CN110241219B - Myom3在黑色素瘤转移中的应用 - Google Patents
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Abstract
本发明公开了MYOM3在黑色素瘤转移中的应用,通过确定MYOM3基因表达差异而指示黑色素瘤是否发生转移,研究发现MYOM3在发生黑色素瘤转移的患者中表达显著下调,进一步研究发现MYOM3能有效抑制黑色素瘤细胞的侵袭和转移。本发明揭示了MYOM3在黑色素瘤转移治疗中具有重要的应用价值,可以作为治疗黑色素瘤转移的药物靶点。
Description
技术领域
本发明涉及疾病诊断领域,具体涉及MYOM3在黑色素瘤转移中的应用。
背景技术
恶性黑色素瘤简称恶黑(Malignant Melanoma,MM),是一种高度恶性肿瘤。其主要起源于黑色素细胞,多见于皮肤及粘膜。皮肤恶性黑色素瘤(Cutaneous MalignantMelanoma,CMM)与其他体表肿瘤相比,侵袭及转移最具特征性,也是影响患者预后最重要的原因。CMM侵袭转移机制异常复杂,是由多种致病因素、多个发病步骤及阶段共同作用的结果。Liotta(1983)提出的恶性肿瘤细胞转移的三步假说即粘附、降解、移动。包括原发灶处肿瘤细胞的脱落,通过各种作用机制进入血运及淋巴管中播散,随后在其他器官组织处粘附,之后通过分泌相关因子加速新生血管生成并增强肿瘤细胞对机体免疫系统的对抗作用,最终形成新的恶性肿瘤病灶。目前有关研究表明其中尤为重要的因素是其相关癌基因的激活及其抑癌基因表达降低或消失,引起癌细胞增殖及侵袭能力增强而致病。针对恶黑的相关癌基因及抑癌基因作为靶点的基因治疗逐渐成为一个有效治疗方法。所以,阐明黑色素瘤侵袭转移的机制,探索有效控制黑色素瘤侵袭和转移的途径,对进一步改善黑色素瘤疗效,防止其复发转移,提高患者生存率,具有重要意义。
发明内容
本发明旨在提供新的黑色素瘤转移诊断性分子标志物,为临床基因检测服务。
为了实现上述目的,本发明采用如下技术方案:
本发明提供了检测MYOM3的试剂在制备诊断黑色素瘤转移的产品中的应用。优选的,所述黑色素瘤转移为皮肤恶性黑色素瘤转移。
进一步,所述制备诊断黑色素瘤转移的产品包括通过测序技术、核酸杂交技术、核酸扩增技术、蛋白免疫技术检测MYOM3表达水平的试剂。
进一步,所述检测MYOM3表达水平的试剂包括特异性识别MYOM3的探针或抗体;或特异性扩增MYOM3的引物。
进一步,所述特异性扩增MYOM3的引物序列如下:上游引物序列如SEQ IDNO.1所示,下游引物序列如SEQ ID NO.2所示。
本发明提供了一种诊断黑色素瘤转移的产品,所述产品包括检测MYOM3表达水平的试剂。
进一步,所述产品包括制剂、试剂盒、芯片或核酸膜条。
本发明中所述试剂盒包括基因检测试剂盒和蛋白免疫检测试剂盒;所述芯片包括基因芯片和蛋白质芯片。
本发明提供了一种药物组合物,所述药物组合物包含MYOM3的促进剂。
进一步,所述促进剂能够促进或增强MYOM3或涉及MYOM3上游或下游途径的物质的表达或活性。
本发明提供了上述药物组合物在制备治疗黑色素瘤转移的产品中的应用。
本发明还提供了MYOM3在筛选治疗黑色素瘤转移的候选药物中的应用。
附图说明
图1显示利用QPCR在mRNA水平上检测MYOM3基因差异表达的统计图。
图2显示利用QPCR在mRNA水平上检测MYOM3基因过表达的统计图。
图3显示利用Transwwell小室检测MYOM3对黑色素瘤细胞侵袭和迁移的影响图;其中图A是MYOM3对黑色素瘤细胞侵袭的影响图;图B是MYOM3对黑色素瘤细胞迁移的影响图。
具体实施方式
本发明经过广泛而深入的研究,通过高通量测序技术,检测黑色素瘤样本中基因在肿瘤组织和肿瘤转移组织中的表达,发现了其中具有明显表达差异的基因,进而筛选出在发生黑色素瘤转移的患者中显著性下调的MYOM3基因,探讨其与黑色素瘤转移之间的关系,从而建立了一种黑色素瘤转移的诊断和治疗途径。
MYOM3基因
根据在NCBI数据库记载的MYOM3的序列信息,可以确定MYOM3是一个定位在1号染色体,accession号为NC_000001.11(24056041..24112135,complement)的基因。
本发明提供了定量鉴定黑素瘤转移以及确定黑素瘤患者治疗方案的方法。这些方法还提供了对患者预后、患者监测和药物开发的辅助。这些方法依赖于分析和测量作为黑色素瘤活检分析的MYOM3的表达水平。
就测量mRNA水平来确定MYOM3基因表达而言,可通过本领域已知的任何手段来进行分析,并且包括诸如PCR、滚环扩增(RCA)、连接酶链反应(LCR)、链置换扩增(SDA)、基于核酸序列的扩增(NASBA)等之类的方法。所涉及的快速分子诊断学最优选的是定量PCR方法,包括QRT-PCR。可通过本领域已知的任何方法进行检测,包括微阵列、基因芯片和荧光。
PCR包括多个扩增步骤或循环,其选择性扩增靶核酸种类。典型的PCR包括三个步骤:变性步骤,其中靶核酸变性;退火步骤,其中一组PCR引物(正向引物和反向引物)退火至互补DNA链;延伸步骤,其中热稳定性DNA聚合物延伸引物。通过重复该步骤多次,DNA片段被扩增而产生对应于靶DNA序列的扩增子。典型的PCR包括20个或更多个变性、退火和延伸循环。通常,退火后延伸步骤可同时进行,在这种情况中该循环仅含两个步骤。
就测量MYOM3蛋白质含量来确定基因表达而言,本领域任何已知的方法都是合适的,只要其可导致足够的特异性和灵敏度。例如,可通过使蛋白质结合至该蛋白质特异性的抗体或抗体片段,并测量抗体结合的蛋白质的量来测量蛋白质含量。可用放射性荧光试剂或其他可检测试剂标记抗体,以方便检测。检测方法包括(但不限于)酶联免疫吸附测定法(ELISA)和免疫印迹技术。
任何给定的基于扩增的分子诊断法的特异性很大程度上(但不是排他地)依赖于引物组的种类。引物组是成对的正向和反向寡核苷酸引物,其退火至靶DNA序列以使得能扩增靶序列,从而产生靶序列特异性的扩增子。引物必须能扩增所关注的疾病状态的标志物。就本发明而言,标志物涉及黑色素瘤。
反应必须还含有检测特异性信号的某些手段。这优选通过使用试剂来完成,这些试剂可检测源于所关注靶序列的聚合反应的DNA序列的区域。当结合至所关注的特异性核酸序列时,用于检测的优选试剂产生可测量的信号差异。通常,这些方法涉及在结合至所关注序列时引起荧光增加的核酸探针。通常,通过针对每种PCR引物对分析扩增子产生的相对速率来监测本发明方法的反应进展。
本发明还包括引物/探针组及其在受权利要求书保护的方法中的用途。这些序列的ID是:SEQ ID NO.1-4。可通过多种检测试剂和方法来监测扩增子产生,包括但不限于荧光引物,以及结合双链DNA的荧光探针和荧光染料。也可使用分子信标、蝎型物和其他检测方案。
在本发明中,所述试剂盒包括检测MYOM3表达量的试剂,试剂包括与MYOM3序列结合的核酸或抗体,核酸包括SYBR Green、TaqMan探针、分子信标、双杂交探针、复合探针检测MYOM3表达量时使用的PCR扩增引物。
所述芯片包括基因芯片、蛋白质芯片;所述基因芯片包括固相载体以及固定在固相载体的寡核苷酸探针,所述寡核苷酸探针包括用于检测MYOM3基因转录水平的针对MYOM3基因的寡核苷酸探针;所述蛋白质芯片包括固相载体以及固定在固相载体的MYOM3蛋白的特异性抗体。
所述核酸膜条包括检测MYOM3表达量的试剂,所述试剂包括与MYOM3序列结合的核酸,所述核酸包括能够检测MYOM3表达量的探针。
本发明的药物组合物包含MYOM3的促进剂,也可以作为医药单独施用或与其它药物一起施用。可以与本发明的药物组合物一起施用的其它药物不受限制,只要它不损害本发明的治疗性或预防性药物组合物的效果即可。优选的,MYOM3的促进剂能够促进或增强MYOM3或涉及MYOM3上游或下游途径的物质的表达或活性。
本发明的药物组合物可根据需要制备成各种剂型。包括但不限于,经皮、粘膜、鼻、口颊、舌下或经口使用的片剂、溶液剂、颗粒剂、贴剂、膏剂、胶囊剂、气雾剂或栓剂。
本发明的药物组合物的施用途径不受限制,只要它能发挥期望的治疗效果或预防效果即可,包括但不限于静脉内,腹膜内,眼内,动脉内,肺内,口服,小泡内,肌肉内,气管内,皮下的,通过皮肤,通过胸膜,局部的,吸入,通过粘膜,皮肤,肠胃,关节内,心室内,直肠,阴道,颅骨内,尿道内,肝内,瘤内。在某些情况下,可以系统地给药。在某些情况下是局部地给药。
本发明的药物组合物的剂量不受限制,只要获得期望的治疗效果或者预防效果即可,可以依据症状、性别、年龄等来恰当的确定。本发明的治疗药物组合物或预防药物组合物的剂量可以使用例如对疾病的治疗效果或者预防效果作为指标来确定。
本发明提供了MYOM3在筛选治疗黑色素瘤转移的候选药物中的应用,包括如下步骤:
用待筛选物质处理表达或含有MYOM3基因的体系;和检测所述体系中MYOM3基因的表达;
其中,若所述待筛选的物质可以促进MYOM3基因的表达水平(优选显著增加,如高20%以上,较佳的高50%以上;更佳的高80%以上),则表明该候选物质是治疗黑色素瘤转移的候选药物。所述体系选自:细胞体系、亚细胞体系、溶液体系、组织体系、器官体系或动物体系。
所述候选物质包括(但不限于):针对MYOM3基因或其上游或下游基因设计的核酸促进物、小分子化合物等。
本发明提供了MYOM3在制备治疗黑色素瘤转移的药物组合物中的应用。所述药物组合物包括了MYOM3的促进剂,以及药学上可接受的载体。所述药学上可接受的载体包括(但不限于)稀释剂、粘合剂、表面活性剂、致湿剂、吸附载体、润滑剂、填充剂、崩解剂。
实施例1黑色素瘤原发病灶和黑色素瘤转移病灶的高通量测序
1、收集样本
3例黑色素瘤原发病灶样本为原发组,3例黑色素瘤转移病灶样本为转移组。
2、提取样品组织的RNA
利用TIANGEN公司的组织RNA提取试剂盒进行总RNA的提取,具体操作详见说明书。
3、RNA样品的质量分析
NanoDrop2000检测RNA的浓度和纯度,OD260/280范围区间为1.8-2.2,AgilentTechnologies 2100Bioanalyzer检测总RNA的浓度、RIN值、28S/18S和片段大小,RNA完整性指数合格,浓度≥20ng/μl,进行mRNA文库构建。
4、高通量转录组测序
应用Illumina Hiseq x-ten第二代高通量测序技术进行mRNA测序。
使用软件cuffquant、cuffdiff进行mRNA表达定量与差异表达分析。Cuffquant是Cufflinks套件中专门用来进行表达量评估和标准化的工具,而cuffdiff则是专门用来计算差异表达的工具,可利用cuffquant的定量结果比较每个基因/转录本在两个分组间的表达差异。显著差异mRNA筛选条件:P<0.05,|log2FC|>1。
5、结果
RNA-seq结果显示,与原发组相比,黑色素瘤转移组中的MYOM3表达水平显著下调。
实施例2 QPCR测序验证MYOM3基因的差异表达
1、样本收集与RNA提取
按照实施例1的方法收集黑色素瘤原发病灶组织36例,黑色素瘤转移病灶组织33例。RNA提取步骤同实施例1。
2、逆转录
利用TIANGEN公司的逆转录试剂盒进行RNA的逆转录,具体反应体系如下:
1)去除基因组DNA的反应体系
将1.0μg的总RNA模板与2.0μl的5×gDNA buffer和RNase Free水混合,终体积为10μl,42℃孵育3min。
2)逆转录PCR反应体系
将2.0μl的10×Fast RT buffer、1.0μl的PrimerScript RT Enzyme Mix I与2.0μl的FQ-RT Primer Mix混合,加入10μl的去除基因组DNA的反应体系和5.0μl的RNase Free水,共20μl,42℃反应15min,之后95℃反应3min。
3、QPCR
1)引物设计
根据GeneBank中MYOM3基因和GAPDH基因的编码序列设计QPCR扩增引物,具体引物序列如下:
MYOM3基因:
上游引物为5’-CTTCAACAACAAGGAGAT-3’(SEQ ID NO.1);
下游引物为5’-AGACAAGTTCTGGATGAT-3’(SEQ ID NO.2);
GAPDH基因:
上游引物为5’-TTTAACTCTGGTAAAGTGGATAT-3’(SEQ ID NO.3);
下游引物为5’-GGTGGAATCATATTGGAACA-3’(SEQ ID NO.4)。
2)按照表1配制PCR反应体系:
SuperReal PreMix Plus试剂盒购自TIANGEN公司。
表1 PCR反应体系
试剂 | 体积 |
2×SuperReal PreMix Plus | 10μl |
正向引物(10uM) | 0.6μl |
反向引物(10uM) | 0.6μl |
50×ROX Reference Dye | 2μl |
DNA模板 | 2μl |
去离子水 | 4.8μl |
3)PCR反应条件:95℃15min,(95℃10s,55℃30s,72℃32s)进行40个循环,之后95℃15s,60℃60s,95℃15s。以SYBR Green作为荧光标志物,在ABI 7300型荧光定量PCR仪上进行PCR反应,通过熔解曲线分析和电泳确定目的条带,2-ΔΔCT法进行相对定量。
4、统计学方法
实验设置3次平行实验,结果数据以平均值±标准差的方式来表示,采用统计软件来进行统计分析,两者之间的差异采用t检验,认为当P<0.05时具有统计学意义。
5、结果
统计结果如图1所示,与黑色素瘤原发组相比,黑色素瘤转移组MYOM3基因表达量显著降低,差异具有统计学意义(P<0.05)。
实施例3 MYOM3基因过表达
1、质粒构建
根据MYOM3基因的编码序列设计扩增引物,引物的设计为本领域技术人员所熟知。从成人胎脑的cDNA文库(clontech公司,货号638831)中扩增全长的MYOM3基因的编码序列,上述cDNA序列插入到真核细胞表达载体pcDNA3.1中,连接获得的重组载体pcDNA3.1-MYOM3用于后续实验。
2、转染
将人黑色素瘤细胞株A357细胞以5×104细胞/孔的密度接种到6孔板,当汇合度达到约50%-80%时,按照脂质体转染试剂Lipofectamine 2000(Invitrogen公司)的说明书进行转染,实验分为阴性对照组(A357)、空白对照组(转染pcDNA3.1-NC)和实验组(转染pcDNA3.1-MYOM3)。
3、QPCR检测过表达效果
3.1提取细胞总RNA
1)称取一定量的样品,液氮下迅速将样品研磨成粉末;
2)加入Trizol,于漩涡振荡器上混匀,室温静置5min;
3)4℃,12000×g离心10min,将上层水相转入新的1.5ml EP管(约400~500μl),室温静置5min,使细胞充分裂解;
4)加入氯仿,盖紧管盖,漩涡振荡器上振荡混匀,室温静置3-5min,使其自然分相;
5)4℃,12000×g离心10min,混合物分成三层:下层红色为苯酚-氯仿有机相,中间蛋白层和无色上层水相,RNA主要集中在水相;
6)转移上层水相(约400~500μl),加入等体积氯仿/异戊醇(24:1),混匀后,冰上静置5min,4℃,12000×g离心10min;
7)必要时重复一次氯仿/异戊醇抽提,至中间层较干净;
8)向转移的上清中加入等体积的异丙醇,-20℃放置1h;
9)4℃,13600rpm离心20min,弃掉上清;
10)RNA沉淀用1.0ml 75%乙醇漂洗两次,每次静置3min,期间颠倒洗涤,4℃13600rpm离心3min;
11)移去残留的乙醇,将沉淀置于超净工作台吹干,用30~50μl Nuclease-freewater溶解RNA沉淀,必要时可于55~60℃下孵育10min助溶。
3.2逆转录
具体步骤同实施例2。
3.3QPCR
具体步骤同实施例2。
3.4统计学方法
实验设置3次平行实验,结果数据以平均值±标准差的方式来表示,采用统计软件来进行统计分析,两者之间的差异采用t检验,认为当P<0.05时具有统计学意义。
4、结果
结果如图2所示,与转染pcDNA3.1-NC的对照组相比,转染pcDNA3.1-MYOM3的实验组中MYOM3的mRNA水平显著上调,差异具有统计学意义(P<0.05)。
实施例4 MYOM3基因对黑色素瘤细胞增殖的抑制作用
1、细胞转染
按照实施例3的方法对黑色素瘤A357细胞进行pcDNA3.1-NC和pcDNA3.1-MYOM3的转染。每组设3个重复,转染48h后分别在实验孔内加入10μl CCK-8溶液,培养2h后测定450nm的吸光度(OD值)。
2、统计学方法
结果数据以平均值±标准差的方式来表示,采用统计软件进行分析,MYOM3过表达组与对照组之间的差异采用t检验,P<0.05时具有统计学意义。
3、结果
结果如表2所示,与转染pcDNA3.1-NC的对照组相比,转染pcDNA3.1-MYOM3的实验组细胞增殖明显减慢,差异具有统计学意义(P<0.05)。
表2黑色素瘤A357细胞OD值
组别 | OD值(光密度) |
pcDNA3.1-NC(对照组) | 1.324±0.005 |
pcDNA3.1-MYOM3(实验组) | 0.841±0.002 |
实施例5细胞侵袭和细胞迁移
1、细胞侵袭
Matrigel无菌冰浴融化后铺在Transwell小室进行包被。将转染的A357细胞以1×108细胞/ml的密度重悬于无血清的DMEM培养基中,接种到上室,下室加入含500μl 10%FBS的DMEM培养基。细胞培养箱中培养24h后,取出transwell小室用磷酸盐缓冲液清洗2次,用棉签擦去上室上层的细胞,将小室在4℃用中性甲醛15min,常温下用20%吉姆萨染色20min,用磷酸盐缓冲液冲洗后,放在正置显微镜观察并计数。
2、细胞迁移
细胞迁移与细胞侵袭步骤基本相同,只是细胞迁移的transwell小室不需要Matrigel包被,直接接种细胞进行后续操作。
3、结果
如图3所示,在黑色素瘤细胞转染重组质粒后,与对照组相比,实验组的侵袭及迁移能力均有明显下降,说明MYOM3能够抑制黑色素瘤的迁移和侵袭。
以上提供了所要求保护的主题的实施例的详细描述以及说明所要求保护的主题的原理的附图。结合此类实施例描述了所要求保护的主题,但其不限于任何特定实施方式。应当理解,所要求保护的主题可以以各种形式体现,并且包含许多替换、修改和等同物。因此,本发明公开的具体细节不应被解释为限制,而是作为权利要求的基础,并且作为代表性基础教导本领域技术人员实质上地在任何适当详细的系统、结构或方式中采用所要求保护的主题。
序列表
<110> 北京泱深生物信息技术有限公司
<120> MYOM3在黑色素瘤转移中的应用
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Claims (3)
1.特异性扩增MYOM3的引物在制备诊断黑色素瘤转移的产品中的应用。
2. 根据权利要求1所述的应用,其中所述特异性扩增MYOM3的引物中上游引物序列如SEQ ID NO.1所示,下游引物序列如SEQ ID NO.2所示。
3.根据权利要求1所述的应用,其中所述产品为试剂、试剂盒、芯片或核酸膜条。
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