研究目的:探讨细胞核因子κB受体活化因子（receptor activator of nuclear factorκB,RANK）/细胞核因子κB受体活化因子配体（receptor activator of nuclear factorκB ligand,RANKL）对人子宫内膜癌细胞转移和上皮间质转化（epithelial-mesenchymal transition,EMT）的影响;进一步研究chemokine ligand 20（CCL20）在RANK/RANKL诱导子宫内膜癌细胞EMT中的作用机制。方法:体外构建针对RANK的过表达质粒载体（p IRES2-3FLAG-EGFP-RANK）,并以空白质粒载体（p IRES2-3FLAG-EGFP-CON236）作为对照。脂质体法转染子宫内膜癌HEC-1A细胞及Ishikawa细胞,上调RANK表达水平。分别给、或不给予RANKL处理子宫内膜癌HEC-1A和Ishikawa细胞。实时荧光定量PCR（q RT-PCR）和Western blot技术分别检测子宫内膜癌细胞RANK的m RNA及蛋白水平。细胞划痕试验、Transwell试验、CCK-8试验分别检测RANK/RANKL对内膜癌细胞迁移、侵袭及增殖能力的影响。q RT-PCR、Western blot技术、细胞免疫荧光技术分别检测EMT相关指标E-cadherin、N-cadherin、Vimentin及Snail、Twist的m RNA及蛋白表达变化。分别采用蛋白芯片技术、酶联免疫吸附试验（ELISA）分析趋化因子CCL20在RANK/RANKL诱导的子宫内膜癌细胞EMT中的表达和分泌水平。建立人子宫内膜癌原位小鼠模型,并采用q RT-PCR及ELISA法体内分析CCL20的表达和分泌水平。免疫组化分析RANK、RANKL、E-cadherin、N-cadherin及Vimentin在各期子宫内膜癌组织切片中的表达情况,并采用Spearman相关分析分别分析RANK与E-cadherin、N-cadherin、Vimentin的表达相关性。结果:1.随着子宫内膜癌分期的升高,其组织中RANK、RANKL的表达水平明显提高,且RANK的蛋白水平与N-cadherin（p=0.0229）、Vimentin（p=0.0398）的水平呈现正相关性,与E-cadherin（p=0.0118）的水平呈现负相关性。2.RANK/RANKL明显促进内膜癌HEC-1A及Ishikawa细胞的迁移、侵袭能力（P＜0.01）,但并不影响细胞的增殖。3.RANK/RANKL上调内膜癌HEC-1A及Ishikawa细胞中EMT相关指标N-cadherin、Vimentin及Snail、Twist的m RNA及蛋白表达（P＜0.05）,下调了E-cadherin的m RNA及蛋白水平（P＜0.05）。4.HEC-1ARANK及IshikawaRANK细胞经RANKL处理后,其CCL20的表达和分泌水平显著提高（P＜0.001）。5.子宫内膜癌原位移植小鼠模型中,RANK/RANKL可增加动物瘤组织及血清中CCL20的表达和分泌水平（P＜0.01）。6.CCL20可促进RANK过表达的内膜癌细胞发生迁移,上调N-cadherin、Vimentin及Twist的蛋白水平（P＜0.05）,下调E-cadherin的蛋白水平（P＜0.01）。结论:1.RANK/RANKL可诱导子宫内膜癌HEC-1A、Ishikawa细胞发生上皮间质转化,促进肿瘤的转移。2.RANK/RANKL通过上调子宫内膜癌细胞中CCL20的表达和分泌水平,从而促进癌细胞发生上皮间质转化、增强其迁移/侵袭能力,加快肿瘤的进展。

目的:明确RANKL促进子宫内膜癌细胞分泌CCL20的分子机制;明确RANKL对调节性T细胞（regulatory T cell,Treg）的调控作用,进而阐明子宫内膜癌RANKL刺激后分泌的CCL20募集Treg细胞所导致的免疫抑制环境,有望为子宫内膜癌的免疫治疗提供新的手段。内容与方法:利用过表达RANK的细胞株进行蛋白芯片分析,筛选出差异最显著的CCL20;结合TCGA,GEO数据库分析RANK,RANKL及CCL20在子宫内膜癌中的表达,并在临床样本中进行验证。利用GEO数据库检测Traf家族的表达水平;利用PCR检测RANKL对Traf6的调控;利用蛋白质互作网络以及Allgen,Qiagen数据库预测CCL20启动子区结合的转录因子;干扰RANK后,利用WB技术验证RANK对转录因子NFκB1的调控。结合TCGA数据库和免疫基因特征,分析与RANKL相关基因的功能以及与Treg细胞的相关性,并在临床样本中进行验证;利用半离体募集实验明确CCL20对Treg细胞的募集功能;利用流式细胞术和ELISA技术检测Treg细胞的功能。结果:1.RANK过表达的Hec-1B细胞经RANKL处理后CCL20表达上调。2.与正常内膜组织相比,RANKL与CCL20在内膜癌组织中表达显著上调（P＜0.001）,且两者蛋白表达呈正相关（R=0.554,P=0.001）CRANKL和CCL20同时高表达与淋巴结阳性相关（P=0.015）。3.干扰Hec-1B细胞中RANK表达可下调Traf6、ESCIT和NFκB1的蛋白水平。4.与正常内膜组织相比,子宫内膜癌组织中Treg细胞数量显著增加（P＜0.05）,其数量与脉管浸润呈正相关（P=0.033）。同时Treg细胞高表达CCL20受体CCR6,且Treg细胞数量与CCL20表达呈正相关（R=0.411,P=0.29）。5.Hec-1B细胞培养基和CCL20皆可促进体外Treg细胞的聚集（P＜0.05）。RANKL处理可上调Treg细胞中抑制因子IL-10,TGF-β和CTLA-4的表达（P＜0.05）。结论:RANKL结合RANK后募集胞内Traf6并上调NFκB1转录活性,促进CCL20表达,后者募集Treg细胞并刺激抑制因子IL-10,TGF-β和CTLA-4的表达,形成免疫抑制环境,促进子宫内膜癌进展。

【研究目的】:体外探究醋酸甲羟孕酮（medroxyoprogesterone acetate,MPA）对人子宫内膜癌（endometrial cancer,EC）细胞凋亡和细胞周期的影响;找到可能介导MPA调控PR+的内膜癌细胞周期和凋亡的基因、分子或通路,为PR-内膜癌治疗提供靶点。【方法】:通过分析子宫内膜癌手术标本病理资料,确定孕激素受体（progesterone receptor,PR）表达和肿瘤肌层浸润及病理分级的相关性。MPA处理Ishikawa内膜癌细胞（PRB+）和KLE内膜癌细胞（PRB-）48h后,利用流式细胞学技术确定MPA对细胞凋亡和细胞周期的影响。LncRNA芯片检测Ishikawa细胞经MPA处理后m RNA和LncRNA表达谱变化。RT-PCR验证芯片数据可信度。共表达分析以及GO、KEGG富集分析预测MPA可能影响的生物学功能。筛选细胞凋亡相关通路、因子和lnc RNA,在Ishikawa和KLE细胞中进行表达水平验证。【结果】:（1）PR+内膜癌标本中肌层浸润＜1/2者显著高于PR-内膜癌标本,PR高表达标本中低病理分级者显著高于PR低表达标本。（2）流式细胞学结果显示:MPA处理Ishikawa后G1/S期细胞显著增加,凋亡细胞水平显著增高;但MPA对KLE细胞周期和凋亡水平无明显影响。（3）基因芯片结果显示MPA处理Ishikawa后,共358个m RNA表达上调,778个m RNA表达下调;292个LncRNA表达上调,655个LncRNA表达下调;q PCR验证结果和芯片数据基本相符;GO、KEGG以及共表达分析发现MPA作用于Ishikawa细胞后可能激活内质网应激途径,内质网应激凋亡分子CHOP表达显著上调,内质网应激相关分子HERPUD1表达显著上调;共表达分析发现lnc-CETP-3和CHOP以及HERPUD1均存在共表达关系,芯片结果显示lnc-CETP-3在Ishikawa-MPA组显著上调,功能分析显示lnc-CETP-3参与细胞周期调控,细胞凋亡,肿瘤途径和内质网应激途径。（4）lnc-CETP-3在试验组Ishikawa显著上调,但在试验组KLE细胞表达未见统计学差异;CHOP和HERPUD1的m RNA和蛋白水平在试验组Ishikawa细胞均显著升高;CHOP的m RNA及蛋白水平在试验组KLE细胞均无统计学差异,HERPUD1的m RNA水平在试验组KLE无统计学差异,HERPUD1蛋白水平在试验组KLE可见轻度升高。【结论】:MPA通过作用于PRB而激活PRB+的子宫内膜癌细胞内质网应激途径,内质网应激关键凋亡分子CHOP可能介导MPA的促凋亡作用,lnc-CETP-3可能参与该过程。

目的:本文主要研究IGF-1R和EGFR在子宫内膜癌中的表达情况、与临床病理参数之间的关系,以及两者之间相互关系,探讨两者与子宫内膜癌进展的相关性及临床意义。方法:用免疫组织化学染色法分析IGF-1R和EGFR在49例正常子宫内膜组织、42例子宫内膜不典型增生组织和156例子宫内膜癌组织中的表达水平,回顾性分析156例子宫内膜癌患者的临床资料,统计分析IGF-1R和EGFR与子宫内膜癌临床病理（分期、分化、分型、脉管浸润、肌层浸润、淋巴结转移、ER、PR等）之间的关系,并通过联合检测评估两者在术前预测淋巴结转移的价值。结果:1.IGF-1R在子宫内膜癌中的表达水平明显高于子宫内膜不典型增生组织及正常子宫内膜组织（P＜0.01）,IGF-1R高表达与子宫内膜癌的分期、分化、分型、肌层浸润、淋巴结转移、脉管浸润、ER表达相关（P＜0.05）,而与年龄、PR表达水平无关（P＞0.05）。2.EGFR在子宫内膜癌中的表达水平较子宫内膜不典型增生/正常子宫内膜组织明显升高（P＜0.01）,EGFR高表达与子宫内膜癌的分期、分化、分型、淋巴结转移相关（P＜0.05）,而与年龄、肌层浸润、脉管浸润、ER、PR表达水平无关（P＞0.05）。3.相关性分析表明IGF-1R和EGFR呈正相关（r=0.618,P＜0.01）。4.IGF-1R和EGFR双分子高表达组的淋巴结转移率明显高于单分子高表达组和双分子低表达组（P＜0.05）。结论:IGF-1R和EGFR与子宫内膜癌的进展尤其是淋巴结转移密切相关,两者联合检测有望作为术前预测淋巴结转移的分子标志物,更好地为子宫内膜癌精准治疗提供指导。

BACKGROUND: Endometrial cancer (EC) is a common gynecological malignancy. and the prognosis of advanced EC is unsatisfactory. The deregulated expression of RNA-binding proteins (RBPs) is closely associated with the occurrence and development of cancer. However, the role of RBPs in EC remains unclear. The aim of this study was to validate the prognostic values of RBPs combined with clinical factors.METHODS: We downloaded the RNA sequencing and clinical data for EC from The Cancer Genome Atlas (TCGA) database. R software was used to identify the differentially expressed RBPs. Univariate and multivariate Cox proportional hazards regression analyses were performed to predict the 4 overall survival (OS)-related RBPs. We then constructed a nomogram combining the 4-RBP signature with clinical risk factors to assess the prognostic power. Furthermore, we validated the expression of 4 RBPs in our patient samples using quantitative real-time polymerase chain reaction (qRT-PCR) and explored the effect of cold-inducible RNA-binding protein (CIRBP) on EC tumor growth using cell proliferation experiments.RESULTS: It is found that Shwachman-Bodian-Diamond syndrome (SBDS), CIRBP, MRPL15, and CELF4 were significantly related to the prognosis of EC patients. In addition. the nomogram showed better performance in OS predictions than the International Federation of Gynecology and Obstetrics (FIGO) stage. The qRT-PCR results showed that low CIRBP expression was associated with cell proliferation.CONCLUSIONS: In our study. we constructed a 4-RBP signature-based nomogram combined with clinical factors in EC that could effectively predict the prognosis of EC patients. The results provide novel insights into the development of treatment targets and prognostic molecular markers in EC.

Cervical cancer is the most common gynecological cancer in women worldwide. Human papillomavirus (HPV) is required but not sufficient for developing cervical cancer. HPV E6 and E7 proteins are able to directly interact with certain nuclear receptors; however, whether steroid hormone receptors mediate cervical carcinogenesis is not completely understood. The present study demonstrated via immunohistochemistry that estrogen receptor alpha (ER alpha) and androgen receptor (AR) expression were decreased in a sequential manner from healthy cervical tissues to cervical intraepithelial neoplasia tissues and further to cervical cancer (CC) tissues, whereas microRNA (miR)-130a-3p expression levels were higher in CC tissues compared with healthy tissues. Both ER alpha and AR were direct targets of miR-130a-3p, as determined by performing luciferase reporter assays and western blotting. Functionally, compared with the corresponding control groups, miR-130a-3p knockdown, ER alpha overexpression and AR overexpression significantly inhibited CC cell proliferation and invasion, as demonstrated by the results obtained from the Cell Counting Kit-8 and Transwell assays in vitro. In addition, antagomiR-130a decreased tumor size and weight in vivo compared with control antagomiR as determined via the xenograft tumor growth assay. Therefore, the results suggested that miR-130a-3p might contribute to tumor progression by suppressing ER alpha and AR, and serve as a promising candidate target for the treatment of patients with CC.

目的基于内源性竞争性网络分析可用于子宫内膜癌(EC)分型及诊治的潜在生物标志物。方法从TCGA数据库中总结EC差异表达的基因,筛选出32个基因来构建内源性竞争性网络。采用基因本体论(GO)和京都基因与基因组数据库(KEGG)富集分析、相关

AMPH1, an abundant protein in nerve terminals, plays a critical role in the recruitment of dynamin to sites of clathrin-mediated endocytosis. Recently, it is reported to be involved in breast cancer and lung cancer. However, the impact of AMPH1 on ovarian cancer is unclear. In this study, we used gain-of-function and loss-of-function methods to explore the role of AMPH1 in ovarian cancer cells. AMPH1 inhibited ovarian cancer cell growth and cell migration, and promoted caspase-3 activity, resulting in the increase of cell apoptosis. In xenograft mice model, AMPH1 prevented tumour progression. The anti-oncogene effects of AMPH1 on ovarian cancer might be partially due to the inhibition of PI3K/AKT signalling pathway after overexpression of AMPH1. Immunohistochemistry analysis showed that the staining of AMPH1 was remarkably reduced in ovarian cancer tissues compared with normal ovarian tissues. In conclusion, our study identifies AMPH1 as a tumour suppressor in ovarian cancer in vitro and in vivo. This is the first evidence that AMPH1 inhibited cell growth and migration, and induced apoptosis via the inactivation of PI3K/AKT signalling pathway on ovarian cancer, which may be used as an effective strategy.

Background: Perineural invasion (PNI) is correlated with negative prognosis in multiple cancers, but its role in endometrial cancer (EC) is still largely unknown; thus, targeted treatment for nerve infiltration is lacking as well.Methods: The interaction between nerve and EC cells were investigated by in vitro neural invasion assay and transwell coculture system. Then the nerve-related receptor gene glutamate ionotropic receptor AMPA type subunit 2 (GRIA2) was detected in EC tissues and cells using PCR array, western blotting, and immunohistochemistry. The role of GluR2 (gene name GRIA2) on EC proliferation, migration and invasion was evaluated by a GluR2 antagonist and shRNA. At the same time, the neurotransmitter effect on GluR2 (glutamate) from the cocultured conditional medium was measured using high-performance liquid chromatography (HPLC).Results: EC cell line Ishikawa (ISK) showed the ability to migrate along neurites in vitro and the numbers of migrated/invaded EC cells in the DRG neuron coculture group were significantly increased. The expression of GluR2 in EC tissue was found to be higher than that in para-carcinoma tissue. After GluR2 antagonist and GluR2 shRNA treatment, the proliferation, migration and invasion of ISK cells was markedly inhibited. Moreover, the ability of DRG neurons to promote the migration and invasion of ISK cells could also be attenuated by downregulation of GIuR2, and the concentration of the neurotransmitter glutamate was notably increased in the coculture conditional medium compared to that in the DRG neuron or ISK cells alone.Conclusions: DRG neurons promote metastasis of EC cells via GluR2, which might be a risk factor for PNI in EC. Moreover, the perineural system may promote tumor invasion and metastasis under certain circumstances.

Ovarian cancer is one of the deadliest gynecological cancer, with a low overall 5-year survival rate. RDM1, RAD52 motif-containing protein 1, is sensitive to cisplatin, a common chemotherapy drug and it has an important role inDNA damage repair pathway. Until now, the effect of RDM1 in ovarian cancer is undiscovered. Here, clinical data shows that the tumour tissues of ovarian carcinoma patients with higher mRNA and protein expression of RDM1. Knockdown of RDM1 in ovarian carcinoma cells reduces cell proliferation and promotes apoptosis, consistent with the role RDM1 in the overexpression experiments. The research of xenograft mouse model shows stable knockdown of RDM1 significantly inhibits ovarian cancer tumour growth. These in vitro and in vivo results conclude that RDM1 plays an oncogenic role in human ovarian carcinoma. Interestingly, p53/RAD51/RAD52 signalling pathway can be regulated by RDM1, and the negative regulation of p53 by RDM1 may be one of major mechanisms for RDM1 to accomplish its oncogenic functions in ovarian carcinoma. Therefore, RDM1 may be a new target for the treatment of ovarian carcinoma.
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