MicroRNA-200b Regulates Cell Proliferation, Invasion, and Migration by Directly Targeting ZEB2
一种细胞间通信的新的信号分子——Microvesicle运输的microRNA
一种细胞间通信的新的信号分子——Microvesicle运输的microRNA梁宏伟;陈熹;曾科;张辰宇【摘要】Microvesicle(MV)是机体内的细胞在正常和病理状态下都会分泌的直径在30~1000 nm之间的微小囊泡.细胞把特异的生物活性分子如蛋白质、mRNA 等包裹到MV中,这些生物活性分子会通过MV被运输到相应的受体细胞以调节受体细胞的生物功能.这种由MV介导的细胞间信息传递在一些生理和病理过程中扮演着十分重要的作用.近期研究表明MV中包含microRNA(miRNA),并且通过MV 的运输,这些miRNA会被运送入靶细胞以调控靶细胞相关基因的表达.本文就MV 运输的miRNA在细胞间通信的功能做一总结.【期刊名称】《分子诊断与治疗杂志》【年(卷),期】2010(002)006【总页数】7页(P417-423)【关键词】Microvesicle;MicroRNA;细胞间通信;外泌体【作者】梁宏伟;陈熹;曾科;张辰宇【作者单位】南京大学医药生物技术国家重点实验室,南京,江苏210093;南京大学医药生物技术国家重点实验室,南京,江苏210093;南京大学医药生物技术国家重点实验室,南京,江苏210093;南京大学医药生物技术国家重点实验室,南京,江苏210093【正文语种】中文【中图分类】R3Microvesicle(MV)是机体内细胞在正常和病理状态下都会分泌的直径在30~1000 nm之间的微小囊泡[1,2]。
多数研究者用MV来指代具有细胞间信号传递功能的囊胞[3,4]。
体内和体外的实验都证明MV可以由网织红细胞[5]、B细胞[6]、T细胞[7]、树突细胞[8]、肥大细胞[9]、上皮细胞[10,11]和肿瘤细胞[12,13]等多种细胞分泌。
细胞把特异的生物活性分子如蛋白质、mRNA等包裹到MV中,被运输到相应的受体细胞并调节受体细胞的生物功能。
这种由MV介导的细胞间信息传递在一些生理和病理过程中扮演着十分重要的作用[14~17]。
微小RNA-200家族对糖尿病及其并发症的影响-中华老年多器官疾病
·100· 中华老年多器官疾病杂志 20xx 年xx 月xx 日 第14卷 第xx 期 Chin J Mult Organ Dis Elderly, Vol.14, No. xx , Xxx 28, 2015收稿日期: 2015−04−23; 修回日期: 2015−06−03基金项目: 国家自然科学基金(81070157、81370303);江苏省自然科学基金(BK2011179);江苏省人事厅“六大人才高峰”第七批高层次项目(006);江苏省医学重点人才资助项目(RC201134) 通信作者: 王如兴, E-mail: ruxingw@·综 述·微小RNA-200家族对糖尿病及其并发症的影响钱玲玲,徐 凤,王如兴*(南京医科大学附属无锡市人民医院心内科,无锡 214023)【摘 要】糖尿病并发症可引起人体心、脑和肾等重要组织和器官损害,严重危害人类健康,但其发病机制并不十分清楚。
近年研究发现微小RNA (microRNA )在糖尿病及其并发症的发生发展中起重要作用。
microRNA 是真核生物中一类长度在22~25个核苷酸左右的内源性非编码小分子RNA ,通过转录后机制调控其下游靶基因的表达,进而影响疾病的病理生理学过程。
其中microRNA-200(miR-200)家族对糖尿病及其并发症的调控研究较多。
MicroRNA-200(miR-200)家族不仅对胰岛素相关信号通路具有调控作用,在糖尿病微血管及大血管并发症中也发挥重要调控作用。
因此,探讨miR-200家族与糖尿病及其并发症之间的关系,可为糖尿病及其并发症的诊断及治疗提供新的思路。
【关键词】微RNAs ;miR-200;糖尿病;并发症【中图分类号】 R342.3; R587.1 【文献标识码】 A 【DOI 】 10.11915/j.issn.1671-5403.2015.xx.xxxRole of microRNA-200 family in diabetes mellitusQIAN Ling-Ling, XU Feng, WANG Ru-Xing *(Department of Cardiology, Wuxi People ’s Hospital Affiliated to Nanjing Medical University, Wuxi 214023, China)【Abstract 】 The complications of diabetes usually cause target organ damage, such as the heart, brain, kidneys and others, and are seriously hazardous to human health. But the underlying mechanism is still not very clear. Recent studies indicated that microRNA (miRNA) plays an important role in the occurrence and development of diabetes and its complications. miRNA, an endogenous small non-coding RNA molecule (containing about 22-25 nucleotides) in eukaryotes, regulates the expression of its downstream target genes at post-transcriptional level, and then influences the pathophysiology of diseases. There are many studies have reported that microRNA-200 (miR-200) family plays certain regulative roles in diabetes and its complications. The family not only regulates insulin related signal pathway, but also plays an important role in diabetic microvascular and macrovascular complications. Therefore, to explore the relationship of miR-200 family with diabetes and its complications would provide a new train of thought for diagnosis and treatment of diabetes and its complications.【Key words 】 microRNAs; miR-200; diabetes mellitus; complicationsThis work was supported by the National Natural Science Foundation of China (81070157, 81370303), the Natural Science Foundation of Jiangsu Province (BK2011179), the Seventh Batch of High-level Project of the “Six -Industry Talent Summit” of Jiangsu Provincial Bureau of Personnel (006) and the Project of Medical Key Talents of Jiangsu Province (RC201134). Corresponding author: WANG Ru-Xing, E-mail: ruxingw@糖尿病是一种以慢性血糖升高为特征的代谢性疾病,已成为危害人类健康的主要慢性病之一[1]。
组织细胞 microRNA提取试剂盒
组织细胞microRNA提取试剂盒microRNA Extraction Kit(Tissue&Cell) Cat.No.:B1801 Size: 25次描述:海基生产的组织细胞microRNA提取试剂盒是目前世界上提取小RNA(<200nt)操作步骤最简单、重复性最好、小RNA产率最高的方法。
使用该试剂盒提取的小RNA (Small RNA)中,长度在15~200nt范围的RNA在95%以上,基本不含有大RNA和DNA。
使用该试剂无需酚氯仿、过柱、去DNA等复杂的步骤,可在2小时内完成小RNA的提取。
该试剂盒广泛适用于动物组织、细胞、全血和多糖多酚含量不高的植物组织样本。
组分:储存:室温可保存1年,密封状态下4℃避光可放置2年。
使用防护建议:miRNA Reagent A/B溶液中含有胍盐,其具有强烈的腐蚀性,试验时请务必佩戴防护眼镜、手套、口罩等防护措施,如有皮肤接触请立即用大量清水冲洗,再另行就医。
样本使用量及小RNA产量自备试剂:异丙醇、乙醇、75%异丙醇操作方法:1. 组织样本提取(1) 向1.5ml EP管中加入300μl miRNA Reagent A。
植物组织则再加入10μlβ-巯基乙醇,混合均匀。
(2) 在液氮条件下充分将组织研磨粉碎,将一定的样品量(见样本使用量表)研磨成粉状的组织加入到上述300μl miRNA Reagent A中,立即手腕用力震荡至组织粉末彻底溶解于裂解液中。
室温静置5min以充分裂解细胞。
注意:将组织加入到miRNA Reagent A后要迅速将组织震散,否则易引起组织成团状,不容易裂解。
如发生成团状,务必用移液器将团状组织吹打松散。
(3) 向上述裂解完毕的裂解液中加入350μl miRNA Reagent B,手腕用力震荡混合均匀,室温放置5min。
(4) 13,000rpm离心5min,吸取550μl上清液,转移到新的1.5ml EP管中。
miR-200a_对宫颈癌细胞系HeLa_增殖、迁移、侵袭调控作用观察及其靶向mRNA_和lncRN
miR -200a 对宫颈癌细胞系HeLa 增殖、迁移、侵袭调控作用观察及其靶向mRNA 和lncRNA 预测分析蔡添娥,陈丽婷,于英上海交通大学医学院附属上海儿童医学中心海南医院产前诊断中心,海南三亚572000摘要:目的 观察微小RNA -200a (miR -200a )对宫颈癌细胞系HeLa 增殖、迁移和侵袭的调控作用,对miR -200a 的靶向mRNA 及靶向长链非编码RNA (lncRNA )进行预测分析。
方法 取人宫颈癌细胞系HeLa 分为1、2、3及4组,分别转染miRNA 模拟物miR -200a mimics 、miRNA 抑制物miR -200a inhibitor 、对照物mimics NC 及inhibitor NC 。
培养48 h 时采用qRT -PCR 法检测各组细胞miR -200a 表达;采用CCK8法检测各组细胞增殖能力,采用Tran⁃swell 侵袭实验检测细胞侵袭能力,采用Transwell 迁移实验和细胞划痕实验检测各组细胞迁移能力。
使用miRNA 数据库在线预测miR -200a 靶向mRNA 和靶向lncRNA ,采用基因本体论功能注释和京都基因和基因组百科全书富集分析法分析宫颈组织中miR -200a 的靶向mRNA 和靶向lncRNA 的生物学功能。
结果 与3组比较,1组细胞miR -200a 相对表达量高;与4组比较,2组细胞miR -200a 相对表达量低(P 均<0.05)。
与3组比较,培养72、96 h 时1组细胞增殖能力低,培养48 h 时侵袭细胞数及细胞穿膜数少,细胞迁移率低(P 均<0.05);与4组比较,培养72、96 h 时2组细胞增殖能力高,培养48 h 时侵袭细胞数及细胞穿膜数多,细胞迁移率高(P 均<0.05)。
miR -200a 靶向mRNA 及lncRNA 分别有83、6个, miR -200a 靶向mRNA 功能主要有与转录调控、自噬体组装、神经元迁移和轴突导向等生物学过程,信号通路主要有Wnt 信号通路、Hippo 信号通路及内质网蛋白加工信号通路等。
非编码RNA来源的小肽:“微不足道”却“功能强大”
第 62 卷第 3 期2023 年 5 月Vol.62 No.3May 2023中山大学学报(自然科学版)(中英文)ACTA SCIENTIARUM NATURALIUM UNIVERSITATIS SUNYATSENI非编码RNA来源的小肽:“微不足道”却“功能强大”*陈晓彤,赵文龙,孙林玉,王文涛,陈月琴中山大学生命科学学院,广东广州 510275摘要:非编码RNA(ncRNA, non-coding RNA)长久以来被认为不具有编码能力。
近年来随着研究技术和生物信息学工具的迅速发展,研究发现在基因组的非编码区域上存在大量小开放阅读框(sORFs,small/short open read‐ing frames),其翻译产物被称作小ORF编码肽(SEPs,sORF encoded peptides)或小肽(micropeptides)。
部分小肽被证实在细胞内稳定存在并独立于其来源RNA发挥重要作用。
本文系统总结了非编码RNA来源小肽的鉴定方法、可编码小肽的RNA类型以及其研究困难和瓶颈,并重点回顾了疾病和植物中发现的功能小肽,以期对小肽的筛选鉴定提供思考,对小肽作为药物研发或者农作物增产的关键靶点提供新的思路和方向。
关键词:非编码RNA;小肽;非经典翻译;鉴定方法;调控机制中图分类号:Q71 文献标志码:A 文章编号:2097 - 0137(2023)03 - 0001 - 13 Micropeptides derived from non-coding RNAs: Tiny but powerful CHEN Xiaotong, ZHAO Wenlong, SUN Linyu, WANG Wentao, CHEN Yueqin School of Life Sciences, Sun Yat-sen University, Guangzhou 510275,ChinaAbstract:It was long presumed that non-coding RNAs (ncRNAs) are lacking in protein-coding poten‐tial. However, recent advances in technology and tools have led to an important finding that a number of small open reading frames (sORFs) were found in different kind of ncRNAs, and their translated products have been termed sORF encoded peptides (SEPs) or micropeptides. Some micropeptides have been confirmed to exist stably in cells and play important roles independently of their source RNA. In this review,we summarize the identification methods of micropeptides derived from ncRNAs,the types of RNA that can encode micropeptides,and focus on the functional micropeptides found in diseases and plants. The purpose of the review is to provide a thought on the screening and identifica‐tion of micropeptides, and provide new ideas for micropeptides as potentials for drug development or crop yield improvement.Key words: non-coding RNA; micropeptide; non-canonical translation; identification methods; regula‐tion mechanism随着人类基因组计划的完成以及ENCODE计划的开展,科学家发现,约75%的基因组可以产生转录本(Derrien et al.,2012;Djebali et al.,2012)。
一种微囊化大肠杆菌口服灭活疫苗的优化制备方法[发明专利]
专利名称:一种微囊化大肠杆菌口服灭活疫苗的优化制备方法专利类型:发明专利
发明人:姚刚,徐新龙,苏艳,黄嘉驷,雷成红,马雪莲,苏战强
申请号:CN201310285124.4
申请日:20130708
公开号:CN103417963A
公开日:
20131204
专利内容由知识产权出版社提供
摘要:本发明提供的一种微囊化大肠杆菌口服灭活疫苗的优化制备方法,在于改变传统大肠杆菌疫苗注射剂型,采用微囊包被技术制备一种新型口服制剂,使大肠杆菌疫苗的免疫接种途径更加简单方便,提供反刍动物用大肠杆菌口服疫苗。
采用微囊包被技术制备口服疫苗与注射疫苗产生同样的体液免疫;制得的产品颗粒饱满粒径均匀、平均粒直径为5.23±2.70μm,包埋率达到75.23%;以海藻酸钠作为包被壁材,制得的微胶囊颗粒,具有抵御胃酸的作用。
申请人:新疆农业大学
地址:830052 新疆维吾尔自治区乌鲁木齐市农大东路311号
国籍:CN
代理机构:乌鲁木齐新科联专利代理事务所(有限公司)
代理人:欧咏
更多信息请下载全文后查看。
小分子抑制剂的新应用[发明专利]
专利名称:小分子抑制剂的新应用专利类型:发明专利
发明人:董涛
申请号:CN202111423070.4
申请日:20211126
公开号:CN114159416A
公开日:
20220311
专利内容由知识产权出版社提供
摘要:本发明涉及小分子抑制剂的新应用。
主要涉及小分子抑制剂在制备抑制霍乱弧菌的药物中的应用,所述小分子抑制剂的化学结构式如式(I)或者式(II)所示:还涉及式(II)所示的小分子抑制剂在制备抑制铜绿假单胞菌、大肠杆菌和金黄色葡萄球菌的药物中的应用。
与传统技术相比,本发明具备如下有益效果:发明人筛选到针对霍乱弧菌有明显抑菌效果的化学结构式如式(I)和式(II)所示的小分子抑制剂,为霍乱弧菌的抑制提供了新的手段。
并且,式(II)所示小分子抑制剂还能杀灭其他多种病原细菌,包括铜绿假单胞菌、大肠杆菌和金黄色葡萄球菌。
申请人:南方科技大学
地址:518055 广东省深圳市南山区桃源街道学苑大道1088号
国籍:CN
代理机构:华进联合专利商标代理有限公司
代理人:王秉丽
更多信息请下载全文后查看。
miRNA-200b通过靶向RhoA抑制宫颈癌细胞增殖、促进细胞凋亡
miRNA-200b通过靶向RhoA抑制宫颈癌细胞增殖、促进细胞凋亡何丽杰陈会佳①王静常丹丹吕丹丹李海娜(天津市第五中心医院检验科,天津300450)中图分类号R737.33文献标志码A文章编号1000-484X(2021)13-1576-06[摘要]目的:探讨miRNA-200b通过靶向调控RhoA对宫颈癌细胞增殖和凋亡的影响。
方法:将宫颈癌HeLa细胞分为5组:空白对照组、阴性对照组、miRNA-200b mimic组、RhoA阴性对照组、RhoA过表达组,转染后48h收集细胞。
RT-PCR检测miRNA-200b表达;双荧光素酶靶标实验验证miRNA-200b与RhoA的靶向关系;RT-PCR和Western blot检测RhoA mRNA和蛋白表达;流式细胞术检测细胞凋亡,CCK8法检测细胞增殖。
RT-PCR和Western blot检测Bax、CyclinD1mRNA和蛋白表达。
结果:双荧光素酶报告基因实验分析结果显示,RhoA是miRNA-200b的靶基因;与空白对照组和miRNA-200b mimic-NC组相比,miRNA-200b mimic组细胞凋亡比例显著升高,细胞增殖被抑制(P<0.05)。
过表达RhoA,细胞凋亡比例降低,细胞增殖能力显著增强(P<0.05)。
结论:miRNA-200b可通过靶向RhoA基因抑制宫颈癌细胞增殖并促进其凋亡。
[关键词]miRNA-200b;RhoA;宫颈癌;增殖;凋亡miRNA-200b inhibits proliferation and promotes apoptosis of cervical cancer cells by targeting RhoAHE Li-Jie,CHEN Hui-Jia,WANG Jing,CHANG Dan-Dan,LYU Dan-Dan,LI Hai-Na.Clinical Laboratory,Tian⁃jin Fifth Central Hospital,Tianjin300450,China[Abstract]Objective:To investigate effect of miRNA-200b on proliferation and apoptosis of cervical cancer cells by targeting RhoA.Methods:HeLa cells of cervical cancer were divided into five groups:blank control group,negative control group,miRNA-200b mimic group,RhoA negative control group,and RhoA overexpression group.Cells were collected48h after transfection.Expres‐sion of miRNA-200b was detected by RT-PCR.Target relationship between miRNA-200b and RhoA was verified by double luciferase target experiment.RhoA mRNA and protein expressions were detected by Western blot and RT-PCR.Flow cytometry to detect apopto‐sis of cells,CCK8method to detect proliferation of cells.mRNA and protein expressions of Bax and CyclinD1were detected by RT-PCR and Western blot.Results:Double luciferase reporter gene assay results showed that RhoA was target gene of ‐pared with blank control group and miRNA-200b mimic-NC group,proportion of apoptotic cells was increased significantly in miRNA-200b mimic group,proliferation of cells was inhibited(P<0.05).Overexpression of RhoA,percentage of apoptotic cells was decreased and ability of cell proliferation was increased significantly(P<0.05).Conclusion:miRNA-200b can inhibit proliferation and promote apoptosis of cervical cancer cells by targeting RhoA gene.[Key words]miRNA-200b;RhoA;Cervical cancer;Proliferation;Apoptosis宫颈癌是世界范围内常见癌症,是严重威胁妇女生命的常见恶性肿瘤之一,女性患者死亡率较高[1]。
MicroRNA-200c、ZEB1及ZEB2在膀胱癌中的表达及意义的开题报告
MicroRNA-200c、ZEB1及ZEB2在膀胱癌中的表
达及意义的开题报告
背景:
膀胱癌是一种常见的泌尿系统恶性肿瘤,其发病率逐年上升,在临
床上具有很高的致死率和复发率。
已有多项研究表明,膀胱癌的发生、
发展和转移可能与分子机制有关,其中包括微小RNA和转录因子的调节。
微小RNA是一类长度约为20 nt的RNA分子,能够通过与mRNA靶标结合而抑制其翻译或降解,从而影响细胞的基因表达。
转录因子是调控基
因表达的重要因素,它们可以促进或抑制基因的转录。
在膀胱癌中,MicroRNA-200c、ZEB1和ZEB2可能会影响转录因子的表达,从而调节细胞的增殖、侵袭和迁移,但这些调节的具体分子机制还不明确。
研究目的:
本研究旨在探讨MicroRNA-200c、ZEB1和ZEB2在膀胱癌中的表达
及其对膀胱癌发生和发展的影响。
研究方法:
从膀胱癌组织和健康人组织中分别提取RNA,并进行RT-qPCR分析MicroRNA-200c、ZEB1和ZEB2的表达水平。
同时,通过Western blot
检测转录因子ZEB1和ZEB2的蛋白表达水平。
采用Transwell实验评估
细胞侵袭性和迁移性,并使用MTT实验评估细胞增殖能力。
最后,对研
究结果进行统计学分析。
预期结果:
本研究预计揭示MicroRNA-200c、ZEB1和ZEB2在膀胱癌中的表达
模式及其对膀胱癌发生和发展的影响。
研究结果可为膀胱癌治疗提供新
的靶向分子,并为探索膀胱癌转化过程提供新的理论支持。
microRNA-200c在高糖诱导腹膜间皮细胞转分化中的作用及分子机制的开题报告
microRNA-200c在高糖诱导腹膜间皮细胞转分化中的作用及分子机制的开题报告题目:microRNA-200c在高糖诱导腹膜间皮细胞转分化中的作用及分子机制研究背景和目的:腹膜间皮细胞是覆盖腹膜的一种特殊细胞,具有良好的吸收和分泌功能。
然而,高糖环境会引起腹膜间皮细胞转分化,从而导致吸收和分泌功能的受损。
因此,了解高糖环境下腹膜间皮细胞转分化的分子机制,对促进腹膜健康具有重要意义。
microRNA是非编码RNA的一种,具有调节基因表达的作用。
先前的研究表明,microRNA-200c参与了多种疾病的发生发展,包括肿瘤和代谢性疾病等。
然而,其在高糖环境下对腹膜间皮细胞转分化的作用及分子机制尚不明确。
本研究旨在探究microRNA-200c在高糖环境下对腹膜间皮细胞转分化的作用及其分子机制,为腹膜健康的促进提供新的理论依据。
研究方法:1.采用化学方法将小鼠腹膜间皮细胞诱导成转分化状态的细胞模型;2.通过qPCR和Western blot检测细胞转分化相关标志物的表达;3.利用miRDB、miRWalk和TargetScan等数据库预测microRNA-200c可能的靶点;4.构建microRNA-200c上调表达质粒并转染到转分化状态的细胞中;5.分析microRNA-200c上调对细胞转分化相关标志物的表达、细胞增殖、凋亡、迁移和侵袭等生物学特性的影响;6.利用Luciferase reporter assay验证microRNA-200c靶向调控的基因。
研究意义:本研究将探究microRNA-200c在高糖环境下对腹膜间皮细胞转分化的作用及分子机制,为深入认识腹膜间皮细胞转分化的分子生物学机制提供依据。
同时,该研究还将为探索潜在的治疗策略提供理论基础,从而防止或减缓糖尿病等代谢性疾病对腹膜的损害。
微小RNA-200c靶向抑制EFNA1基因对胃癌细胞增殖、凋亡及侵袭的影响
微小RNA-200c靶向抑制EFNA1基因对胃癌细胞增殖、凋亡及侵袭的影响李鹰飞;高健伟;王红;周永健;聂玉强【摘要】Objective To evaluate the effect of microRNA (miR)-200c on regulating cell proliferation,apoptosis and invasion by targeting ephrin A1 (EFNA1) in gastric cancer cells.Methods SGC7910 cells were cultivated and randomly divided into miR-200c mimics group and mimics control group,which were then transfected by miR-200c mimics and mimics oligonucleotides.Cell proliferation was detected by CCK-8,apoptosis was determined by flow cytomet~,and cell invaswn was assessed using Transwell assay.The target relationship between miR-200c and EFNA1 was measured by the dual luciferase reporter gene system.Cancer and adjacent non-cancerous tissues were obtained from 48 patients who underwent gastrectomy at Guangzhou First People's Hospital,and the protein expression of EFNA1 was tested by immunohistochemistry.The relationship between EFNA1 expression and gender,age,smoking status,alcohol drinking,histological type,depth of invasion,lymph node and liver metastasis was assessed.Results Compared with the mimics control group,the proliferative activity of SGC7910 cells in the miR-200c mimics group was decreased.the whole apoptosis rate was increased and the invasive ability was decreased (all P < 0.05).The luminescence intensity was significantly decreased in miR-200c mimic transfected cells than in mimics control cells (P < 0.05),and the luminescence intensity wassignificantly increased in miR-200c inhibitor transfected cells than in inlhibitor control cells (P < 0.05).EFNA1 was significantly up-regulated in the gastric cancer tissues as compared with that noted in the adjacent non-cancerous tissues.The high expression of EFNA1 was associated with lymph node metastasis (P < 0.05),bat not associated with other clinicopathological characteristics (all P > 0.05).Conclusion EFNA1 gene was highly expressed in the gastric cancer.miR-200c can inhibit gastric cancer cell proliferation and invasion,and enhance cell apoptosis by targeting EFNA1 gene.%目的探讨微小RNA (miR)-200c靶向抑制肝配蛋白A1(EFNA1)基因对胃癌细胞增殖、凋亡及侵袭的影响.方法选取胃癌细胞株SGC7901,随机分为miR-200c模拟物组、模拟物对照组、miR-200c抑制物组、抑制物对照组,分别转染miR-200c模拟物、模拟物对照寡核苷酸、miR-200c抑制物、抑制物对照寡核苷酸,采用CCK-8法检测细胞增殖,流式细胞术检测细胞凋亡,Transwell法检测细胞侵袭.采用双荧光素酶实验验证miR-200c对EFNA1基因的靶向抑制作用.取本院手术切除的胃癌和相应癌旁组织标本48例份,通过免疫组化法检测EFNA1蛋白表达,分析胃癌组织中EFNA1蛋白表达与患者性别、年龄、吸烟、饮酒、病理类型、浸润深度、淋巴结及远处转移、肿瘤部位间的关系.结果与模拟物对照组相比,miR-200c模拟物组胃癌细胞SGC7901增殖活性降低(P<0.05),总凋亡率升高(P<0.05),侵袭能力降低(P<0.05).miR-200c模拟物组的荧光素酶活性低于模拟物对照组(P<0.05),而miR-200c抑制物组的荧光素酶活性高于抑制物对照组(P<0.05).EFNA1蛋白在胃癌组织中表达高于癌旁组织,其高表达与胃癌淋巴结转移有关(P<0.05),与其他临床病理特征均不相关(P均>0.05).结论胃癌组织中EFNA1基因呈高表达,miR-200c可通过靶向EFNA1基因抑制胃癌细胞增殖及侵袭,促进凋亡.【期刊名称】《山东医药》【年(卷),期】2017(057)009【总页数】4页(P16-19)【关键词】胃癌;微小RNA-200c;肝配蛋白A1;细胞增殖;侵袭【作者】李鹰飞;高健伟;王红;周永健;聂玉强【作者单位】广州医科大学附属广州市第一人民医院,广州510180;广州医科大学附属广州市第一人民医院,广州510180;广州医科大学附属广州市第一人民医院,广州510180;广州医科大学附属广州市第一人民医院,广州510180;广州医科大学附属广州市第一人民医院,广州510180【正文语种】中文【中图分类】R735.2Effect of Micro-200c on cell proliferation, apoptosis and invasion of癌症研究机构近年数据显示,胃癌的发病率居全球恶性肿瘤的第5位,病死率高居第3位[1],而我国每年胃癌新发病例占世界新发病例40%以上。
MicroRNA对药物转运蛋白和代谢酶中的调控研究
MicroRNA对药物转运蛋白和代谢酶中的调控研究
海英民;陈丁丁;朱怀军;葛卫红
【期刊名称】《临床合理用药杂志》
【年(卷),期】2014(7)26
【摘要】微小RNA(miRNA)是一类内源性长18~25个核苷酸的非编码小RNA分子,在进化过程中具有高度保守性,在转录后水平调控基因的表达,从而在生物过程中发挥重要的作用。
生物信息学分析表明人类全部基因的1/3都受到miRNA的调控。
目前的研究表明,miRNA广泛调控机体各种生理和病理过程,可能是药物个体差异的原因,在将来可作为个体化给药分子标志物或指导新药开发。
【总页数】2页(P172-173)
【关键词】MiRNA;药物转运蛋白;药物代谢酶
【作者】海英民;陈丁丁;朱怀军;葛卫红
【作者单位】中国药科大学;南京大学医学院附属鼓楼医院
【正文语种】中文
【中图分类】R968
【相关文献】
1.中药调控药物代谢酶和转运体活性及表达研究进展 [J], 韩向晖;马越鸣
2.虚寒状态下药物代谢酶CYP3A和转运蛋白P-gp变化的体内外实验研究 [J], 薛春苗;张冰;李连珍;林志健
3.MicroRNA 对药物转运蛋白和代谢酶调控的研究进展 [J], 郑志伟;劳海燕;余细
勇;钟诗龙
4.孕烷X受体介导的代谢酶和转运体转录调控及其在化疗药物耐药中的作用 [J], 赵其锦;査伟斌;周芳;吴晓兰;曹蓓;张经纬;王广基
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微小RNA(microRNA)与雄性生殖
微小RNA(microRNA)与雄性生殖鲍坚强;徐晨【期刊名称】《生殖与避孕》【年(卷),期】2008(028)009【摘要】作为非编码RNA家族的一类重要成员,目前越来越多的证据显示microRNA(miRNA)分子广泛参与细胞增殖、分化、凋亡、信号转导、器官形成、脂肪代谢、造血、胚胎发育、病毒复制,甚至肿瘤发生等多种细胞生命活动进程的调控.对人与鼠睾丸组织的小RNA克隆文库测序发现了大量表达的miRNA分子,研究显示这些睾丸高丰度或特异表达的miRNAs可能发挥极其重要的对生殖细胞性别决定、自我更新、精原细胞有丝分裂增殖、精母细胞减数分裂以及圆形精子细胞变态等过程的调控作用.【总页数】4页(P554-557)【作者】鲍坚强;徐晨【作者单位】上海交通大学医学院组织胚胎学教研室,上海市生殖医学重点实验室,上海,200025;上海交通大学医学院组织胚胎学教研室,上海市生殖医学重点实验室,上海,200025【正文语种】中文【中图分类】R321.1【相关文献】1.天牛科雄性外生殖器的新类型:—狭天牛属雄性外生殖器的研究 [J], 吴蔚文;蒋书楠2.猪龙根三部曲──生殖:赵宝沟文化雄性野猪龙的浪漫主义神话礼治:伏羲氏“龙师”是红山文化的特定产物崇祖:殷商甲骨文的“龙”字都是雄性野猪龙 [J], 布谷3.雄性小鼠肾虚对其雄性仔鼠血清生殖激素含量的影响 [J], 孙洁;周安方;周艳艳;方婷4.雌激素受体在雄性生殖系统中的分布及其对雄性生殖的作用 [J], 蒋春霞;潘连军;黄宇烽5.渔游蛇(Natrix piscator)的雄性生殖周期及其肾性节的生殖生理学研究:Ⅰ.雄性生殖周期及肾性节的组织学和组织化学 [J], 张宏;吴美锡因版权原因,仅展示原文概要,查看原文内容请购买。
microRNA 在预防医学中的研究进展
microRNA 在预防医学中的研究进展
张迎建;李文超;杨红
【期刊名称】《东南大学学报(医学版)》
【年(卷),期】2013(000)005
【摘要】微小RNA( microRNA,miRNA)是一类由内源基因编码的长度约为22个核苷酸的非编码单链RNA分子,它们在动植物中参与转录后基因表达调控。
作者对miRNA在职业毒理学、环境毒理学、食品毒理学、放射毒理学和卫生学方面的国内外研究进展作一综述。
【总页数】6页(P606-611)
【作者】张迎建;李文超;杨红
【作者单位】东南大学公共卫生学院环境医学工程教育部重点实验室,江苏南京210009;东南大学公共卫生学院环境医学工程教育部重点实验室,江苏南京210009;东南大学公共卫生学院环境医学工程教育部重点实验室,江苏南京210009
【正文语种】中文
【中图分类】R114
【相关文献】
1.外泌体中的microRNAs在神经胶质瘤中的研究进展 [J], 徐峰;牛万祥;谢时帅;牛朝诗
2.尿液中microRNA在膀胱癌诊断中的研究进展 [J], 吴海超;陈振杰;丁明霞
3.胃液中microRNA检测在胃癌筛查及诊断中的研究进展 [J], 王君辅;谢勇;胡林;李昌荣;李红浪
4.Krüppel样因子相关microRNAs在肺癌中调控作用的研究进展 [J], 龚海英;何芳;黄语嫣;庞敏;姜永杰(综述);蒋莉;邓太兵(审校)
5.microRNA在心搏骤停后脑损伤中的研究进展 [J], 王静(综述);谢吐秀(审校);吕菁君(审校)
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miRNA-152和miRNA-200s在IL-6诱导的肝胰岛素抵抗中的作用
miRNA-152和miRNA-200s在IL-6诱导的肝胰岛素抵抗中的作用窦琳;黎健【期刊名称】《中国动脉硬化杂志》【年(卷),期】2013(21)9【摘要】目的胰岛素抵抗是2型糖尿病的主要发病机制之一。
microRNA (miRNA)是-簇长度约为20—24个核苷酸的单链非编码的小分子RNA。
它们在转录后水平对下游靶基因进行调控。
miRNA广泛表达于各种组织器官中,对组织器官的功能和发育具有重要的作用。
miRNA参与糖尿病及其并发症的发生。
它们调控胰岛素的合成、分泌、胰岛素信号通路和糖脂代谢等过程。
目前,与肝胰岛素抵抗相关的miRNA报道尚少。
研究证明miRNA.152和miRNA-200s与肿瘤的发生密切相关,但miR—NA-152和miRNA-200s是否在肝胰岛素抵抗发生中起重要作用?目前尚不清楚。
本研究通过建立IL-6诱导的胰岛素抵抗的细胞和动物模型,分析细胞或肝脏组织中miRNA-152和miRNA-200s的表达变化,探讨miRNA-152和miRNA-200s在IL-6诱导的肝胰岛素抵抗中的作用,明确miRNA-152和miRNA.200s调节P13K/AKT信号通路的分子机制。
方法(1)以12周龄db/db小鼠作为胰岛素抵抗模型小鼠,收集小鼠肝脏组织,用miRNA芯片分析肝脏中miRNA表达谱变化,并用Real—timePCR进一步验证芯片结果。
(2)10斗∥LIL-6处理小鼠肝细胞系NCTCl469和C57BL/6小鼠原代肝细胞24h,建立胰岛素抵抗的细胞模型,检测P13K/AKT信号和miRNA.152、miRNA-200s表达水平;在c57BL/6J小鼠背部皮下埋泵缓释注射IL-61周,建立IL-6诱导的胰岛素抵抗动物模型,观察肝脏中P13K/AKT信号通路和miRNA-152、miRNA-200s表达水平。
(3)合成miRNA-152、miRNA-200smimics和inhibitors,并转染入NCTCl469细胞,用Real—timePCR测定细胞中miRNA-152和miRNA.200S的水平,并且检测P13K/AKT信号通路和糖原水平。
MicroRNA在葡萄膜炎发病过程中的调控作用研究进展
MicroRNA在葡萄膜炎发病过程中的调控作用研究进展孙园园;郭大东;毕宏生【期刊名称】《眼科新进展》【年(卷),期】2016(036)011【摘要】微小RNA(microRNA,miRNA)是生物体内源性的、约19 ~ 24个核苷酸构成的非编码单链RNA分子.miRNA可以与靶标mRNA基因的3'端非编码区域(3'UTR)互补结合,从而在转录后水平负调控靶基因的表达.miRNA通过降解靶基因或者抑制转录后的翻译水平,进而影响细胞的分化、增殖和凋亡,并在生物生长发育和疾病发生发展过程中发挥重要的调节作用.近年来研究表明,miRNA在葡萄膜炎的发生发展进程中同样发挥重要的调控作用.本文就miRNA在葡萄膜炎发生发展过程中的调控作用作一综述,为深入研究葡萄膜炎的发病机理提供新思路.【总页数】4页(P1082-1085)【作者】孙园园;郭大东;毕宏生【作者单位】250014 山东省济南市,山东中医药大学;250002 山东省济南市,山东中医药大学眼科研究所;250002 山东省济南市,山东中医药大学眼科研究所【正文语种】中文【中图分类】R774.1【相关文献】1.rno-miR-30b-5p在葡萄膜炎发病过程中对白细胞介素-10和Toll样受体4表达的调控作用 [J], 孙园园;郭大东;陈美清;李少玉;刘滨;唐凯;毕宏生2.MicroRNA在非酒精性脂肪性肝病发病过程中作用的研究进展 [J], 何占娣;刘迎娣3.非编码RNA在葡萄膜炎发生发展过程中的调控作用研究进展 [J], 魏慧霞; 殷学伟; 毕宏生; 郭大东4.脑出血后继发性神经损伤发生过程中microRNAs调控作用机制的研究进展 [J], 曹洪涛;邹伟;于学平5.microRNAs调控MKP-1在抑郁症发病机制中的研究进展 [J], 赵霞;王慧颖;冯来鹏;谷景阳;刘聪;耿梦军;王长虹因版权原因,仅展示原文概要,查看原文内容请购买。
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ORIGINAL ARTICLE–TRANSLATIONAL RESEARCH AND BIOMARKERSMicroRNA-200b Regulates Cell Proliferation,Invasion,and Migration by Directly Targeting ZEB2in Gastric CarcinomaJunji Kurashige,MD,Hidenobu Kamohara,MD,PhD,Masayuki Watanabe,MD,PhD,Yukiharu Hiyoshi,MD,PhD,Masaaki Iwatsuki,MD,PhD,Youhei Tanaka,MD,Koichi Kinoshita,MD,Seiya Saito,MD,Yoshifumi Baba,MD,and Hideo Baba,MD,PhD,FACS Department of Gastroenterological Surgery,Graduate School of Medical Sciences,Kumamoto University,Kumamoto, JapanABSTRACTBackground.The microRNA-200(miR-200)family has been reported to induce epithelial differentiation and sup-press epithelial–mesenchymal transition(EMT)by inhibiting translation of zincfinger E-box-binding homeobox(ZEB)1 and2mRNAs in several types of cancers.This study aimed to clarify the role of miR-200b in regulating EMT and promoting cellular proliferation,invasion,and migration in gastric cancer.Methods.The relationships among the expression levels of miR-200b,ZEB1and ZEB2,and E-cadherin mRNAs were analyzed by quantitative real-time reverse transcrip-tion–polymerase chain reaction in frozen tissue samples from40gastric cancer patients who underwent gastrec-tomy from2008to2010.The effects of miR-200b on EMT in gastric cancer cells in vitro were also analyzed. Results.Diffuse histologic type,depth of tumor,tumor size,lymph node metastasis,and lymphatic invasion were significantly higher in the low-miR-200b expression group compared with the high expression group.There was a strong correlation between the levels of miR-200b,and ZEB2and E-cadherin mRNAs in gastric cancer patients. Upregulation of miR-200b in gastric cancer cells changed the cell morphology fromfibroblast-to epithelial-like, associated with localization of E-cadherin to the plasma membrane.ZEB2mRNA levels fell,while E-cadherin expression levels increased in gastric cells overexpressing miR-200b,associated with significantly reduced cellular proliferation,and inhibition of cellular migration and invasion.Conclusions.miR-200b regulates ZEB2expression and thus controls metastasis in gastric cancer.Gastric cancer is one of the most common malignancies worldwide.1Improvements in its diagnosis and treatment have resulted in excellent long-term survival in patients with early gastric cancer,although the prognosis of patients with advanced disease still remains poor.Gastric cancers can be divided into two major histologic types with dif-ferent biologic behaviors:intestinal and diffuse type.2 However,details of the mechanisms underlying gastric cancer progression remain unclear.Gastric cancer cells withfibroblastic morphologic changes show increased migration and invasiveness as a result of decreased cell–cell adhesion.3These changes are reminiscent of the epithelial–mesenchymal transition(EMT)that occurs during embryonic development.4During EMT,epithelial cell–cell adhesion is decreased through the downregulation of adhesion molecules such as E-cadherin,and the cells then acquire a spindle-shaped,highly motilefibroblast phenotype.5 EMT plays a key role in invasion and metastasis during car-cinogenesis,as well as in gastrulation and neurulation during embryogenesis.Several mechanisms for E-cadherin tran-scriptional silencing during EMT have been proposed, including direct inhibition by transcriptional repressors such as Snail,Twist,and zincfinger E-box-binding homeobox1 and2(ZEB1and ZEB2).6–9Among these molecules,ZEB1 and ZEB2bind to paired ZEB-type E boxes within target gene promoters,repressing their transcription.10,11 Electronic supplementary material The online version of thisarticle(doi:10.1245/s10434-012-2217-6)contains supplementarymaterial,which is available to authorized users.ÓSociety of Surgical Oncology2012First Received:26April2011H.Baba,MD,PhD,FACSe-mail:hdobaba@kumamoto-u.ac.jpAnn Surg OncolDOI10.1245/s10434-012-2217-6MicroRNAs(miRs)are small noncoding RNAs that mediate posttranscriptional repression through sequence-spe-cific binding to their target mRNAs,resulting in translational inhibition or,in some cases,destruction of the targets.12Sev-eral recent studies have shown that the miR-200family(miR-141,miR-200a,miR-200b,miR-200c,miR-429)inhibits translation of ZEB1and ZEB2mRNAs,thus inducing epi-thelial differentiation and suppressing EMT in several types of cancers.13–16However,to our knowledge,no studies have determined the role of the miR-200family in the regulation of EMT in gastric cancer.The present study therefore aimed to clarify the involvement of miR-200b in EMT in gastric cancer.MATERIALS AND METHODSPatients and Tissue SamplesPrimary gastric carcinoma tissue and matched normal gastric epithelium were obtained from40patients who underwent gastric resection without preoperative treatment at the Department of Gastroenterological Surgery,Kumamoto University Hospital,from2008to2010,after receiving ade-quate informed consent.All tissue samples were immediately frozen in liquid nitrogen and stored at-80°C until the extraction of RNA.Clinicopathologic information,including age,gender,pathology,differentiation,and tumor–node–metastasis classification,was available for all patients.The study was approved by the medical ethics committee of Kumamoto University.Cell Culture and TreatmentWe selected the human gastric cancer cell lines NUGC3 and AGS because they both show lower expression of miR-200b than other cell lines(MKN1,MKN7,MKN45,MKN74, NUGC4).These cell lines were provided by the Cell Resource Center for Biomedical Research Institute of Development, Aging,and Cancer,Tohoku University,Japan.All cells were grown in RPMI1640(Cambrex Bioscience,Walkersville, MD)supplemented with10%fetal bovine serum(Sigma-Aldrich,St.Louis,MO),and incubated at37°C in a humidified chamber supplemented with5%CO2.RNA IsolationTotal RNA,including miRNA,was isolated from tissue samples and cell lines with a mirVana miRNA Isolation Kit (Ambion,Austin,TX),andfinally eluted into100l l of heated elution solution,according to the manufacturer’s protocol.The purity and concentration of all RNA samples were quantified with a NanoDrop ND-1000spectropho-tometer(NanoDrop Technologies,Rockland,DE).Quantitative Real-time Reverse Transcription–Polymerase Chain ReactionThe expression levels of miR-200b were determined by TaqMan quantitative real-time reverse transcription–poly-merase chain reaction(qRT-PCR)with TaqMan microRNA assay kits(Ambion)according to the manufacturer’s protocols, as described previously.17miR-200b expression was normal-ized to that of RNU6B,small nuclear RNA expression.The expression levels of E-cadherin and ZEB1and ZEB2were quantified by SYBR Green qRT-PCR with a LightCycler 480SYBR Green I Master(Roche Diagnostics,Mannheim, Germany)and normalized to glyceraldehyde-3-phosphate dehydrogenase.All qRT-PCR reactions were run with the LightCycler480System II(Roche Diagnostics).The relative amounts of miR-200b,E-cadherin,and ZEB1and ZEB2were measured with the2-DD CT method.All qRT-PCR reactions were performed in triplicate.Western Blot AnalysisTo isolate the proteins,cells collected from6-well plates were washed once in phosphate-buffered saline and lysed in lysis buffer[Tris–HCl(pH7.4)25mmol/l,NaCl100mmol/l, EDTA2mmol/l,Triton X-1001%,with10l g/ml aprotinin, 10l g/ml leupeptin,and1mmol/l Na3VO4,1mmol/l phe-nylmethylsulfonylfluoride].Each protein sample(15l g)was resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis,transferred onto a polyvinylidene difluoride membrane,and incubated with a monoclonal antibody against E-cadherin(1:200,BD Bioscience,San Jose,CA)or b-actin (1:2000;Sigma-Aldrich).The signals were detected by incubation with secondary antibodies labeled with the ECL Detection System(GE Healthcare,Little Chalfont,UK). Transfection of miRNACells were transfected with20nM pre-miR miRNA precursor molecule pre-200b(pre-miR-200b)(Applied Bio-systems,Foster City,CA)with Lipofectamine2000 transfection reagent(Invitrogen,Carlsbad,CA),according to the manufacturer’s instructions.The specificity of the trans-fection was verified with pre-miR miRNA precursor molecule negative control#1(pre-miR-n.c.)(Applied Biosystems).The expression levels of miR-200b were quantified48h after transfection,and the cells were subsequently used for cell invasion and wound-healing assays,and for Western blot and immunofluorescence analyses.Immunofluorescence Microscopy and Stainingfor E-cadherinNUGC3cells in6-well plates were transfected with pre-miR-200b.The medium was aspirated48h afterJ.Kurashige et al.transfection,and the cells werefixed in4%paraformal-dehyde.Nonspecific protein binding sites were blocked with3%bovine serum albumin(Sigma,Tokyo,Japan). Sections were permeabilized in0.1%Triton X-100and probed with mouse–anti-E-cadherin(1:50;BD Bioscience) for1h at room temperature.The primary antibody was detected with rabbit–anti-mouse IgG labeled with Hilyte Fluor555(1:50;AnaSpec,CA,USA)for1h at room temperature.Nuclei were detected by costaining with 40-6-diamidino-2-phenylindole(Invitrogen,Carlsbad,CA). Cells were observed and photographed with a FV300fluorescence microscope(Olympus,Tokyo,Japan).Images were analyzed by Olympus Cell software.Luciferase Reporter AssayNUGC3cells in96-well plates were transfected with Lipofectamine2000(Invitrogen)with the LightSwitch ZEB230untranslated region(UTR)reporter GoClone plasmid(SwitchGear Genomics,Menlo Park CA)or empty vector(SwitchGear Genomics,Menlo Park CA),and pre-miR-n.c.or pre-miR-200b(Applied Biosystems).Reporter assays were performed at24h after transfection with LightSwitch Luciferase Assay reagents(SwitchGear Genomics).All transfection experiments were conducted in triplicate.Cell Proliferation,Wound Healing,and Invasion AssaysCell proliferation assays were carried out in96-well plates using the WST-8assay with a Cell Counting Kit-8 (Dojin Laboratories,Japan)at24,48,72,and96h after transfection,as previously described.17Absorbance was measured at450nm.Cell invasion was assessed by using Matrigel Invasion Chambers(BD Biosciences),in triplicate.At48h after transfection,Cells were plated in transwell chambers pre-coated with Matrigel Invasion Chamber medium,and containing5%fetal bovine serum in the lower chamber as the chemoattractant.After incubation for22h at37°C in a humidified incubator with5%CO2,the noninvading cells were removed with cotton swabs.The invasive cells that attached to the lower surface of the membrane insert were fixed in100%methanol at room temperature for2min and stained with toluidine blue.The number of invasive cells on the lower surface of the membrane was counted under a microscope.Wound-healing assays were performed with AGS cells at48h after transfection with20nM pre-miR-200b or pre-miR-n.c.The cells were trypsinized and plated into6-well plates at high densities.When the cells had reached about 90%confluence(the next day),a wound was made through the cells with a1000-l l micropipette tip.Photographs were taken immediately and after24h.The area of migrating cancer cells was measured by ImageJ software. Statistical AnalysisAll experiments were repeated at least three times. Continuous variables were expressed as the mean±stan-dard deviation.The results of PCR,and of the proliferation, invasion,and wound-healing assays were analyzed by Student’s paired t-tests.The relationship between the expression of miR-200b and clinicopathologic factors was analyzed by v2tests.The associations between the expression levels of miR-200b,ZEB1and ZEB2,and E-cadherin mRNAs were analyzed by Pearson correlation coefficients.Thefindings were considered significant at a P value of\0.05.All statistical analyses were performed by SPSS software,version13.0(SPSS,Chicago,IL). RESULTSClinicopathologic Significance of miR-200b in Gastric Cancer PatientsThe miR-200family includesfive miRNAs located on chromosome1(miR-200a,miR-200b,and miR-429)and chromosome12(miR-200c and miR-141).miR-200b and miR-200c were chosen for the current study because each represents a distinct subclass,according to seed sequence and genomic location.There was no significant difference in miR-200b expression levels between gastric cancer tis-sue and normal mucosa(Fig.1a).However,there were differences in the tumor/normal tissue(T/N)miR-200b ratios of among the cases,and we therefore compared clinicopathologic parameters between the groups,divided according to T/N ratio of miR-200b.The high-expression group was defined as cases with a T/N ratio above the median value,while the remainders were included into the low-expression group.As shown in Table1,diffuse his-tologic type,tumor invasion beyond the muscularis propria,positive lymph node metastasis,positive lymphatic invasion,and progressive stage were all associated with low expression of miR-200b.The current study focused on histologic type because diffuse-type histology is frequently associated with EMT-like morphology.As shown in Fig.1b,diffuse-type tumors exhibited significantly lower miR-200b T/N ratios than intestinal-type tumors.However, there was no significant difference in levels of miR-200c expression between gastric cancer tissue and normal mucosa,and no significant difference in miR-200c T/N ratios between diffuse and intestinal-type tumors(data not shown).Function of miR-200b in Gastric CancerCorrelations among miR-200b,E-cadherin,and ZEB1and ZEB2Expression Levels in Gastric Cancer PatientsThe expression levels of miR-200b,E -cadherin,and ZEB1and ZEB2were analyzed in clinical samples from 40gastric cancer patients via RT-PCR.There was no signifi-cant correlation between miR-200b and ZEB1(Fig.2a),but significant correlations were observed between miR200b and both ZEB2(Fig.2b,P \0.05,r =-0.32)and E -cadherin (Fig.2c,P \0.05,r =0.38).There was also a significant correlation between ZEB2and E -cadherin mRNA levels (Fig.2d,P \0.05,r =-0.37).Upregulation of miR-200b Changed the Morphology in Gastric Cancer Cell LinemiR-200b expression levels in two gastric cancer cell lines known to express low levels of miR-200b were upregulated by transfection with miR-200b precursor.miR-200b upregulation in NUGC3cells was associated with a change from a fibroblast-like to a more epithelial-like morphology,and with increased cell–cell adhesion (Supplemental Fig.S1).miR-200b Inhibits ZEB2Expression and Enhances E-cadherin Expression and miR-200b Directly Binds the 30UTR of ZEB2Total RNA was isolated from cells collected 48h after transfection,and the expression levels of miR-200b andtarget mRNAs were investigated.NUGC3and AGS cells transfected with pre-miR-200b showed a 19-fold and 25-fold increase in miR-200-b expression,compared with the pre-miR-n.c.(Fig.S2).ZEB2mRNA was significantly decreased and E -cadherin mRNA was significantly increased in NUGC3and AGS cells transfected with pre-miR-200b,compared to the pre-miR-n.c.(Fig.3a,b).E-cadherin protein expression demonstrated by Western blot testing was upregulated in NUGC3cells transfected with pre-miR-200b (Fig.3c).Immunofluorescence staining with anti-E-cadherin antibody also revealed that E-cad-herin was overexpressed in cells transfected with pre-miR-200b and was localized to the plasma membrane.TheTABLE 1miR-200b expression and clinicopathologic factors FactorHigh expression Low expression P (n =20)(n =20)Age (mean ±SD)75.2±9.174.5±6.20.967Sex 0.107Male1814Female 26Histologic grade 0.004*Intestinal type 156Diffuse type514Size0.112\50mm (small)138C 50mm (large)712Depth of tumor invasion0.024*m,sm,mp 114s,se,si916Lymph node metastasis 0.010*Absent 146Present 614Liver metastasis 1Absent 1919Present11Peritoneal dissemination 0.282Absent1917Present13Lymphatic invasion 0.025*Absent 147Present 613Venous invasion 0.073Absent 83Present 1217Stage 0.026*I,II 158III,IV512m Tumor invasion of mucosa,sm submucosa,mp muscularis propria,s subserosa,se penetration of serosa,and si invasion *P \0.05J.Kurashige et al.staining pattern was typical of epithelial cells(Fig.3d).By means of a luciferase reporter assay in NUGC3cells,we found that the luminescence intensity was significantly decreased in ZEB230UTR reporter and pre-miR-200b transfected cells(Fig.3e;P\0.001)than others,sug-gesting that miR-200b has target sites in the30UTR of ZEB2mRNA.miR-200b Suppresses Cell Growth,Migration,and InvasionThe effects of miR-200b upregulation on cell growth, migration,and invasion were determined via several assays.Transfection of pre-miR-200b significantly reduced cell proliferation in both NUGC3and AGS cell lines, compared to control transfections(Fig.4a).The effects of miR-200b on cell migration were determined with scratch wound-healing assays.Both NUGC3and AGS cells treated with pre-miR-200b were distinctively less migratory than negative control cells(Fig.4b).AGS cells were used in invasion assays because parental AGS cells demon-strated significant invasion into Matrigel membranes.Upregulation of miR-200b in AGS cells significantly inhibited cell invasion(Fig.4c).These results indicate that upregulation of miR-200b suppressed cell growth and inhibited both the migration and invasion of gastric cancer cells in vitro.DISCUSSIONThe results of the present study provide what is to our knowledge thefirst evidence for a negative association between miR-200b expression and EMT in gastric cancer. miR-200b expression was significantly related to histologic type,as well as to other clinicopathologic parameters. Tumors with low expression of miR-200b frequently demonstrate diffuse-type morphology,suggestive of EMT, and we therefore investigated the expression levels of ZEB1and ZEB2and E-cadherin in these samples.There was a significant inverse correlation between miR-200b and ZEB2expression,and a significant positive relation-ship between miR-200b and E-cadherin expression. Upregulation of miR-200b also reduced the expression of ZEB2and increased the expression of E-cadherin inthe Function of miR-200b in Gastric Cancerplasma membrane.Increased expression of pre-miR-200b in gastric cancer cells was associated with a change in their morphology to a more epithelial-like one,and with inhi-bition of cellular invasion,migration,and proliferation.The miR-200family has recently been shown to regulate EMT and cancer cell migration by targeting ZEB1and ZEB2,which act as a transcriptional repressor of E-cad-herin.13,14,16Deregulation of the miR-200family has been observed in several types of cancers;it is downregulated in some types of tumors and upregulated in others.16,18–22Thesefindings suggest that the miR-200family can act as either an oncogene or as a tumor-suppressor gene.We have demonstrated that miR-200b,but not other members of the miR-200family(miR-141,miR-200a,miR-200c,miR-429),affect EMT through targeting ZEB1and ZEB2. Several studies have reported associations between the expression levels of various metastasis-related proteins, such as Snail,Twist,ZEB1and ZEB2,vimentin,and E-cadherin,and tumor metastasis and poor prognosis in gastric cancer.23–27ZEB2,which is also called SIP1E-cadherin-actin pre-miR-n.c.non-transfectedDAPI E-cadherin Mergedpre-miR-2bJ.Kurashige et al.Function of miR-200b in Gastric CancerNUGC3pre-miR-n.c.pre-miR-200bAGSpre-miR-n.c.pre-miR-200bcpre-miR-n.c.pre-miR-200b(Smad-interacting protein1),is a transcriptional repressor of E-cadherin.A previous report revealed that ZEB2was overexpressed,and that ZEB2and Slug may act synergis-tically in gastric tumors with intestinal-type histology.25,28 Additionally,E-cadherin tissue status may be a powerful aid in evaluating the metastatic potential or prognosis of patients with gastric cancer.29,30The results of the current study demonstrated that these molecules play an important role in EMT in gastric cancer cells under the control of miR-200b.Some recent reports have revealed roles for other miRs as promoters or sup-pressors of metastasis by means of a variety of approaches. For example,miR-126and miR-335were shown to act as metastasis suppressors in human breast cancer,while miR-9inhibited the expression of E-cadherin and regulated metastasis,also in breast cancer.31–33In contrast,miR-10b was identified as a prometastatic miR in breast cancer,and miR-10b antagomir appears to offer a promising new an-timetastatic strategy.34Our data suggest that miR-200b negatively regulates EMT and may thus reduce the risk of metastasis in gastric cancer.The miR-200family has also demonstrated a strong relationship with the functions of cancer stem cells.35,36Some reports have shown a corre-lation between the expression of the miR-200family and resistance to chemotherapy.37–39The restoration of miR-200c expression decreased the expression of class III b-tubulin and thus increased sensitivity to several antitu-mor agents.35The miR-200family may therefore have the therapeutic potential to increase chemosensitivity,as well as to reduce metastatic activity in gastric cancer.It is likely that further research into miR-200b may open up new opportunities for the development of drugs and therapeutic strategies for the treatment of gastric cancer. However,a limitation of this study was the fact that other miR-200family members,such as miR-200a,miR-141, and miR-429,have similar but not identical functions.In addition,the sample size in this study was small.Further studies are needed to investigate the correlation between miR-200b expression levels and patient survival.This study,however,provides what is to our knowledge thefirst report of miR-200b as an important regulator of EMT through inhibition of gastric cancer cellular growth, migration,and invasion via targeting the mRNAs of ZEB2. In addition,the results demonstrated correlations between the levels of miR-200b and ZEB2,and E-cadherin mRNA in gastric cancer patients.Restoration of miR-200b expression therefore represents a potential candidate for miRNA-based therapy against gastric cancer. 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