CN111996251B - 一种恶性胶质瘤生物标志物的应用 - Google Patents
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Abstract
一种恶性胶质瘤生物标志物的应用,如SEQ ID No:1所示的RHOJ mRNA和如SEQ ID No:2所示的RHOJ蛋白在作为检测靶标在制备诊断、治疗或预后判断恶性胶质瘤试剂盒中的应用。我们发现沉默RHOJ表达显著抑制胶质瘤细胞增殖和侵袭等恶性表型,RHOJ可作为恶性胶质瘤诊断与预后判断的分子标志物,靶向RHOJ的抗体或小分子抑制剂等治疗策略有望改善恶性胶质瘤治疗。
Description
技术领域
本发明属于生物技术,病理检验和肿瘤学领域,具体涉及一种恶性胶质瘤生物标志物的应用。
背景技术
胶质母细胞瘤(GBM)是中枢神经***肿瘤中最常见的致命性颅内原发恶性肿瘤,胶质瘤的发病率为5/10万~8/10万,5年病死率在全身肿瘤中仅次于胰腺癌和肺癌,位列第三。其主要特征是丰富血管生成和弥漫性浸润生长,难以病理学上全切除,术后易复发且对于放化疗抗拒。脑胶质瘤可按肿瘤细胞在病理学上的恶性程度分为:Ⅰ~Ⅳ级。低级别胶质瘤(LGG) 主要是WHO II和III级胶质瘤,而高级别胶质瘤(GBM)主要是WHO IV级胶质瘤。随着分子生物学和分子病理诊断的发展,脑胶质瘤的分子分型越来越重要,比如IDH突变,MGMT 启动子甲基化,1p/19q共缺失,TERT启动子突变,EGFR扩增和EGFRvⅢ重排和ATRX突变等。高度血管化和侵袭性生长导致GBM预后差,因此,锁定介导GBM血管生成和侵袭关键分子对于开发有效的分子标志物和治疗靶点具有重要临床意义。
近年研究发现GBM血管生成和内皮屏障完整性与侵袭转移密切相关,涉及多种病理生理状态如细胞骨架及细胞间连接调控,而Rho-GTP酶在其中起着核心作用。Rho-GTP酶是GBM侵袭调控中介导受体起始信号的关键信号元件,可调节细胞形态和肌动蛋白空间结构的动态变化,并刺激通过神经元的狭窄细胞外空间挤压细胞。Rho-GTP酶是小G蛋白的一个家族成员,也是Ras超家族的一部分,它能够调节肌动蛋白,参与细胞骨架重排、囊泡运输和细胞黏附等生理过程。在哺乳动物中目前已经发现20种Rho-GTP酶,被分为8个亚家族。Rho-GTP酶在血管内皮屏障的完整性中起着核心作用,Rho-GTP酶及其调节剂是细胞骨架与细胞间连接的重要调节因子。Rho-GTP酶家族分子对血管内皮屏障的完整性具有双重影响。一方面Rho-GTP酶参与炎性因子诱导的血管内皮稳定性丧失的过程,另一方面参与维持血管内皮屏障稳定性,并且在受损后诱导血管内皮完整性的修复。Rho-GTP酶的双重作用与其亚细胞定位密切相关。
RHOJ是CDC42的同系物,被认为是细胞形态、肌动蛋白细胞骨架、细胞运动、细胞周期的关键调控因子。其高表达于内皮细胞,对于内皮细胞的迁移和血管生成有着重要的调节作用。以往研究显示RHOJ在黑色素瘤中高度表达,并通过其下游的PAK-BAD信号通路调控肿瘤的转移和进展,RHOJ也通过抑制DNA损伤的途径调控黑色素瘤的耐药性。RHOJ 作为一种富含于内皮细胞的小G蛋白,在肿瘤血管生成、肺癌转移和自发乳腺癌模型中发挥了重要作用。RHOJ高表达是胃癌生存结果的独立阴性预后因素,并与胃癌进展和转移有关。然而,RHOJ在恶性胶质瘤中的表达,诊断、治疗和预后判断中的应用目前并无相关报道。
发明内容
解决的技术问题:本发明提供一种恶性胶质瘤生物标志物的应用,明确了RHOJ在调控胶质瘤细胞与肿瘤和正常血管内皮细胞中的作用差异及其调控机制;阐明RHOJ调控胶质瘤增殖、凋亡、侵袭转移和血管生成等恶性表型的作用及其分子机制;如何靶向RHOJ或其结合的关键靶分子并阐明其介导GBM恶性表型的机制及信号通路。制备了用于检测RHOJ 表达的抗体和试剂盒的特异性,基于RHOJ蛋白与晶体结构结合电脑预测的小分子化合物设计合成,以及通过噬菌体展示技术等抗体筛选方法筛选靶向RHOJ的治疗性抗体。
技术方案:如SEQ ID No:1所示的RHOJ mRNA作为检测靶标在制备诊断、治疗或预后判断恶性胶质瘤试剂盒中的应用。
如SEQ ID No:2所示的RHOJ蛋白作为检测靶标在制备诊断、治疗或预后判断恶性胶质瘤试剂盒中的应用。
抑制RHOJ蛋白表达的化合物在制备治疗恶性胶质瘤产品中的应用。
有益效果:本发明通过二代测序,基因芯片,实时荧光定量RT-PCR和免疫组化对恶性胶质瘤组织中Rho-GTP酶家族分子表达分析,发现RHOJ在恶性胶质瘤中过表达并与病人预后差相关,锁定其对恶性胶质瘤诊断与预后判断的重要价值。沉默RHOJ表达抑制GBM 细胞增殖,并诱导细胞周期G2/M期阻滞,稳定敲低RHOJ显著抑制GBM细胞侵袭,迁移与致瘤性,并影响细胞形态和EMT表型,提示RHOJ在GBM中起重要作用。明确RHOJ调控差异靶分子介导恶性表型的信息或证据,为胶质瘤分子诊断与靶向治疗提供新型分子标志物和干预靶点。
附图说明
图1为RHOJ在高级别胶质瘤(GBM)中过表达与病人复发和预后差显著相关;其中 a为正常脑组织和GBM组织中RHOJ的IHC染色表达差异,b为在脐带血内皮细胞(HUVEC) 和人GBM细胞系中RHOJ表达的Western印迹分析,c-e为Kaplan-Meier分析显示GBM中高表达RHOJ与所有GBM(c),原发性GBM(d)和复发性GBM(e)患者预后差显著相关, f为GBM与LGG中RHOJ相对表达差异,g为复发性GBM与原发性GBM的相对表达差异;图2为沉默RHOJ表达抑制GBM细胞的增殖并诱导了G2期的细胞周期停滞;其中a-b为RT-qPCR或蛋白质印迹检测显示siRNA抑制RHOJ mRNA和蛋白表达的作用,c-d和e-f为 siRNA抑制RHOJ表达后分别通过细胞计数(c-d)和Cell Counting Kit-8(CCK-8试剂盒)(e-f) 分析GBM细胞系的生长曲线,g为通过Western印迹分析LN229细胞中稳定敲低和过表达 RHOJ具有拯救作用,h-i为LN229细胞中稳定敲低和过表达RHOJ后细胞计数(h)和CCK-8 分析(i)的细胞生长曲线,j-k为LN229细胞中RNAi敲除RHOJ诱导细胞周期停滞在G2期,l 为LN229细胞中稳定敲低RHOJ后蛋白质印迹显示Cyclin B1的表达显著降低,所有数据通过t检验对P值进行了分析,作为三个独立实验的平均值±SEM。*P<0.05;**P<0.01; ***P<0.001;
图3为稳定敲低RHOJ表达抑制小鼠GBM细胞的致瘤性和血管生成;其中a为RHOJ稳定敲低(shRHOJ)减少了U251和LN229细胞的集落数,b为代表性肿瘤球形成试验中U251 细胞(500细胞/孔)的shRHOJ和RHOJ过表达(RHOJ-oe)的图像,以及培养9天后肿瘤球数量的量化;比例尺,50μm,c-d为将500万表达shRHOJ或对照-shRNA(shNC)的U87 细胞皮下接种到BALB/c裸鼠皮下,每4-5天测量一次肿瘤大小(n=6),注射后26天,对小鼠和肿瘤拍照并保存以进行IHC(免疫组织化学)和蛋白质印迹,e为在对照或RhoJ-shRNA 的异种移植肿瘤组织中H&E染色和IHC分析增殖标记物Ki-67的表达,比例尺,500μm,f 为RHOJ,p-Cofilin(Ser3),总Cofilin在异种移植肿瘤组织中表达的蛋白质印迹分析。数据代表通过三个独立实验的t检验分析的均值±SEM,*P<0.05,**P<0.01;
图4为沉默RHOJ表达可抑制GBM细胞的体外迁移和侵袭能力;其中a-b为代表图像(a)和统计图(b)显示在GBM细胞稳定敲低和过表达RHOJ后细胞迁移能力分析,比例尺,50μm,c为在GBM细胞稳定敲低和过表达RHOJ后细胞伤口愈合试验的代表性图像和相对迁移面积进行了统计,d为在GBM细胞稳定敲低和过表达RHOJ后细胞侵袭测定的代表性图像和统计的侵袭细胞数,都进行了三个独立的体外测定实验,分别包括细胞迁移和侵袭测定以及伤口愈合测定,数据表示为三个实验的平均值±SEM,***P<0.001;
图5为恶性胶质瘤细胞RHOJ表达与GBM细胞形态和EMT表型有关;其中a-c为U251(a) 和U87(c)GBM细胞中显示RHOJ(绿色),F-actin(红色),DAPI(蓝色)和Merge染色, b-d为RHOJ和F-actin的荧光强度定量分析,比例尺:200μm,e为U251GBM细胞中E- 钙粘蛋白(绿色),波形蛋白(红色)和DAPI(蓝色)的代表性免疫荧光染色,比例尺:50 μm,f为检测RHOJ和EMT标记E-钙粘蛋白和波形蛋白表达的代表性免疫印迹法,数据表示为平均值±SEM,t检验分析了P值,*P<0.05,**P<0.01。
图6为GBM细胞中RHOJ表达可受转录因子c-Jun表达调控;其中a为RHOJ上游区域中-1kb至0kb之间的预测转录因子c-Jun结合位点,WT包含c-Jun结合位点,推定的结合序列中的突变列为Mut1,Mut2和Mut3,b为使用转染了PGL3Basic或pGL3-RHOJ(含有RHOJ DNA启动子区域[–1000/0]的pGL3-质粒)的HEK-293T细胞建立的萤光素酶测定,将萤光素酶活性标准化为海肾荧光素酶活性,c为双重荧光素酶测定,pGL3Basic, pGL3B-RHOJ或突变质粒Mut1、2或3用于转染c-Jun过表达或空载体对照U251细胞,萤光素酶活性被标准化为海肾萤光素酶活性,d-e为Jun抑制剂SP600125抑制了U87细胞中RHOJ mRNA(d)和蛋白质(e)的表达,并显示出显着的剂量依赖性,f为shRNA靶向的JNK证实了RHOJ表达的下调,结果表示平均值±SEM,是通过三个独立实验的Student t检验进行分析的,*p<0.05,**##p<0.01,***###p<0.001。
图7为RHOJ与ERM家族蛋白moesin相互作用驱动其下游Rac1-PAK-cofilin交换,细胞迁移和增殖。其中a为用蛋白质印迹法进一步验证了RHOJ和moesin之间的相互作用,然后进行了co-IP,b为代表性的荧光图像显示RHOJ和moesin在U87细胞中的共定位。比例尺:50μm,c为阴性对照(NC),RHOJ shRNA和稳定的过表达细胞经过免疫印迹处理,并带有识别下游蛋白质的抗体,d为在表达阴性对照或RHOJ-shRNA或shRHOJ和RHOJ过表达质粒的U251细胞中制备PAK2/4活性,Rac1和RHOJ表达的免疫印迹测定法,微管蛋白用作上样对照,e为d中蛋白免疫印迹的量化分析,f为用RHOJ或Rac1siRNA转染U251 细胞72小时,通过免疫印迹分析细胞裂解物中的RHOJ和Rac1,g显示了每组RHOJ和Rac1 的相对定量,该实验已经独立重复了3次,h为描绘可能有助于GBM细胞迁移和增殖的RHOJ 上游和下游信号总结图。数据代表通过三个独立实验的2尾学生t检验分析的均值±SEM。* #p<0.05;**##p<0.01;***p<0.001。
具体实施方式
下面的实施例可使本专业技术人员更全面地理解本发明,但不以任何方式限制本发明。
实施例1
我们对收集的恶性胶质瘤患者临床组织切片进行RHOJ抗体(Sigma,HPA003050)免疫组织化学染色:将收集到的病理切片置于二甲苯I中5min,更换二甲苯II浸泡5min,无水乙醇 3min,95%乙醇3min,80%乙醇3min,70%乙醇3min,自来水3min进行脱蜡、水化,置于抗原修复液(柠檬酸盐缓冲液,pH6.0)中95-98℃煮沸15min,自然冷却至室温进行抗原修复,PBST缓冲液洗3次,每次3min。滴加山羊血清封闭液,室温30min。甩去封闭液,加一抗(RHOJ,1:400),4℃过夜。PBST缓冲液洗3次,每次3min,滴加二抗,室温1-2h, PBST缓冲液洗3次,每次3min,DAB显色(DAB辣根过氧化物酶显色试剂盒,碧云天生物),自来水冲洗10min,苏木精(索莱宝生物)复染20秒左右,迅速用自来水冲洗,酒精梯度脱水(70%,80%,95%,100%,二甲苯I,二甲苯II),中性树脂封片。倒置显微镜拍照。染色结果显示RHOJ在恶性胶质瘤患者高表达。
通过二代测序和实时荧光定量RT-PCR对恶性胶质瘤患者临床组织样本检测,用染料法或Taqman探针法对RHOJ进行实时定量荧光PCR定量测定,结合临床分析其表达水平与胶质瘤分子分型,微血管形成,病人存活和预后等的关系,探究其作为分子诊断或预后判断的标志物和干预治疗靶点的应用前景。发现RHOJ在GBM中表达明显高于LGG,在复发GBM 中表达明显高于非复发GBM,而且高表达RHOJ与原发和复发GBM患者预后差都显著相关。
分别提取人内皮细胞HUVEC及恶性胶质瘤细胞LN229,U251,U87,T98G,KNS81 和LN18总蛋白,进行western blot检测RHOJ蛋白表达情况,发现RHOJ在各种恶性胶质瘤细胞株中表达上调,而在对照组细胞株HUVEC细胞中表达丰度相对较低。
实施例2
特异性抑制RHOJ的siRNA,根据RHOJ序列及invivogen公司提供的siRNA在线设计网站 (https://link.zhihu.com/?target=http%3A//www.invivogen.com/sirnawizard/siRNA.php)设计两条 siRNA以防止脱靶效应。siRNA由上海吉玛制药技术有限公司合成及提供。
特异性抑制RHOJ的shRNA和过表达RHOJ质粒的构建。特异性shRNA序列由sigma 公司,官网获得,并***PLKO.1质粒载体,构建shRHOJ表达质粒。Pubmed获得RHOJ基因CDS区,设计引物,引物由金斯瑞生物科技有限公司合成提供,进行PCR克隆获得RHOJ 的全长序列,并***真核表达载体plenti-CMV-GFP-puro中。将shRHOJ质粒及RHOJ表达质粒转化大肠杆菌,培养、挑选单克隆后摇菌培养,使用天根生化无内毒素质粒小提中量试剂盒提取质粒后送擎科生物公司测序,选取构建正确的质粒使用。RHOJ-flag标签质粒为在已构成功的RHOJ过表达质粒基础上设计flag标签引物,进行克隆扩增获得。
siRNA,shRNA和RHOJ-oe表达质粒转染及稳转细胞株筛选,RHOJ特异性的siRNA 序列及阴性对照(negative control,siNC)均购自上海吉玛公司。siRNA使用Exfect 2000转染试剂(南京诺唯赞生物公司)。将细胞接种于6孔板,过夜后细胞密度达到70%-80%时,分别取5μL Exfect 2000与125μL无血清DMEM混匀,100pmol siRHOJ/siNC与125μL无血清DMEM混匀,室温静置5min,将孵育好的Exfect 2000与siRNA混合。静置15min后加入6 孔板中,并加入1.75mL无血清培养基补足到2mL,6h后更换为DMEM完全培养基。转染 48h后,进行后续实验。ShRNA及plenti表达质粒用293T细胞进行慢病毒包装,293T细胞接种于6孔板,目的质粒与病毒包装质粒pRSV-Rev,pMDLg/pRRE和pMD2.G(购自addgene) 以合适比例进行转染48h收取病毒上清,用于感染U87,LN229和U251细胞株,感染24h 后,加入嘌呤霉素进行稳转细胞株筛选,约一周后获得shRNA或RHOJ表达质粒稳定转染细胞株。
siRHOJ序列如下:
#si-RHOJ-1:5′-AGAAACCUCUCACUUACGAG-3′;
#si-RHOJ-2:5′-CCACUGUGUUUGACCACUA-3′;
shRHOJ序列如下:
#sh-RHOJ-1:5'- CCGGCAACACUUGCUCGGACUGUAUCUCGAGAUACAGUCCGAGCAAGUGUUGUUUU UG-3';
#sh-RHOJ-2:5'- CCGGGCCCGUUUGCUGUAUAUGAAACUCGAGUUUCAUAUACAGCAAACGGGCUUUU UG-3';
RHOJ表达质粒引物如下:
上游引物:5'-CGGAATTC-GCCACC-ATGAACTGCAAAGAGGGAAC-3';
下游引物:5'-CGGGATCC-TCAGATAATTGAACAGCAG C-3';
Flag引物序列:
上游引物:5'-GCTCTAGAGCCACCATGGGAGACTACAAGGACGATGATGACAAGATGAACTGCAAAGAGGGAAC-3';
下游物:5'-CGGAATTCGCCACCATGGGAGACTACAAGGACGATGATGACAAGATGAACTGCAAAGAGGGAAC-3';
细胞生长曲线实验,U87细胞在siRNA转染48h后,接种于6孔板,每孔20万,siNC(5’–UUCUCCGAACGUGUCACGUUU–3’)和siRHOJ(两条siRNA,siJ-1-5′-AGAAACCUCUCACUUACGAG-3′和siJ-2-5′-CCACUGUGUUUGACCACUA-3′)每组3 个复孔,完全培养基培养,每天计数,持续3d。计数结果通过Graphpad软件作图。
裸鼠皮下成瘤实验,为了进行体内动物研究,将已稳转成功的表达shNC或shRHOJ的U87细胞消化,计数,将500万细胞重悬于100μL无血清培养基后,与100μL的Matrigel 胶(Corning)混匀,注射入5至6周龄雄性BALB/C裸鼠(购自中国南京医科大学动物研究中心)皮下,每组6只。注射后每4-5天测量一次肿瘤大小,并根据下式计算:(长×宽2) /2。在注射26天后处死小鼠。所有动物实验均在中国卫生研究院的批准下进行。
细胞周期检测(LN229细胞株),LN229细胞转染siNC或siRHOJ(siJ-1)48h后,收集50万细胞,重悬于1.5mL的75%乙醇中于-20℃固定过夜。800rpm离心3min,用PBS洗 3次,碘化丙锭(PI,propidium iodide)处理30min,然后用流式细胞仪(BD bioscience,美国)进行检测,并使用Modfit软件进行数据分析,发现干扰RHOJ表达后LN229细胞周期发生G2期阻滞图2j-k)。通过细胞生长曲线实验和裸鼠皮下成瘤实验发现敲减RHOJ后,明显抑制GBM细胞的生长和皮下肿瘤生长,流式细胞检测还发现细胞周期进程受到影响。
实施例3
Transwell迁移实验,细胞在无血清培养基中饥饿过夜后胰酶消化,用无血清DMEM培养基悬浮细胞,计数后将浓度调整为2×104/mL;将600μL含有10%血清的DMEM完全培养基加入24孔板底部,200μL的细胞悬液加入上室,之后放入培养箱中培养;24h后将小室用镊子小心取出,吸除上室液体,加入约600μL 4%多聚甲醛,室温下固定30min;吸除上室的4%多聚甲醛,加入约600μL结晶紫,室温染色30min;PBS轻轻冲洗数次后取出小室,吸除上室液体,用湿棉签小心擦去上室底部膜表面的细胞;在40倍光镜下随机选取5 个视野进行观察、计数并进行统计结果分析发现敲减RHOJ抑制细胞迁移。
Transwell侵袭实验,将Transwell小室中铺上约30μL Matrigel稀释胶,即无血清培养基:Matrigel胶=3:1,于37℃培养箱中凝固1-2h;细胞在无血清培养基中饥饿过夜后胰酶消化,用无血清DMEM培养基悬浮细胞,计数后将浓度调整为2×104/mL;将600μL含有10%血清的DMEM完全培养基加入24孔板底部,200μL的细胞悬液加入上室,之后放入培养箱中培养;24h后将小室用镊子小心取出,吸除上室液体,加入约600μL 4%多聚甲醛,室温下固定30min;吸除上室的4%多聚甲醛,加入约600μL结晶紫,室温染色30min; PBS轻轻冲洗数次后取出小室,吸除上室液体,用湿棉签小心擦去上室底部膜表面的细胞;在40倍光镜下随机选取5个视野进行观察、计数并进行统计结果分析发现RHOJ敲减后抑制细胞侵袭。
划痕愈合实验,为了进行愈合分析,用marker笔在6孔板背后用直尺均匀划横线,每孔划3条线;消化细胞后,以无血清的DMEM培养液2mL/孔、5×105个细胞/孔铺到6 孔板过夜,培养至细胞基本融合;次日用10μL微量枪头在6孔板内垂直划痕,用PBS洗细胞3次,去除划下的细胞加入无血清培养基;放入含5%CO2的37℃培养箱内培养。分别于 0h、24h、48h在倒置显微镜取6个100×视野内观察,比较划痕空隙的距离。发现敲减RHOJ 抑制细胞迁移,体外功能实验发现敲减RHOJ表达抑制恶性胶质瘤细胞迁移与侵袭。
实施例4
细胞免疫荧光实验,将稳定表达shRHOJ的U251和LN229细胞接种到铺有盖玻片的6孔板中,每孔细胞2.0×105个。72h后吸去培养液,用PBS清洗细胞3次,然后每孔加入1mL4%的多聚甲醛固定30min,吸除多聚甲醛,用PBS清洗细胞两次,每孔加入1mL含 0.5%Triton X-100的封闭液室温封闭1h,PBS清洗细胞两次。加入一抗4℃孵育过夜,PBS 清洗细胞3次,每次5min。加入相应荧光二抗,室温孵育2h后,PBS清洗3次,加入 rhodamine-conjugated phalloidin(熠圣生物)染细胞骨架30min,PBS清洗3次,加入DAPI染液,10min后PBS清洗细胞3次,90%的甘油封片后在荧光显微镜下观察。Phalloidin免疫荧光染色发现敲减RHOJ后细胞变大,Phalloidin标记的细胞骨架蛋白F-actin荧光密度降低。
双荧光素酶报告基因实验,JASPAR和ALGGEN-PROMO数据库在线预测RHOJ启动子上游区域有3个转录因子c-Jun的结合位点,接下来通过双荧光素酶报告实验进行验证,方法如下:1)pGL3-RHOJ报告基因质粒构建:通过NCBI查询RHOJ序列全长构建质粒,合成野生型序列(WT)和3个c-Jun结合位点突变序列(Mut1,Mut2和Mut3),分别***到pGL3-Basic报告基因质粒,构建PGL3-WT和pGL3-Mut1/Mut2/Mut3质粒。C-jun表达质粒购自Sigma公司。2)细胞转染:转染前一天将293T细胞或U251细胞按一定密度接种于96孔酶标板中,每组设置6个复孔,转染方法同siRNA,转入载体质粒的量为0.25μg,同时转入Renilla,转染24h后收集细胞。3)荧光素酶活性测定:弃去培养基,用PBS轻轻洗2遍,每孔加入20μL细胞裂解液(1ⅩPLB),置摇床上振摇30min以保证细胞充***解;加入100μL Luciferase Assay ReagentII混匀后立即放入仪器中读数;加100μL预先混好的 Stop&Glo Reagent,混匀后放入仪器中读数,记录Firefly-Luc和Renilla-Luc的数值,比较分析荧光活性。双荧光素酶报告实验发现c-Jun能够增强Luciferase活性,促进RHOJ的表达。
进一步给予c-jun小分子抑制剂SP600125(购自MedChemExpress公司),细胞按适度密度接种于6孔板中,第二天按0,5,10,20μM浓度梯度的SP600125处理细胞,48h后收取细胞分别提取总RNA和蛋白。行qPCR和western blot验证发现,SP600125明显抑制RHOJ 的mRNA和蛋白水平并且呈明显的剂量依赖性(图6d和e)。JNK属于MAPK通路,是c-Jun 的上游调节因子,通过构建shRNA靶向JNK抑制JNK的表达,同样发现RHOJ的表达得到有效抑制(图6f)。
RHOJ qPCR引物如下:
上游引物:5'-CCTGAGTGACAGAGAAAGAACC-3'
下游引物:5'-GGAGTGTGTGCGTATGAAAGA-3'
Jun qPCR引物如下:
上游引物:5'-AAGAACTCGGACCTCCTCA-3'
下游引物:5'-CCGTTGCTGGACTGGATTAT-3'
实施例5:
免疫共沉淀(Co-IP)实验,用1%BSA封闭磁珠30min(4℃),用PBS洗两遍,加入1μLFlag antibody(Sigma)或mouse IgG(abclonal),磁珠加抗体混于700μL IP buffer中于4℃偶联1-2h,弃buffer,收集细胞,PBS洗两遍,加入约800μL IP buffer置于摇床上裂解10min,14000rpm 离心10min后取上清,将蛋白上清分别加入偶联好的磁珠加抗体中4℃孵育过夜,IP buffer洗5遍,弃buffer,加入SDS loading buffer,95℃煮10min用于westernblot检测。Co-IP实验发现RHOJ与moesin之间有相互作用,并且进一步通过免疫荧光实验验证两者之间存在共定位。Western blot检测发现RHOJ敲减后PAK通路下调,骨架相关蛋白Rac1及cofilin磷酸化水平降低,并且EMT相关蛋白CDH1上调,VIM下调。
综上申请人获得RHOJ在临床胶质瘤病人组织与血液样品的表达水平与胶质瘤病人存活和预后的关系的临床证据;阐明RHOJ调控胶质瘤增殖、凋亡、侵袭转移和血管生成等恶性表型的作用及其分子机制。明确RHOJ调控差异靶分子介导恶性表型的信息或证据,为胶质瘤分子诊断与靶向治疗提供新型分子标志物和干预靶点。绘制了“转录因子c-Jun能够调控RHOJ的表达,RHOJ并与moesin相互作用激活PAK-Rac1通路促进细胞骨架动态和神经胶质瘤细胞的增殖和迁移,RHOJ也被证明在上皮-间充质转化过程中起到关键作用。”的机制图。首次证明RHOJ在恶性胶质瘤诊断、治疗和预后判断中的重要作用,接下来开发检测RHOJ表达的特异性抗体和试剂盒以及免疫组化或酶联免疫吸附检测(ELISA)技术及其应用,研发治疗恶性胶质瘤的药物组合物,包括抑制或沉默人RHOJ表达的核酸分子,RHOJ小分子抑制剂或治疗性抗体等及其应用,都应该涵盖在本发明范围之内。
序列表
<110> 皖南医学院第一附属医院(皖南医学院弋矶山医院)
<120> 一种恶性胶质瘤生物标志物的应用
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cugcuucucu gucguaaacc cugccucuua ccacaauguc caggaggaau ggguccccga 780
gcucaaggac ugcaugccuc acgugccuua uguccucaua gggacccaga uugaucuccg 840
ugaugaccca aaaaccuugg cccguuugcu guauaugaaa gagaaaccuc ucacuuacga 900
gcauggugug aagcucgcaa aagcgaucgg agcacagugc uacuuggaau guucagcucu 960
gacucagaaa ggucucaaag cgguuuuuga ugaagcaauc cucaccauuu uccaccccaa 1020
gaaaaagaag aaacgcuguu cugaggguca cagcugcugu ucaauuaucu gagguugucu 1080
gggaccugcc uccaccccau ccagggauga gaauggcagc caaucucugu ggccaagcuc 1140
cagccaaaaa ggagggcacg accagaaagg aacucccuuu gcacggaggc uugccccauc 1200
acccucugag cccucccaac acagcacacu agucagccca cugccacgac cucccugcca 1260
gccagaagca uccguacugc acgcugucug agaaugcugg gccuggauug cagacagugc 1320
cgcugcugau cgcaucaaaa acaaagucaa aggccaucuc acauuuuaca aauccccagc 1380
ucaugaacgu gaagcugaua ggaaaucacc ccagggaacc cgaaaaagaa acuugauucc 1440
ucuauugcug gccuuacuug augucuuuua uaaaacuugg gacuacaaua cuaaccuuuu 1500
uuucugaauc ugcuguucua cccauguguc ucacauucau uuguauuauu ucaagaaaug 1560
uacuaauuuc caguucacuc aggccuuacu aauccauacc aaauuagccu aaagacaagg 1620
cauuuuauau ucauuucuau uuucagcaug uuucuaccaa agcuauuaga accaacacgu 1680
accucugaau gcccgauuau aagaagacau gagaagacuu uaaaaguuuu ggaaauuuac 1740
agagccauga uuuuugaacc uaauugaaag aaaaccaucu gaauuguugc agguccacau 1800
uuuugccaaa gauacacucu auagaugcuu aguaguggcc ugauuuuuuu ccauguauug 1860
ccacgacaaa cuaaaaauga acuguguuua agaauguagu auuucuguuu uucauccaag 1920
uugauugggg gaagaauaug gcaggaucca ucuuuuacag uauuuuguau ucaguaaagu 1980
ggacauuccu gcuccucccu ucccccauug caugcccucu uccucccuug auuucacuuu 2040
cucucaugcc cggauccuuu uauucucccc aguuauaacc caguuauaaa agaaagaucu 2100
gagcauaaag auacguguuu aaaaauaacu aaaaguaaag gaaagugccu uaauuuuucu 2160
auuugcuuca acugaaagug cuucucagcu cgccccaugu aaguucucau uccauguaaa 2220
ugacauuuuc caguuacaac ugguacugag auuuugccuc ucucuuuccu uacucauccu 2280
cccaaauguc uuugugggag ccauaucagu ggauaccaag cucuguaucc auuugucccc 2340
ugcccuccac aaugugugac auagaacagg gacuuuggcc cugggaaagc aaaagcuccc 2400
aguaaggaau ccugugccca augauguaaa acaauuccaa acauccagga auuuuuguau 2460
cauagagcga auuacuuccu aucuuuucau uagaggcuau gaggacuucu aauuagucuu 2520
aguugcuuau aagugcccug gaaucaccca gguaggcacu uaauuuuuuu uucaguugca 2580
ugagcaaagu gcuucuuagu agugugaaau uacaacaacu uuaagacuuu ccagauucaa 2640
gcucccacug uuggaaaaag ccagccuuuc uaaucucuuc ugcuacugga auaagcacuu 2700
aagaauugcg ugauagccag gcaccguggc ucaugccugu aaucccaaca cuuagggagg 2760
cugagguggg ugggccgcuu gagcucagga guucaagacc agccugggua auauagugag 2820
auccuguguc ucuauaaaaa aauuaaaaau uagucaguug uagugacaca uaccuguagu 2880
cccagcuacu caggaggcug agguggaagg aucacuugag cccagaaggu aaggcugcag 2940
ugagcuguga cugugccacu acacuccagc cugagugaca gagaaagaac cugucaaaaa 3000
aaaaaaaaaa acaaccuaca uuucaaguac uauuucccuu cucucccauc uaauugcuaa 3060
agauuuucuu ucauacgcac acacuccagu gacuggaaaa acgggaguuu ucagucaaag 3120
cuugacauuu agagaaaaca aggacuuucu gccuuuauaa auggaaauca acuguguaug 3180
aacuauaacu cugcagaggu uaugaauuca uccuuuacaa acaauaauga acuuuuaguc 3240
cuguaauaaa ugaaauguua uuaggcagcu uuguugcaug auugcauagu uauaucuugc 3300
uaacgggcca cucauuucuc acugaugugg augaaaaaau gagagcagua uguuuccagg 3360
ugugugcacu caacaggcaa auagcucccg aggucaccac uucccuaaug ggccacagga 3420
aguaaguuga ucuugauggg gagaucacgu cacccagaac cagcaacugg auagagacug 3480
uuguuagugu cuggguagag cacaggcucc caggggucuu aagagcuaau uacugaauaa 3540
aacaaucuag aacaaa 3556
<210> 2
<211> 214
<212> PRT
<213> 人工序列(Artificial Sequence)
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Met Asn Cys Lys Glu Gly Thr Asp Ser Ser Cys Gly Cys Arg Gly Asn
1 5 10 15
Asp Glu Lys Lys Met Leu Lys Cys Val Val Val Gly Asp Gly Ala Val
20 25 30
Gly Lys Thr Cys Leu Leu Met Ser Tyr Ala Asn Asp Ala Phe Pro Glu
35 40 45
Glu Tyr Val Pro Thr Val Phe Asp His Tyr Ala Val Thr Val Thr Val
50 55 60
Gly Gly Lys Gln His Leu Leu Gly Leu Tyr Asp Thr Ala Gly Gln Glu
65 70 75 80
Asp Tyr Asn Gln Leu Arg Pro Leu Ser Tyr Pro Asn Thr Asp Val Phe
85 90 95
Leu Ile Cys Phe Ser Val Val Asn Pro Ala Ser Tyr His Asn Val Gln
100 105 110
Glu Glu Trp Val Pro Glu Leu Lys Asp Cys Met Pro His Val Pro Tyr
115 120 125
Val Leu Ile Gly Thr Gln Ile Asp Leu Arg Asp Asp Pro Lys Thr Leu
130 135 140
Ala Arg Leu Leu Tyr Met Lys Glu Lys Pro Leu Thr Tyr Glu His Gly
145 150 155 160
Val Lys Leu Ala Lys Ala Ile Gly Ala Gln Cys Tyr Leu Glu Cys Ser
165 170 175
Ala Leu Thr Gln Lys Gly Leu Lys Ala Val Phe Asp Glu Ala Ile Leu
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Thr Ile Phe His Pro Lys Lys Lys Lys Lys Arg Cys Ser Glu Gly His
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Ser Cys Cys Ser Ile Ile
210
<210> 3
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<213> 人工序列(Artificial Sequence)
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agaaaccucu cacuuacgag 20
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<213> 人工序列(Artificial Sequence)
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ccacuguguu ugaccacua 19
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<213> 人工序列(Artificial Sequence)
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ccggcaacac uugcucggac uguaucucga gauacagucc gagcaagugu uguuuuug 58
<210> 6
<211> 58
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<213> 人工序列(Artificial Sequence)
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ccgggcccgu uugcuguaua ugaaacucga guuucauaua cagcaaacgg gcuuuuug 58
<210> 7
<211> 34
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<213> 人工序列(Artificial Sequence)
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cggaattcgc caccatgaac tgcaaagagg gaac 34
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<213> 人工序列(Artificial Sequence)
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cgggatcctc agataattga acagcagc 28
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<213> 人工序列(Artificial Sequence)
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gctctagagc caccatggga gactacaagg acgatgatga caagatgaac tgcaaagagg 60
gaac 64
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cggaattcgc caccatggga gactacaagg acgatgatga caagatgaac tgcaaagagg 60
gaac 64
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<213> 人工序列(Artificial Sequence)
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uucuccgaac gugucacguu u 21
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<211> 20
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 12
agaaaccucu cacuuacgag 20
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<213> 人工序列(Artificial Sequence)
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ccacuguguu ugaccacua 19
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cctgagtgac agagaaagaa cc 22
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<212> DNA
<213> 人工序列(Artificial Sequence)
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ggagtgtgtg cgtatgaaag a 21
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<212> DNA
<213> 人工序列(Artificial Sequence)
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aagaactcgg acctcctca 19
<210> 17
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ccgttgctgg actggattat 20
Claims (1)
1.敲除如SEQ ID No:1所示RHOJ mRNA的siRNA在制备体外降低人高级别胶质瘤细胞系增殖和迁移产品中的应用。
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