CN113801935B - 索拉非尼耐药标志物及其应用 - Google Patents
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Abstract
本发明涉及一种索拉非尼耐药标志物及其应用,属于生物医学技术领域。该索拉非尼耐药标志物为CCT6A。该标志物的研究发现,一方面可通过深入研究鉴定CCT6A/B55γ通路调控细胞自噬与肝癌细胞索拉非尼耐药的关系,为靶向该通路治疗肝癌的新途径提供科学依据。另一方面还可对索拉非尼耐药人群进行提示,在肝癌治疗或癌症治疗中起到个性化治疗、精准治疗的作用。
Description
技术领域
本发明涉及生物医学技术领域,特别是涉及一种索拉非尼耐药标志物及其应用。
背景技术
肝癌是最致命的癌症之一,是导致全球癌症死亡的第三大常见原因。根据世卫组织公布最新全球癌症数据显示;2020年肝癌新发病例为91万,死亡病例为83万,呈现明显上升趋势,严重危害人类健康。由于肝癌发病隐匿,普查或筛查工作推进不足,大多数患者确诊时已到晚期,目前靶向药物索拉非尼是晚期肝癌的首选药物,索拉非尼是一种口服多激酶抑制剂,可抑制肿瘤细胞增殖和血管生成,促进肿瘤细胞凋亡,2007年批准其作为治疗晚期HCC的首个靶向药物。然而,由于肝癌内部和个体之间具有高度异质性,相当数量的患者对索拉非尼治疗无应答,即使有应答的患者,产生治疗应答的往往最多4个月左右(中位PFS和TTP均为3.7个月),随后就发生耐药,耐药后出现疾病进展。因此,深入探讨索拉非尼耐药机制,寻找联合用药策略,对于提高肝癌患者预后,降低死亡率具有重要意义。
发明内容
基于此,有必要针对上述问题,提供一种索拉非尼耐药标志物,采用该标志物可对索拉非尼耐药人群进行提示,促进肝癌精准治疗发展。
一种索拉非尼耐药标志物,该标志物为CCT6A。
本发明人在研究癌症治疗机制中了解到,自噬是一种细胞内的保守过程,可以选择性降解各种蛋白和细胞器,自噬与许多疾病有关,包括神经元变性疾病和癌症。溶酶体作为自噬过程中的不可或缺的重要组成部分,在包括癌症在内的各种生理病理条件下的生物学功能越来越得到大家的认识。溶酶体的功能依赖于溶酶体水解酶和整合溶酶体膜蛋白,蛋白酶、肽酶、磷酸酶等50多种分解酶都储存在溶酶体的酸性腔内,而这一重要的酸性环境主要由液泡H+-ATPase(V-ATPase)复合物控制。此外,溶酶体的功能可以通过转录依赖的方式进行调节,转录因子EB(transcription factorEB,TFEB)从细胞质转位到细胞核,在细胞核中它与被称为“协调溶酶体表达和调控的特定基因网络”(coordinated lysosomalexpression and regulation,CLEAR)的启动子结合。TFEB通过增加溶酶体和自噬前体的生成和功能来促进自噬,促进自噬底物的降解。
本发明人通过全球开放的肿瘤基因芯片数据库并结合自身工作中临床肝癌组织标本、队列随访数据和体外细胞模型研究发现:含无尾复合多肽分子伴侣CCT的第六个亚单位(CCT6A)在肝癌组织中呈高表达,并与肝癌不良预后相关;且蛋白质互作分析提示CCT6A与PP2A-B55γ亚基之间的存在直接关系,经进一步免疫荧光实验显示CCT6A与B55γ共定位于细胞质,经免疫共沉淀证实了CCT6A和B55γ存在结合;敲低CCT6A/B55γ后细胞mTOR磷酸化水平增加、自噬减弱,而自噬减弱后可增加药物敏感性。
据此,我们推测分子伴侣CCT6A通过促进B55γ折叠,增强PP2A的磷酸酶活性,加速下游mTOR通路相关分子的去磷酸化而促进肝癌细胞自噬发生,从而导致肝癌对索拉非尼产生耐药。
在其中一个实施例中,所述CCT6A通过促进细胞自噬发生,从而降低细胞对索拉非尼的敏感性。
在其中一个实施例中,所述CCT6A通过与B55γ结合,加速细胞mTOR通路去磷酸化水平促进细胞自噬发生,从而降低细胞对索拉非尼的敏感性。
在其中一个实施例中,所述细胞为肝癌细胞。
本发明还公开了上述索拉非尼耐药标志物在非治疗目的的索拉菲尼用药研究中的应用。
本发明还公开了检测生物样本中CCT6A的试剂在制备索拉非尼耐药评估的试剂或诊断设备中的应用。
可以理解的,上述CCT6A既可以包括CCT6A蛋白本身,也可以包括用于转录翻译CCT6A蛋白的DNA或RNA。
在其中一个实施例中,所述索拉非尼耐药为索拉非尼治疗肝癌过程中产生的耐药。
在其中一个实施例中,所述生物样本为组织。
本发明还公开了一种索拉非尼耐药评估试剂盒,包含检测CCT6A含量的试剂。
可以理解的,上述CCT6A既可以包括CCT6A蛋白本身,也可以包括用于转录翻译CCT6A蛋白的DNA或RNA。
在其中一个实施例中,所述检测CCT6A含量的试剂包括以下引物对:
CCT6A正向引物:5′-TGACGACCTAAGTCCTGACTG-3′(SEQ ID NO.1)
CCT6A反向引物:5′-ACAGAACGAGGGTTGTTACATTT-3′(SEQ ID NO.2)。
与现有技术相比,本发明具有以下有益效果:
本发明的一种索拉非尼耐药标志物,具体为CCT6A,本发明人通过全球开放的肿瘤基因芯片数据库并结合自身工作中临床肝癌组织标本、队列随访数据和体外细胞模型研究发现:含无尾复合多肽分子伴侣CCT的第六个亚单位(CCT6A)在肝癌组织中呈高表达,并与肝癌不良预后相关;且蛋白质互作分析提示CCT6A与PP2A-B55γ亚基之间的存在直接关系,经进一步免疫荧光实验显示CCT6A与B55γ共定位于细胞质,经免疫共沉淀证实了CCT6A和B55γ存在结合;敲低CCT6A/B55γ后细胞mTOR磷酸化水平增加、自噬减弱、并对索拉非尼的敏感性增加。据此,我们推测分子伴侣CCT6A通过促进B55γ折叠,增强PP2A的磷酸酶活性,加速下游mTOR通路相关分子的去磷酸化而促进肝癌细胞自噬发生,从而导致肝癌对索拉非尼产生耐药。
该标志物的研究发现,一方面可通过深入研究鉴定CCT6A/B55γ通路调控细胞自噬与肝癌细胞索拉非尼耐药的关系,为靶向该通路治疗肝癌的新途径提供科学依据。另一方面还可对索拉非尼耐药人群进行提示,在肝癌治疗或癌症治疗中起到个性化治疗、精准治疗的作用。
附图说明
图1为实施例1中ONCOMINE数据库检索结果示意图;
图2为实施例1中UALCAN数据库检索结果示意图;
图3为实施例1中qRT-PCR检测结果;
图4为实施例1中Western blot检测结果;
图5为实施例1中代表性病例的免疫组化染色照片;
图6为实施例1中病理积分比较示意图;
图7为实施例1中CCT6A表达水平与总生存期关系;
图8为实施例1中CCT6A表达水平与无瘤生存期关系;
图9为实施例2中B55γ基因PPP2R2C与CCT6A家族互作模拟图;
图10为实施例2中GEPIA数据库中CCT6A、B55γ基因PPP2R2C表达相关性示意图;
图11为实施例2中免疫荧光显示CCT6A(红色荧光)与B55γ(绿色荧光)共定位于细胞质照片;
图12为实施例2中免疫共沉淀显示外源表达CCT6A和B55γ存在相互结合作用示意图;
图13为实施例3中Western bloting检测CCT6A表达变化示意图;
图14为实施例3中mRFP-GFP-LC3荧光报告系统检测自噬水平示意图;
图15为实施例3中敲低CCT6A自噬水平统计图;
图16为实施例3中Western bloting检测自噬相关指标LC3-I、LC3-II、Beclin-1以及p-mTOR等表达变化示意图;
图17为实施例3中CCK-8检测不同浓度索拉非尼处理肝癌细胞MHCC-97L后的存活率;
图18为实施例4中索拉菲尼耐药与索拉菲尼敏感的临床肝癌患者组织标本中CCT6A的水平比较;
图19为实施例4中索拉菲尼耐药与索拉菲尼敏感的临床肝癌患者组织标本中CCT6A的的mRNA水平比较;
图20为实施例4中索拉菲尼耐药与索拉菲尼敏感的临床肝癌患者组织标本免疫组化结果代表性示意图(标尺为200μm):
图21为实施例4中CCT6A在索拉菲尼敏感组和耐药组中蛋白印迹水平比较的代表性示意图。
具体实施方式
为了便于理解本发明,下面将参照相关附图对本发明进行更全面的描述。附图中给出了本发明的较佳实施例。但是,本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
以下实施例所用试剂,如非特别说明,均为市售可得;以下实施例所用方法,如非特别说明,均为常规方法可实现。
实施例1
CCT6A在肝癌组织中表达水平及与肝癌患者预后关系研究。
1、方法:
1.1利用肿瘤基因数据库ONCOMINE(https://www.oncomine.org/resource/login.html)和UALCAN(http://ualcan.path.uab.edu/)和GEPIA(http://gepia.cancer-pku.cn/)分析各基因、蛋白等在肝癌中的表达情况。
1.2利用人肝癌组织标本检测CCT6A的水平和组织中的分布情况
(1)收集200例经手术切除后的肝癌及配对癌旁组织,分别提取总RNA、总蛋白,并制备组织切片;搜集各项临床数据,随访资料等。
(2)免疫组化方法:
实验步骤如下:首先设置60℃烘箱烤片30min脱蜡;再用二甲苯浸泡溶蜡,2缸,10min/次,共2次。接下来水化:100%酒精浸泡,2缸,10min/次,共2次;梯度酒精浸泡:依次按照95%、90%、80%、70%、60%每缸,5min/次,共5次。ddH2O浸泡,1缸,5min/次。除去内源性过氧化物酶:3%浓度的H2O2浸泡,1缸,10min。抗原修复:配制pH8.0 EDTA,先煮沸,然后浸泡其中,放到高压锅煮沸修复2min。取出片子,甩干,用免疫组化笔对组织切片画圈,快速插入准备好的PBST,浸泡1min后取出,轻轻甩干,加入CCT6A一抗(sigma公司,货号HPA049949;浓度为1:400稀释),4℃过夜孵育。室温平衡30min,甩掉一抗;PBST浸泡,2~3次,5min/次;加通用二抗,37℃孵育30min,PBST浸泡,2~3次,5min/次。DAB染色(时间控制在1-5min内),甩掉染色液,PBST浸泡终止染色;苏木素染核浸泡1.5min,自来水洗掉苏木素,苏木素回收;盐酸-酒精浸泡,1-2s,快速取出浸泡于ddH2O中,缓水流慢慢冲洗冲完甩干37℃烘干;用中性树脂封片,通风橱中吹干,完成制片。
(3)qPCR方法:采用qRT-PCR对34例匹配的肝癌与癌旁组织进行mRNA检测。
所采用的引物:
CCT6A正向引物:5′-TGACGACCTAAGTCCTGACTG-3′(SEQ ID NO.1)
CCT6A反向引物:5′-ACAGAACGAGGGTTGTTACATTT-3′(SEQ ID NO.2)
qPCR体系:
初始变性:5min,95℃;
45个循环:变性15sec,95℃、退火20sec,55℃、延伸15sec,72℃;
最后延伸:5min,72℃。
(4)蛋白印迹(western bloting)检测并比较肝癌组织和癌周非癌组织中CCT6A表达的差异。
(5)分析CCT6A与肝癌患者预后的相关性。
结合患者临床数据,SPSS 21.0统计软件对CCT6A的表达与预后进行相关性分析;建立Cox比例风险回归模型,探讨CCT6A表达对肝癌预后的影响。
生存分析方法:
邀请2名病理医生对CCT6A免疫组化结果进行评分,将表达面积(0-4分)、表达强度(0-3分)的乘积记作总分。评分后的CCT6A表达数据以平均数(或中位数)为界线分为CCT6A高表达和CCT6A低表达两组。生存分析采用Kaplan-Meier方法,使用GraphPad Prism7.0软件辅助进行。
2、结果:
2.1通过公共数据库挖掘,发现CCT6A在癌组织表达显著高于正常组织,如下表和图1-2所示,图1为ONCOMINE数据库分析结果,其中分析条件为:基因=CCT6A,分析类型为:癌vs正常,肿瘤类型为:肝癌,P值=0.05,倍数变化=2,基因排序=前10%,符合条件的共3个子集(见下表1);图2为UALCAN数据库检索结果,表示CCT6A分别在癌旁(Normal)组织中和肝癌(Primary tumor)组织的表达。
表1.ONCOMINE数据库中3个不同子集中CCT6A mRNA的表达情况
根据上述初筛结果,随即展开进一步的实验验证研究。
2.2人肝癌组织标本中CCT6A的水平和组织中的分布情况
(1)qRT-PCR检测结果。
qRT-PCR检测结果如图3所示,图中N表示癌旁组织,T表示肝癌组织。结果显示,肝癌组织中CCT6A表达高于癌旁组织,且具有统计学上的显著差异,P=0.0317(P<0.05)。
(2)Western blot检测结果。
Western blot检测结果如图4所示,7例样本中均显示出肝癌组织中CCT6A表达高于癌旁组织。
(3)免疫组化染色结果。
代表性病例的免疫组化染色结果如图5所示,其中case表示不同病例,N表示癌旁组织,T表示肝癌组织,N+T指将二者放在同一视野下观察。从图中可以看出CCT6A在肝癌组织中的表达显著高于配对的癌周非癌组织,提示CCT6A在癌组织中高表达。
(4)病理积分结果。
病理积分结果如图6所示,其中,N表示癌旁组织,T表示肝癌组织,纵坐标为病理医生评分。从图中可以看出,CCT6A在肝癌组织较癌旁组织的表达水平明显上调。
(5)CCT6A表达对肝癌预后的影响结果。
对本实施例纳入200例样本中133例样本(其中67例因随访数据不全而未被纳入)验证结果如图7-8所示,图7为CCT6A表达水平与总生存期的关系,图8为CCT6A表达水平与无瘤生存期的关系。
上述结果表明,肝癌组织中CCT6A表达水平与肝癌病理分级、临床分期和恶劣预后相关。
实施例2
CCT6A与B55免疫共沉淀和共定位研究。
1、方法:
1.1为进一步探究CCT6A影响肝癌进展的潜在调控因素,我们通过STRING以及数据库(https://string-db.org/)以及GEPIA数据库分别预测与CCT6A互作的蛋白网络。
1.2构建带有Flag标签的CCT6A质粒和带有Ha标签的B55γ质粒,通过外源表达系统转染肝癌细胞HepG2,利用Western blotting检测标签表达情况。
1.3将上述1.2中构建好的质粒共同转染HepG2细胞株,48小时后收集细胞,以免疫共沉淀技术检测CCT6A与B55γ结合情况,免疫荧光检测CCT6A和B55γ在细胞质中的共定位情况。利用UniProt(https://www.uniprot.org/)数据库分析CCT6A和B55γ蛋白结构,预测两者潜在结合位点并构建结构域缺失质粒,通过免疫共沉淀和免疫荧光实验鉴定两者结合关键区域。
(1)免疫荧光方法:
取对数期生长的肝癌细胞均匀接种于腔室培养皿,每孔接种1×105个。待细胞贴壁后,弃去上清,使用PBS洗两遍,每孔加入200μl预冷的甲醇溶液固定15min。弃去甲醇,PBS清洗两遍,加入0.1%的TritonX-100破膜5min。PBS清洗两遍,加入适量CCT6A一抗(sigma公司,货号HPA049949;1:100稀释)至每孔(以完全覆盖细胞为宜),置于4℃孵育过夜。取出样品并回收一抗,PBS清洗两遍,加入荧光二抗(1:1000稀释)室温孵育45min(加入二抗后全程避光操作)。弃去二抗,PBS清洗两遍,加入DAPI(1:5000稀释)染色5min。再用PBS洗去残留的DAPI染料,拆除腔室培养皿的外部装置,自然晾干后使用防淬灭剂封片,荧光显微镜下观察。
(2)免疫共沉淀和共定位方法如下:
取对数期生长的肝癌细胞接种于六孔板,待细胞贴壁后观察汇合度,于汇合度约60%时进行转染操作,每孔转染2μg带有Flag标签的pENTR-CCT6A质粒,每天观察细胞状态。转染48h后弃去培养基,用预冷的PBS清洗两遍,每孔加入800μl NETN裂解液(含蛋白酶抑制剂和磷酸酶抑制剂),冰上裂解15min,将蛋白转移至1.5mL EP管中,4℃,14000rpm离心20min,轻轻吸取750μl上清液转移至新的EP管。每管蛋白分别吸取40μl(5%)作为input样品,加入20μl 3×SDS Loading Buffer,充分混合,于100℃金属浴中变性10min。向剩下710μl蛋白管中每管加入10μl带Flag偶联的琼脂糖珠子,4℃旋转仪孵育过夜。将样品取出,于4℃,5000rpm离心1min洗去未结合的蛋白,弃上清,再加入1ml NETN清洗珠子,重复5次。清洗完毕后弃去上清,小心吸净残余液体,每管各加入60μl 1×SDS Loading Buffer,于金属浴加热10min,将input样品于IP样品一同使用western blotting进行检测。
2、结果:
2.1蛋白互作预测。
通过STRING数据库预测与CCT6A互作的蛋白网络;结果发现,PPP2R2C基因与CCT家族成员具有相互作用关系,并且PPP2R2C基因(表达B55γ蛋白)与CCT6A结合最为紧密如图9所示。
随后,应用GEPIA数据库对CCT6A与PPP2R2C进行相关性分析,结果显示在肝癌患者中两者表达量呈显著正相关(p<0.001),如图10所示。
2.2免疫荧光定位结果。
结果如图11所示,图11为CCT6A和B55γ免疫共定位结果图,图中,DAPI(蓝色)为细胞核染色,B55γ组表示细胞质中可见B55γ发出的绿色荧光,CCT6A组表示细胞质中可见CCT6A发出的红色荧光,Merge组表示三者混合后,可见细胞核染色蓝色荧光,B55γ发出的绿色荧光和CCT6A发出的红色荧光合并为黄色荧光。即利用免疫荧光检测到CCT6A与B55γ在肝癌细胞HepG2中存在共定位信号,显示CCT6A(红色荧光)与B55γ(绿色荧光)共定位于细胞质。
2.3免疫共沉淀结果。
构建带有Flag标签的CCT6A和带Ha标签的B55γ过表达质粒,转染HepG2细胞,待质粒表达后(24h)裂解蛋白,通过flag-agarose珠子对CCT6A-Flag进行富集,westernblotting检测B55γ-Ha蛋白表达情况。
结果如图12所示,从左到右依次为泳道1-4,其中,泳道1:单独转染B55γ-Ha后总蛋白(Input)检测结果;泳道2:CCT6A-Flag与B55γ共转染的总蛋白(Input)检测结果;泳道3:单独转染B55γ后,对总蛋白中Flag标签进行IP,拉下来的IP蛋白检测的结果;泳道4:CCT6A-Flag与B55γ共转染后,对总蛋白中Flag标签进行IP,拉下来的IP蛋白检测结果。以上免疫共沉淀实验进一步显示两者存在结合作用。
上述结果提示,B55γ可能是CCT6A影响肝癌进展的潜在靶点。
实施例3
CCT6A敲减后促进肝癌细胞自噬并提供索拉非尼敏感性研究。
1、方法
1.1构建CCT6A敲低肝癌细胞株
通过慢病毒转染系统构建CCT6A敲低肝癌细胞株(MHCC-97L和HepG2),以无义序列和空载体作为阴性对照,并通过Western bloting检测CCT6A表达变化进行验证。
慢病毒感染表达构建方法:
将pLKO.1载体(Sigma-Aldrich,USA)用限制性内切酶ageⅠ和EcoRⅠ行双酶切,将酶切产物用低熔点琼脂糖胶回收并和上述寡核苷酸退火后的产物混合,然后经T4 DNA连接酶连接过夜。shCCT6A序列为:
shCCT6A-1#(MHCC-97L),5′-CCGGCCAGACATCTCTTCGTACTACTCGAGTAGTACAGAGATGTTCTGGTTTTTG-3’(Forward)(SEQ ID NO.3);
shCCT6A-2#(HepG2)5′-CCGGGCACACACTCACTCAGATCAACTCGAGTTGATCT GAGGTGTGCTTTTTG-3′(Forward)(SEQ ID NO.4)。
将上述shCCT6A序列插入慢病毒载体pLKO.1中,构建pLKO.1-CCT6A重组载体。连接后的重组质粒转化感受态DH5α细菌(Takara,日本),经含氨苄青霉素培养基筛选阳性克隆后,挑取单菌落扩大培养。按分子克隆实验指南用碱裂解法手工抽提质粒。使用Axygen试剂盒抽提质粒并通过DNA测序进一步鉴定。将shCCT6A质粒和包装质粒与脂质体2000共转染293T细胞。转染后24小时和48小时收集细胞上清,然后分别用于感染HCC细胞(HepG2和MHCC97L细胞)。用嘌呤霉素筛选1周后,用WB法(Western bloting)验证CCT6A的敲除作用。
1.2细胞自噬情况研究
在敲低CCT6A稳定株中,Western bloting检测mTOR磷酸化水平,自噬相关蛋白(LC3-I、LC3-II、Beclin-1等)变化。
2.3CCT6A敲低表达肝癌细胞稳株索拉非尼敏感性实验
利用上述第2项中构建好的CCT6A敲低表达肝癌细胞稳株进行实验,分别加入不同浓度的索拉非尼处理24h、48h,以CCK-8实验检测肝癌细胞的存活率,计算IC50。
(1)CCK-8实验具体操作如下:
取对数期生长的CCT6A敲低肝癌细胞株,消化计数,并用含10%FBS的DMEM培养基稀释至每毫升10000个细胞。吸取稀释好的细胞悬液按照每孔100μl的体积将细胞接种于96孔板,并围绕板的边缘加一圈灭菌的双蒸水,置于细胞培养箱培养过夜。按照比例配制CCK-8工作液体(1:100),从培养箱取出细胞,小心弃去上清,向每孔加入100μL稀释好的CCK-8工作液,置于37℃培养4h。孵育结束后将96孔板置于酶标仪中,读取450nm波长下的吸光值。每天于固定时间进行检测,以天数为横坐标,吸光值为纵坐标绘制生长曲线。
(2)我们进一步检测敲低CCT6A对索拉非尼敏感性的影响,使用不同浓度的Sorafenib处理细胞,24h后通过CCK-8测定细胞增殖情况。
(3)通过透射电镜观察肝癌细胞稳定株在索拉非尼处理下自噬小体生成情况,利用mRFP-GFP-LC3慢病毒系统感染CCT6A敲低稳定株,免疫荧光检测细胞中红色和黄色点状荧光表达情况,统计并分析各组阳性信号差异;Western bloting检测索拉非尼处理下,CCT6A稳定株自噬相关蛋白(LC3-I、LC3-II、Beclin-1等)变化。
2.实验结果:
2.1构建CCT6A敲低肝癌细胞株。
结果如图13所示,其中CCT6A即表示CCT6A的表达水平,通过Western bloting检测CCT6A表达变化了两株肝癌细胞稳定表达株(稳株)构建成功。
2.2敲低CCT6A的肝癌株MHCC-97L自噬水平。
我们构建了稳定敲低CCT6A的肝癌株MHCC-97L,通过转染mRFP-GFP-LC3腺病毒载体(市售自噬双标腺病毒)检测自噬产生水平。
结果如图14-15所示,图14为mRFP-GFP-LC3荧光报告系统检测自噬水平结果,DAPI(蓝色)为细胞核染色,GFP(绿色)、mRFP(红色)为LC3阳性信号。
没有自噬发生时,LC3分布于细胞浆中,荧光信号呈现弥散分布。当自噬发生时,胞浆的LC3形成自噬小体并被包裹其中,因此,在荧光显微镜下观察到点状荧光信号,黄色点状荧光为自噬小体(红色信号与绿色信号叠加呈现黄色),单独红色荧光信号为自噬溶酶体(自噬小体与溶酶体结合,由于溶酶体酸性环境,绿色荧光信号淬灭,导致只有红色信号保留)。该图中,shNC组可见大量黄色点状荧光,定位于核外,可判断为自噬小体,在shCCT6A1#和shCCT6A 2#组,黄色点状荧光数量显著下降,说明敲低CCT6A可抑制自噬小体生成。
图15为敲低CCT6A自噬水平统计图,体现对图14的视野下黄色和红色点状荧光数量进行统计,以柱状图形式呈现结果。结果显示敲低shCCT6A 1#和shCCT6A 2#组自噬小体(黄色荧光)数量较shNC显著下降(p<0.01),而自噬溶酶体(红色荧光)数量变化不大,说明敲低CCT6A主要抑制自噬小体的产生,而不是影响自噬小体降解。
Western bloting结果如图16所示,用western blotting方法检测自噬相关蛋白的变化,结果显示:
1)mTOR是自噬的负向调节因子,当mTOR激活时(磷酸化水平上升),自噬减弱。
2)Beclin-1是Ⅲ类PI3K复合物的亚基,Beclin-1与自噬前体的结合启动自噬体的形成,因此该蛋白是自噬体形成所必须的,其表达水平与自噬水平正相关。
3)LC3参与自噬的形成,自噬形成时,胞浆型LC3(即LC3-I)会酶解掉一小段多肽,转变为膜型(即LC3-II),LC3-II升高代表自噬的启动,LC3-I升高代表自噬被抑制。
图中显示敲低CCT6A后,mTOR磷酸化(p-mTOR)水平上升,Beclin-1水平下降,LC3-I表达上升,说明敲低CCT6A后,自噬形成受到抑制。
结果显示,自噬相关蛋白Beclin-1表达减少,LC3-I对LC3-II转换减少,mTOR磷酸化水平增加。
2.3索拉非尼敏感性影响结果。
使用不同浓度的Sorafenib(5,10,20,40μmol/L)处理MHCC-97L细胞,24h后通过CCK-8测定细胞增殖情况如图17所示,图中:*p<0.05,**p<0.01。
结果显示,敲低CCT6A后,细胞对索拉非尼的敏感性增加,IC50分别为shNC:17.58μM、shCCT6A 1#:12.46μM、shCCT6A 2#:12.47μM(shCCT6A1#和shCCT6A 2#由于结果相近,在图中曲线呈现近似重合状态),这些结果表明,抑制CCT6A表达后通过抑制肝癌细胞自噬增加对索拉非尼敏感性。也即CCT6A的表达可促进索拉非尼耐药。
实施例4
临床真实病例中CCT6A与索拉菲尼耐药的相关性。
1、方法:
我们选取了两组肝癌患者:(1)索拉菲尼敏感组(n=27);(2)索拉菲尼耐药组(n=33)。对两组患者肝癌标本分别进行CCT6mRNA水平和蛋白质水平的检测,同时利用免疫组化检测敏感组和耐药组的组织中CCT6A的表达水平,分析CCT6A对肝癌患者治疗的影响。
2、结果:
结果如图18所示,图中显示为CCT6A的mRNA水平在索拉菲尼耐药组与敏感组中的差异,结果提示CCT6A的mRNA水平在耐药组中显著升高,P=0.0001。
同样地,发明人对上述组织标本进行蛋白印迹实验,发现耐药的肝癌组织中CCT6A蛋白表达水平显著高于其在药物敏感的肝癌组织中,如图19所示,两组具有显著差异,P=0.021。
免疫组化检测敏感组和耐药组中CCT6A的表达结果如图20所示,其中深色点表示CCT6A,该免疫组化代表图显示CCT6A在索拉菲尼耐药中的分布显著多于其在索拉菲尼敏感组中的分布;图21为免疫印迹代表图,同样地显示索拉菲尼敏感组中CCT6A水平较低。
通过以上研究提示,CCT6A可通过PP2A-B55γ对下游mTOR基因通路起调控作用,进而增强肝癌细胞自噬和促进索拉非尼耐药,其成果将为开发靶向该通路联合索拉非尼治疗或者治疗索拉非尼耐药的肝癌患者新策略提供科学依据。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
序列表
<110> 中山大学附属第三医院(中山大学肝脏病医院)
<120> 索拉非尼耐药标志物及其应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tgacgaccta agtcctgact g 21
<210> 2
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
acagaacgag ggttgttaca ttt 23
<210> 3
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ccggccagac atctcttcgt actactcgag tagtacagag atgttctggt ttttg 55
<210> 4
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ccgggcacac actcactcag atcaactcga gttgatctga ggtgtgcttt ttg 53
Claims (2)
1.一种检测生物样本中CCT6A含量的试剂在制备索拉非尼治疗肝癌过程中产生的耐药评估的试剂或诊断设备中的应用。
2.根据权利要求1所述的应用,其特征在于,所述检测生物样本中CCT6A含量的试剂包括以下引物对:
CCT6A正向引物:5ʹ-TGACGACCTAAGTCCTGACTG-3ʹ(SEQ ID NO.1)
CCT6A反向引物:5ʹ- ACAGAACGAGGGTTGTTACATTT-3ʹ(SEQ ID NO.2)。
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