芯片技术，检测AhR在正常子宫内膜组织、子宫内膜增生组织、子宫内膜癌组织中的表达与分布规律，并对AhR与病理信息间的关系进行分析。同时，应用RT-PCR法和Western blot法检测AhRmRNA和蛋白在人子宫内膜癌和癌旁正常组织中的表达情况，探讨AhR蛋白与子宫内膜癌发生、发展的关系，及其在临床中的应用意义。结果显示：AhR在正常增生子宫内膜组织、子宫内膜单纯及复杂性增生组织、子宫内膜不典型增生组织和子宫内膜癌组织中的阳性表达率分别为4％、12％、20％和52％。从正常子宫内膜组织、子宫内膜增生组织至子宫内膜癌组织，AhR蛋白的阳性表达率呈显著下降的趋势，差异具有统计学意义（P<0.05）。在子宫内膜癌组织中，AhR的表达强度与其手术—病理分期、组织学分级、淋巴结转移相关（P<0.05），而与其病理学分型、肌层浸润深度无明显相关性（P>0.05）。经RT-PCR及Western blot法检测，AhR mRNA和蛋白在子宫内膜癌和癌旁正常组织中均有表达。AhR蛋白的分子量大小约95kDa，经与内参GAPDH校准后，AhR蛋白在肿瘤组织中的表达量约高于正常组织中AhR蛋白表达量的2倍（P <0.05）。因此，AhR在子宫内膜癌组织中高表达，可能在子宫内膜癌发生发展中起着重要作用，具有一定的临床意义。2. AhR对子宫内膜癌细胞增殖和侵袭能力的影响研究本实验部分将在子宫内膜癌细胞系（Ishikawa细胞和ECC-1细胞）中，检测AhR基因和蛋白的表达情况，同时应用RNA干扰技术、MTT法、Tanswell等方法，明确AhR对子宫内膜癌细胞增殖和侵袭能力的影响。结果显示：AhR在Ishikawa和ECC-1两种子宫内膜癌细胞系中均有表达，为一条95kDa的蛋白条带。经与GAPDH标准化校正后，AhR在两种细胞系中的表达量与在第一部分子宫内膜癌组织中表达相当（p＞0.05），高出正常组织2倍（p <0.05）。分别应用特异性AhR siRNA和混杂siRNA作用于Ishikawa和ECC-1细胞后，AhR siRNA组AhR蛋白表达在24h、48h和72h都降低，有统计学差异（P＜0.05）。应用MTT法检测AhR siRNA对子宫内膜癌细胞增殖的影响发现，在Ishikawa和ECC-1细胞中，分别经特异性AhR siRNA和混杂siRNA作用后，两种肿瘤细胞的增殖程度相一致，并无统计学差异（p＞0.05）。应用Transwell法检测AhR siRNA对子宫内膜癌细胞侵袭的影响发现，经特异性AhR siRNA和混杂siRNA作用后，两种肿瘤细胞的侵袭程度相一致，并无统计学差异（p＞0.05）。因此，无配体激活且AhR基因敲除后，子宫内膜癌细胞的增殖和侵袭能力未受到明显影响。3. AhR配体3-MC对子宫内膜癌细胞增殖和侵袭能力的影响。本实验部分将检测AhR配体3-MC对子宫内膜癌细胞增殖和侵袭的影响，同时采用特异性siRNA将AhR基因敲除验证肿瘤细胞增殖与侵袭情况是否与AhR通路相关。结果显示：分别经不同剂量的3-MC处理后，Ishikawa和ECC-1两种肿瘤细胞的呈现出不同程度的增殖抑制现象，增殖抑制程度与3-MC剂量呈正相关关系（P＜0.05）。分别经特异性AhR siRNA和混杂siRNA作用后，3-MC抑制的肿瘤细胞增殖过程被阻断，各种肿瘤细胞增殖无统计学差异（p＞0.05）。3-MC处理后的两种肿瘤细胞的侵袭能力未发生明显变化，并无统计学差异（p＞0.05）。因此，3-MC抑制EC细胞增殖是由AhR通路介导的。更多还原

【Abstract】 Endometrial cancer (EC) is the most prevalent gynecologic malignancy amongwomen worldwide. EC has been traditionally divided into two types based on thehistopathology and clinical behavior: type I and type II. Women with these cancersoften have risk factors such as long-term unopposed estrogen therapy, obesity,tamoxifen therapy, polycystic ovarian syndrome (PCOS), obesity, diabetes mellitus, andhypertension. Over the past decades, great advances in our understanding ofendometrial cancer biology, the patients still have an extremely poor prognosis.Undoubtedly, a better understanding of molecular pathogenesis of EC will assist thedevelopment of new therapeutic methods for this deadly disease.The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor of thePER-ARNT-SIM (PAS) family, mediates multiple biological processes includingimmune system homeostasis, cell growth and differentiation, vasular remodeling.Unliganded AHR exists in the cytoplasm as part of a multimeric, upon ligand binding,AhR translocates to the nucleus where it associates with ARNT to form a functionaltranscription factor complex, binds to specific enhancer sequences adjacent to targetpromoters termed dioxin responsive elements and subsequently regulates the expression of Cytochrome P4501A1(CYP1A1) and other xenobiotic target genes to combat theeffects of environmental contaminants such as3-methylcholanthrene (3MC).For the last decades, AhR has been widely studied as a receptor for environmentaltoxicants. Recently, increasing evidence has disclosed the potential role of AhR in thetumor development. The important role of AhR in carcinogenic pathways is evidencedin AhR knockout mice, which are refractory to polycyclic aromatic hydrocarbons(PAHs)-induced carcinogenesis. Increased expression of AhR has been observed inbreast, skin, and lung cancers in humans. Meanwhile, several studies have clearlydemonstrated that AhR-mediated responses are closely correlated with proliferation indifferent types of cancer cells, suggesting that AhR might be as a potential target forcancer treatment. However, the precise role of AhR in EC tumorigenesis is still lacking.In this study, we evaluated AhR expression in EC tissues as well as cell lines, andinvestigated the effects of AhR protein levels and3-MC on endometrial cancer cellgrowth using Ishikawa and ECC-1as cell models. Our data clearly showed thatknockdown of AhR did not alter EC cells proliferation and invasion. However,3-MCdose-dependently inhibited EC cells proliferation via AhR-mediated pathway. Thecontents of present study are as follows:(1) distribution and clinical significance ofAhR expression in endometrial carcinoma;(2) the function of AhR in proliferation andinvasion of endometrial carcinoma cell line;(3) the effect of3-MC on endometrialcarcinoma cell proliferation and invasion.1. Distribution and clinical significance of AhR expression in endometrialcarcinoma. Human endometrial cancer tissue microarray was used for immunolocalization ofAhR in endometrial cancer tissues. the expression and location of AhR protein wasexamined in normal endometrium， proliferative endometrium and endometrialcarcinoma. Furthermore RT-PCR and Western-blot techniques were used to evaluatethe mRNA and protein expression of AhR in fresh tissues of endometrial cancerobtained from5patients compared with their adjacent normal ones. It was demonstratedthat the positive rate of AhR expression was4％,12％,20%and52%in normalendometrium， proliferative endometrium, atypical proliferative endometrium andendometrial carcinoma．The positive rate of AhR expression in endometrial carcinomawas higher than that of the other groups，with a statistical significance(P<0.05)．Inendometrial carcinoma，the expression intensity of AhR was related to surgical stage，the degree of differentiation and metastasis of lymph nodes， with a statisticalsignificance(P<0.05)， but it was not obviously related to pathological type andmyometrial invasion(P>0.05)．The over-expression of AhR was well related with thegeneration，development and metastasis of endometrial carcinoma，and AhR may serveas an clinical diagnosis indicator and a target for chemotherapy of endometrialcarcinoma．2. The function of AhR in proliferation and invasion of endometrial carcinoma cellline.In this part of study, we investigated the function of AhR in proliferation andinvasion of endometrial carcinoma using Ishikawa and ECC-1cells lines. Both mRNAand protein levels of AhR in human endometrial cancer tissues and cell lines were determined by RT-PCR and Western blot analysis. Our data showed that the AhRantibody detected a band approximately at95kDa. After normalized to GAPDH, AhRprotein levels in cancer tissues were2.0fold higher (p <0.05) than those in normaltissue. Also, we detected high protein levels of AhR in both Ishikawa and ECC-1cells.Given AhR is highly expressed in the human endometrial cancer tissues and cell lines,we investigate whether knockdown of AhR would alter the malignant features of ECcells. As shown in Western blot results, the transfection of AhR siRNA reduced theAhR protein levels in both Ishikawa and ECC-1cells in comparison with the cellstreated with the scrambled siRNA. And this inhibitory effect maintained at least72hrs.To gain further insight into the potential role of AhR in the proliferation and invasion ofEC cells, we performed cell proliferation and invasion assays after tranfected with AhRsiRNA. The transfection of AhR siRNA did not change cell proliferation and invasionin EC cells in comparison with the cells treated with the scrambled siRNA. Theseresults suggested that AhR protein levels may not be involved in the features ofmalignant in endometrial cancer.3. The effect of3-MC on endometrial carcinoma cell proliferation and invasion.In this part of study, we investigated the effect of3-MC on endometrial carcinomacell proliferation and invasion. Cells were cultured in the complete growth media withdifferent concentrations of3-MC for48hrs. Additional cells were transfected withscrambled and AhR siRNA for determining their proliferation responses to3-MC.Meanwhile, cells were cultured in complete growth media in the absence or presence of3-MC for24hours. Cells were subjected to the invasion assay. After16hours, the numbers of invaded cells were stained and counted. Results demonstrated that3-MCdose-dependently inhibited EC cell proliferation, but not invasion. Importantly,knockdown of AhR using AhR specific siRNA completely blocked3-MC-nihibited ECcell proliferation, indicating that this proliferative suppression is mediated by AhRpathway.更多还原