CN117286251A - 生物标志物nedd4在小细胞肺癌中的应用 - Google Patents
生物标志物nedd4在小细胞肺癌中的应用 Download PDFInfo
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Abstract
本发明公开了一种生物标志物NEDD4在小细胞肺癌中的应用,本发明研究发现,在SCLC细胞系中消耗NEDD4能显著减少细胞迁移和侵袭,同时还能影响细胞增殖。基于本发明的研究结果,泛素相关基因NEDD4在小细胞肺癌进展中发挥着重要的作用,其表达水平增高会促进小细胞肺癌的迁移、侵袭和增殖,可用于小细胞肺癌患者的预后;而抑制泛素相关基因NEDD4的表达可治疗小细胞肺癌。NEDD4可能成为治疗小细胞肺癌或延缓小细胞肺癌化疗耐药的新靶点,亦可作为筛选小细胞肺癌药物的药物靶点。基于此,本发明提供了生物标志物NEDD4在小细胞肺癌的诊断、预后预测或治疗中的应用,为小细胞肺癌的临床治疗提供新的策略。
Description
技术领域
本发明属于生物医药技术领域,具体涉及生物标志物NEDD4作为新靶点在小细胞肺癌中的应用。
背景技术
肺癌作为全球癌症相关死亡的主要原因之一,其5年生存率低于20%。小细胞肺癌(Small-cell lung cancer,SCLC)是起源于支气管黏膜或腺体的一类肺恶性肿瘤,其是一种侵袭性神经内分泌癌,占所有肺癌恶性肿瘤的10-15%。相比于肺癌的其他分型,小细胞肺癌之所以具有极强的侵袭性,是因为其分化程度低、转移范围广,并且对容易对化疗产生耐药性。
目前治疗SCLC的一线标准化疗方案一直采用依托泊苷结合铂类进行双药化疗,这对于大多数小细胞肺癌患者有效,但该病在首次治疗后不久就会出现化疗耐药的表型复发。因此,亟需开发一种能克服化疗耐药并为患者带来显著生存益处的治疗新策略。
泛素化是一种常见的蛋白质翻译后修饰,参与调控各种生理功能和病理过程,如信号转导和癌症进展等。在这一途径中,蛋白质通过一系列酶促反应进行泛素化和去泛素化,其中涉及泛素激活酶(E1)、泛素结合酶(E2)、泛素连接酶(E3)、含泛素结合域的蛋白(UBD)、含泛素样域的蛋白(ULD)和去泛素酶(DUB)。在此过程中,E3泛素连接酶将泛素分子从E2泛素结合酶转移到特定的蛋白质底物上,使反应具有特异性。据报道,泛素连接酶E3表达突变或失调在肝细胞癌、结直肠癌、肺腺癌和鼻咽癌等多种癌症中影响着肿瘤的发生和发展,并与较差的生存率和预后相关,但泛素化在小细胞肺癌中的研究较少,因此开展泛素化相关基因的研究为小细胞肺癌的临床治疗提供新的策略。
发明内容
有鉴于此,本发明有必要提供一种生物标志物NEDD4在小细胞肺癌中的应用,发明人在SCLC细胞系中对NEDD4的功能进行研究,发现NEDD4与SCLC细胞系的侵袭和迁移能力呈正相关,其表达高低会影响肿瘤细胞的增殖能力,以及对顺铂的敏感性;消耗或抑制NEDD4能够减少转移、延缓化疗耐药从而提高小细胞肺癌患者的存活率,从而为小细胞肺癌的临床治疗提供新的策略。
为了实现上述目的,本发明采用以下技术方案:
本发明第一个方面提供了能够检测生物标志物NEDD4或其mRNA的检测试剂的用途,其特征在于,所述用途为以下A~C中的至少一种:
A:制备小细胞肺癌诊断的产品;
B:制备小细胞肺癌预后预测的产品;
C:制备治疗小细胞肺癌的药物。
NEDD4是一种E3泛素连接酶,发明人通过对小细胞肺癌细胞系泛素化基因进行差异分析,确定了泛素相关基因NEDD4可作为小细胞肺癌(SCLC)的诊断或预后预测的生物标志物。并通过基因敲除实验,验证了在SCLC细胞系中消耗NEDD4能显著减少细胞迁移和侵袭,同时还能影响细胞增殖。基于本发明的研究结果,泛素相关基因NEDD4在小细胞肺癌进展中发挥着重要的作用,其表达水平增高会促进小细胞肺癌的迁移、侵袭和增殖,可用于小细胞肺癌患者的预后;而抑制泛素相关基因NEDD4的表达可治疗小细胞肺癌。NEDD4可能成为治疗小细胞肺癌或延缓小细胞肺癌化疗耐药的新靶点,亦可作为筛选小细胞肺癌药物的药物靶点。
进一步方案,在本发明的一些具体的实施例中,所述用途为制备小细胞肺癌诊断或预后预测的产品。
进一步方案,所述产品包括能够检测生物样本中生物标志物NEDD4或其mRNA表达水平的检测试剂。
进一步方案,本文中所述的检测试剂可以是通过本领域中常规的检测技术检测生物标志物NEDD4或其mRNA表达水平的试剂,具体可提及的实例包括但不限于核酸测序技术、聚合酶链式反应、实时荧光定量聚合酶链式反应或蛋白质印迹法(Western blotting)。在本发明的一些具体的实施方式中,所述产品包括能够检测生物样本中NEDD4的mRNA表达水平的检测试剂,通过将mRNA反转录成cDNA后,通过qPCR方式检测。
进一步方案,所述检测试剂为引物或探针。在本发明的一些具体的实施方式中,所述检测试剂为能够特异性扩增基因NEDD4的引物。在本发明的另一些具体的实施例方式中,所述检测试剂为能反转录基因NEDD4的mRNA的引物。在本发明的另一些具体的实施方式中,所述检测试剂为特异性结合基因NEDD4的探针。
进一步方案,本文中所述的生物样本为小细胞肺癌患者的肿瘤组织。
进一步方案,所述产品为制剂、芯片或试剂盒。该产品根据实际需要可用于对小细胞肺癌进行诊断或对小细胞肺癌患者预后进行预测。
本发明第二个方面提供了一种用于小细胞肺癌诊断或预后预测的产品,所述产品包括能够检测生物样本中生物标志物NEDD4或其mRNA表达水平的检测试剂。
进一步方案,所述生物样本为小细胞肺癌患者的肿瘤组织。
进一步方案,所述检测试剂可以是通过本领域中常规的检测技术检测生物标志物NEDD4或其mRNA表达水平的试剂,具体实例包括但不限于核酸测序技术、聚合酶链式反应和/或实时荧光定量聚合酶链式反应或蛋白质印迹法(Western blotting),优选的,所述试剂通过实时荧光定量聚合酶链式反应(qPCR)检测生物标志物NEDD4的mRNA表达水平。
进一步方案,所述检测试剂为引物或探针。在本发明的一些具体的实施方式中,所述检测试剂为能够特异性扩增基因NEDD4的引物。在本发明的另一些具体的实施例方式中,所述检测试剂为能反转录基因NEDD4的mRNA的引物。在本发明的另一些具体的实施方式中,所述检测试剂为特异性结合基因NEDD4的探针。
可以理解的是,本文中所述的产品主要指的是体外检测产品,包括但不限于为制剂、芯片或试剂盒。所述产品还可以根据检测技术的不同而包括相应检测技术所需要的试剂,比如总RNA抽提试剂、逆转录试剂、实时荧光定量聚合酶链式反应试剂等。
在本发明的一些典型的实施例中,所述产品为芯片或试剂盒,所述芯片或试剂盒执行如下方法:获取受试者的生物样本,并检测所述生物样本中生物标志物NEDD4或其mRNA表达水平。
进一步方案,所述方法还包括根据生物标志物NEDD4或其mRNA的表达水平,判断其表达量是否增高或降低的步骤;在本发明的一些具体的实施方式中,所述判断根据生物标志物NEDD4或其mRNA表达水平的阈值进行,高于阈值的判断为表达量高,低于阈值的判断为表达量低。其中,所述阈值是相应基因在给定人群中的平均表达水平;或者,所述阈值是同一患者的正常组织或癌旁组织中相应基因的表达水平。
在本发明的一些具体的实施方式中,该产品用于对小细胞肺癌进行诊断,则当生物样本中,生物标志物NEDD4或其mRNA表达水平高于阈值时,可判断为小细胞肺癌患者。
在本发明的另一些具体的实施方式中,该产品用于对小细胞肺癌患者预后进行预测,则当生物样本中,生物标志物NEDD4或其mRNA表达水平高于阈值时,则判断为高风险患者,视为预后不良。
本发明第三个方面提供了一种治疗小细胞肺癌的药物,所述药物包含NEDD4或其下游AKT的抑制剂。
进一步方案,所述抑制剂能够抑制NEDD4或其下游AKT的表达或活性。
进一步方案,所述药物为抗顺铂耐药性小细胞肺癌药物。
进一步方案,所述药物还包含小细胞肺癌化疗药物,所述化疗药物为顺铂。
可以理解的是,所述药物还包括药学上可接受的载体或辅料,具体可根据药物的剂型等进行选择,这里不再具体阐述。
本发明的有益效果:
本发明从泛素的角度对SCLC的肿瘤发生和发展提供了新的见解,在SCLC中发现了89个差异表达的URGs,包括52个上调URGs和37个下调URGs。通过生物信息学技术对差异表达的URGs进行Cox回归分析,共发现了4个与小细胞肺癌患者预后相关的URGs,其中,NEDD4是最具肿瘤生物学意义的基因。本发明通过基因敲除实验证明,在SCLC细胞中消耗NEDD4能显著减少细胞迁移和侵袭,同时还能影响细胞增殖。并且,NEDD4耗竭会影响AKT的磷酸化,这说明NEDD4是一种潜在的肿瘤蛋白和PI3K/AKT信号通路促进因子。
这些结果可能会应用到临床实践中,这提示NEDD4可用作诊断SCLC或SCLC患者预后预测的生物标志物,以及治疗SCLC的新靶点。
附图说明
图1为实施例1差异表达基因的火山图和热图和基于URGs的预后特征的构建;
图2为实施例2中KM生存曲线分析;
图3为实施例3中NEDD4相关临床特征分析的箱线图和GO/KEGG功能富集分析;
图4为实施例4中NEDD4在SCLC细胞系中广泛表达和验证si-NEDD4的敲低效率;
图5和图6为实施例5中消耗NEDD4可抑制SCLC细胞的生长、迁移和侵袭能力;
图7为实施例6中NEDD4抑制剂可促进细胞凋亡并抑制SCLC的化疗耐药性。
具体实施方式
下面详细描述本发明的实施例,下面描述的实施例是示例性的,仅用于解释本发明,而不能理解为对本发明的限制。
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方式的目的,不是旨在于限制本发明。另外,如无特别说明,未具体记载条件或者步骤的方法均为常规方法。
实施例1对小细胞肺癌细胞系泛素化相关基因进行差异分析并构建预后模型
1、泛素化相关基因的差异分析
从iUUCD数据库(http://iuucd.biocuckoo.org/)收集泛素化过程涉及的六种酶的家族,包括9种E1、43种E2、919种E3、126种DUB、387种UBD和113种ULD,共涉及4132个泛素化相关基因(URGs)。
从GDSC数据库(https://www.cancerrxgene.org/)下载癌症细胞系抗癌药物敏感性数据和细胞系基因组学数据。从该数据库中筛选出53个与小细胞肺癌相关的SCLC细胞系。根据细胞株对顺铂的IC50值,将它们分为顺铂敏感组和顺铂耐药组。
随后分析顺铂敏感细胞系与顺铂耐药细胞系之间表达水平的差异。使用"Affy"软件包中的RMA方法对微阵列数据进行背景调整和归一化。随后,用"limma"软件包对数据进行差异分析,将|Log FC|>0.2和P值<0.05视为差异表达的URGs,初步筛选得到89个差异基因,绘制了火山图和热图,见图1A和图1B。
2、构建和评估临床样本中与URGs相关的预后特征
结合Geroge J数据集中的临床数据,通过单因素Cox、LASSO回归分析和多因素Cox回归分析,最终确定了与预后最相关的4个URGs,包括UBR5、PAN2、GNB1L和NEDD4。见图1C、图1D、图1E和图1F。最后,根据风险分数的值,采用最佳截断法,对队列中的患者进行高低风险分组。风险评分按以下公式计算:
Risk score=Exp gene1×Coef gene1+Exp gene2×Coef gene2+Exp gene3×Coef gene3+Exp gene4×Coef gene4。
KM生存分析的结果表明,高风险组患者的生存期显著短于低风险组,见图1G。
实施例2对预后基因进一步筛选
根据多因素Cox回归分析筛选出的四个基因的表达量分成高低表达组,通过KM生存分析,探究表达量高低与SCLC患者总生存期之间的关系。
其中,Kaplan-Meier曲线见图2,结果显示,在训练集(数据来源于Geroge J数据集)和验证集(数据集来源于GSE60052数据集)中,只有NEDD4的结果一致,且具有统计学意义。这说明NEDD4高表达患者的生存时间明显短于NEDD4低表达患者。
上述结果表明,NEDD4是SCLC的潜在预后预测因子。
实施例3NEDD4在临床不良临床特征分析和功能富集分析的应用
为了探讨基于NEDD4的预后特征的临床价值,本实施例根据不同的临床特征(包括年龄、性别、肿瘤分级、肿瘤分期、T期、N期和M期)对训练队列和验证队列中的SCLC患者进行了分层。结果表明,恶性肿瘤的远处转移和淋巴结转移与NEDD4的表达水平呈正相关(图3A、3B),表明NEDD4的表达水平可用于预测SCLC患者的预后。
为了研究NEDD4在SCLC中差异表达的分子功能和生物学通路,使用"clusterProfiler"软件包对NEDD4高表达组和低表达组进行了GO和KEGG富集分析。
生物过程分析表明,NEDD4的高表达主要集中在淀粉样细胞迁移、细胞组分生物发生的正调控、有丝分裂细胞周期阶段、Ras蛋白信号转导和染色体分离等方面。(图3C)。
细胞成分分析表明,NEDD4的高表达主要集中在细胞-细胞交界处、细胞前缘、纺锤体、细胞-基质交界处和病灶粘附处(图3C)。
分子功能分析显示,NEDD4的高表达主要集中在蛋白丝氨酸/苏氨酸/酪氨酸激酶活性、蛋白丝氨酸/苏氨酸激酶活性、粘附蛋白结合和微管蛋白结合方面(图3C)。
从KEGG分析来看,高表达的NEDD4主要集中在PI3K-Akt信号通路、人乳头瘤病毒感染、肌动蛋白细胞骨架调控和Ras信号通路(图3D)。
实施例4NEDD4在SCLC细胞系中的表达情况
1、细胞培养
将小细胞肺癌NCI-H82、NCI-H69、NCI-H526、DMS273、SBC-2、H446、SHP77和DMS114细胞系,于5%CO2,37℃的恒温培养箱中进行培养,培养基条件为:RPMI1640细胞培养基中加10%胎牛血清以及1%青霉素/链霉素双抗。悬浮细胞:如H82/H69/H526直接收集培养瓶中的所有培养基到15ml离心管中,1100rpm/3min离心后弃上清,用1ml新鲜培养基重悬细胞,转移到新培养瓶中,轻轻晃匀;贴壁细胞:如H446/SHP77/DMS273/DMS114则先用含0.25%EDTA的胰酶进行常规消化后,同悬浮细胞同样转移到离心管中,1100rpm3min离心后弃上清,用1ml新鲜培养基重悬细胞,转移到新培养瓶中,轻轻晃匀。
2、提取RNA
采用TIANGEN试剂盒:300g离心收集细胞后,加入裂解液350μL和10μL蛋白酶K,涡旋1min充分裂解细胞后,于台式低温高速离心机中12000rpm离心5min,收集上清。将上清加入DNA基因组去除柱中,12000rpm离心30s,保留滤液。向上述含滤液的收集管中加入等体积的70%乙醇,充分混匀后转入无RNA酶的吸附柱CR4中,12000rpm离心30s,弃滤液。随后每管加入700μL的去蛋白液RW3,12000rpm离心30s。再用500μL的RW漂洗液,室温静置2min后,12000rpm离心30min,再重复一遍漂洗步骤。最后用30μL的ddH2O洗脱得到RNA,测定浓度。
3、反转录获取cDNA
按照反转录试剂盒使用手册中的提示,20μL反转录体系里需要500ng~1000ngRNA作为模板,根据所测得的RNA浓度,计算出所需RNA的体积,具体的反转录体系如下:
试剂 | 使用剂量(μL) |
Total RNA | x |
R-mix(2×TS Reaction Mix) | 10 |
E-mix | 1 |
Oligo dt | 1 |
ddH2O | 8-x |
反应条件为:42℃,30min;85℃,5s;4℃,∞。
4、实时荧光定量PCR(qPCR)
扩增引物:
名称 | 序列信息(5’-3’) |
ACTIN-F | catgtacgttgctatccaggc |
ACTIN-R | ctcttaatgtcacgcacgat |
NEDD4-F | tccaaacaccacagcatcag |
NEDD4-R | tgcttcagttcacatggctg |
将反转录得到的cDNA产物稀释10倍后作为qRT-PCR反应的模板,根据qPCR试剂盒使用手册,按如下体系进行qPCR,以20μL体系为例:
试剂 | 使用剂量(μL) |
cDNA模板 | 4 |
引物-F | 1 |
引物-R | 1 |
2×ChamQ Universal SYBR qPCR Master Mix | 10 |
ddH2O | 4 |
反应过程中每个样品设置3个重复。反应完成后,比较相同模板下目的基因和内参基因的Ct值(Cyclethreshold,循环阈值),得出相应的比值。对照组这一比值标准化处理为1,同时其它各组的比值与之相比较,从而得出各组mRNA表达水平的相对变化。
5、蛋白质免疫印迹法(Western Blot)
(1)配胶:配制浓度为10%的分离胶和5%的浓缩胶。配方如下:
10%分离胶 | 5%浓缩胶 |
4.8ml ddH2O | 3.15ml ddH2O |
2.5ml 40%Ac/Bis | 0.5ml 40%Ac/Bis |
2.5ml Tris(pH8.8) | 1.25ml Tris(pH6.8) |
100μL 10%SDS | 50μL 10%SDS |
100μL AP | 50μL AP |
10μL TEMED | 10μL TEMED |
灌入制胶架后,插入十孔梳后等待30~40min后,待凝胶凝固后,于纯水中4℃保存。
(2)以15~30μg的蛋白量配制总体积为20μL蛋白样品,100℃金属浴中煮样5min。
(3)电泳:组装电泳装置,上蛋白样品,80V低压稳定缓速电泳30min后待蛋白样品跑出浓缩胶后,调节电压为120V高压快速电泳60~90min。
(4)转膜:甲醇激活PVDF膜后,转膜前先用甲醇活化60s。调整电压为100V,置于冰水浴上转膜60分钟。
(5)封闭:配制5%脱脂牛奶作为封闭液,室温摇床封闭约两个小时。
(6)一抗:封闭结束后,用TBST轻柔清洗,用5%BSA配制一抗。将相应条带的膜放入抗体盒中,4℃冰箱摇床孵育过夜。
(7)二抗:次日回收一抗,于4℃冰箱保存。用TBST摇床上洗10min,重复三次。加入配好的二抗,室温摇床孵育1小时,结束后TBST洗10min,重复三次。
(8)显影:取出ECL化学发光试剂盒,将A液、B液等比例混合,滴加于膜上,使用成像仪曝光,保存结果并分析。
通过Western印迹和qRT-PCR的方法对NEDD4的蛋白和RNA水平的表达量检测,NCI-H82、NCI-H69、NCI-H526、DMS273、SBC-2、H446、SHP77和DMS114等一系列SCLC细胞系的NEDD4表达水平各不相同。可以看出,敏感细胞与耐药细胞的NEDD4蛋白表达量不同。由于SBC-2、DMS114和H446细胞系的NEDD4表达量较高,因此被选中进行随后的siRNA介导的敲除实验(图4A、4B)。
6、siRNA介导的敲除实验
通过脂质体转染法,将siRNA转染至小细胞肺癌H446、DMS114和SBC-2中。具体步骤如下:
(1)将待转染的细胞接种到六孔板中,细胞密度控制在70~90%为宜;
(2)次日转染:a:准备实验材料:转染试剂Lipofectamine 2000、siRNA和opti-MEM;b:分别将5μL siRNA与250μL opti-MEM混合,5μL lipo2000与250μL opti-MEM混合,静置5min;c:将含siRNA的opti-MEM与含lipo2000的opti-MEM的混合,静置20min;d:均匀且轻柔地滴加到六孔板中,6h后更换成完全培养基。
siRNA序列如下:
名称 | 序列信息(5’-3’) |
siRNA-NEDD4-NC | uucuccgaacgugucacgutt |
siRNA-NEDD4-519(si-NEDD4-1) | guggcucagaagaugauaatt |
siRNA-NEDD4-1359(si-NEDD4-2) | ggaggccuuucuuauugatt |
siRNA-NEDD4-2317(si-NEDD4-3) | gggauucuuugaacuaauatt |
通过Westernblot和qPCR分别从蛋白水平和转录水平来检测转染后细胞中NEDD4的表达情况,以乱序的对照siRNA为阴性对照。结果如图所示,转染了siRNA-NEDD4的小细胞肺癌细胞的NEDD4无论是在蛋白水平(图4C)还是在转录水平(图4D)都显著低于对照,这表明,siRNA-NEDD4显著抑制了癌细胞中NEDD4的表达。
实施例5NEDD4耗竭在阻碍SCLC细胞增殖、迁移和侵袭中的应用
本实施例探讨了NEDD4是否会影响SCLC细胞的生长和迁移能力。将转染siRNA 48小时后的细胞分别收集,台盼蓝法细胞计数。按照每100μL 1000个细胞,配制成细胞悬液,均匀地种到96孔板中,在96小时内,实时监测上述小细胞肺癌细胞转染siRNA后的细胞增殖状况,与对照siRNA进行对比,得到图5A和图5B。结果显示,siRNA-NEDD4能够显著抑制癌细胞的增殖。同样,在六孔板中每孔种1000个细胞,每三天换液,10天左右形成克隆,甲醇固定30min后,用0.5%结晶紫染色。通过集落形成实验,我们观察到敲除NEDD4抑制细胞的集落形成能力明显降低(图5C)。上述结果表明,NEDD4的缺失会对SCLC细胞的增殖产生负面影响。
本实施例还研究了去除了NEDD4的SCLC细胞的迁移潜力,从而研究NEDD4在SCLC细胞转移中的作用,为此进行了伤口愈合实验。转染siRNA48小时后,用10μL的枪头在培养孔板中制造一条划痕,同时将培养基更换成无血清培养基,排除细胞增殖造成的迁移影响。48小时后的结果表明,与对照组相比,siRNA-NEDD4转染的SCLC细胞的迁移能力明显下降(图5D、5E)。
此外,本实施例同时还通过基质胶进行了Transwell试验,以研究NEDD4是否能够促进SCLC细胞的侵袭能力。将基质胶按说明书指导的1:40的稀释比例,用无血清培养基进行稀释,铺入Transwell小室中,与37℃烘箱放置4~5小时形成基底膜。用无血清培养基水化基底膜30min,将转染后的细胞计数后种于铺好基质胶的Transwell上室中,下室加入600μL含20%FBS的培养基。迁移和侵袭实验结果表明,与对照组相比,siRNA-NEDD4后细胞的迁移能力(图6A、6B)和侵袭能力(图6C)明显下降。因此,去除了NEDD4会降低SCLC细胞的增殖、迁移和侵袭能力,而这些能力都是肿瘤发生和发展的关键特征。
实施例6NEDD4在调节顺铂敏感性和细胞凋亡的应用
本实施例研究了NEDD4是否是导致小细胞肺癌耐药的关键基因,采用梯度浓度的顺铂处理小细胞肺癌细胞进行了验证。与NC对照组相比,NEDD4敲除细胞表现出明显的药物敏感性(图7A)。这些结果表明,NEDD4是导致耐药性的关键基因。
此外,本实施例进一步验证了沉默NEDD4是否会导致SCLC细胞凋亡。用siRNA转染细胞48h后,收集培养孔内的上清培养液,同时用不含EDTA的胰蛋白酶消化培养皿中的细胞,收获消化后的细胞。预冷的PBS清洗收集的细胞两次,并用FITC Annexin V细胞凋亡检测试剂盒,检测细胞凋亡。向流式管中分别加入5μL Annexin V和5μL PI,对照组为:NC组细胞Annexin V和PI均不染色、NC组细胞单染Annexin V、NC组细胞单染PI;实验组为NC组和三个siRNA组Annexin V和PI双染色。避光染色30分钟,上机检测。使用流式细胞仪检测凋亡细胞,并使用CytExpert软件分析这些结果,结果如图7B所示,与si-NC组相比,si-NEDD4组凋亡细胞的比例明显增加。
实施例7NEDD4在调节SCLC细胞中的PI3K/AKT信号通路中的应用
已有研究报道,NEDD4可能是通过PI3K/AKT通路参与HCC恶性转化过程的一个重要因素。本实施例进一步研究了NEDD4的表达水平是否对SCLC中的AKT有影响,本研究测定了AKT标志物。结果显示,敲除NEDD4对总AKT(t-AKT)的表达水平没有影响,但降低了p-AKT的表达(图4C)。
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。
Claims (10)
1.能够检测生物标志物NEDD4或其mRNA的检测试剂的用途,其特征在于,所述用途为以下A~C中的至少一种:
A:制备小细胞肺癌诊断的产品;
B:制备小细胞肺癌预后预测的产品;
C:制备治疗小细胞肺癌的药物。
2.如权利要求1所述的用途,其特征在于,所述用途为制备小细胞肺癌诊断或预后预测的产品;
优选的,所述产品包括能够检测生物样本中生物标志物NEDD4或其mRNA表达水平的检测试剂;
优选的,所述检测试剂为通过核酸测序技术、聚合酶链式反应、实时荧光定量聚合酶链式反应或Western blot法检测生物样本中生物标志物NEDD4或其mRNA表达水平的试剂;
优选的,所述检测试剂为引物或探针;
优选的,所述生物样本为小细胞肺癌患者的肿瘤组织;
优选的,所述产品为制剂、芯片或试剂盒。
3.一种用于小细胞肺癌诊断或预后预测的产品,其特征在于,所述产品包括能够检测生物样本中生物标志物NEDD4或其mRNA表达水平的检测试剂;
优选的,所述生物样本为小细胞肺癌患者的肿瘤组织。
4.如权利要求3所述的产品,其特征在于,所述检测试剂为通过核酸测序技术、聚合酶链式反应、实时荧光定量聚合酶链式反应或Western blot法检测样本中所述生物标志物NEDD4或其mRNA表达水平的试剂。
5.如权利要求3所述的产品,其特征在于,所述检测试剂为引物或探针。
6.如权利要求3~5任一项所述的产品,其特征在于,所述产品为制剂、芯片或试剂盒;
优选的,所述产品为芯片或试剂盒。
7.如权利要求6所述的产品,其特征在于,所述芯片或试剂盒执行如下方法:获取受试者的生物样本,并检测所述生物样本中生物标志物NEDD4或其mRNA的表达水平;
优选的,所述方法还包括根据生物标志物NEDD4或其mRNA的表达水平,判断其表达量是否增高或降低的步骤;
优选的,所述判断根据生物标志物NEDD4或其mRNA表达水平的阈值进行,高于阈值则判断为表达量高,低于阈值则判断为表达量低;
优选的,所述阈值是生物标志物NEDD4在给定人群中的平均表达水平;或者,所述阈值是同一患者的正常组织或癌旁组织中生物标志物NEDD4的表达水平。
8.一种治疗小细胞肺癌的药物,其特征在于,所述药物包含NEDD4或其下游AKT的抑制剂;
优选的,所述抑制剂能够抑制NEDD4或其下游AKT的表达或活性。
9.如权利要求8所述的药物,其特征在于,所述药物为抗顺铂耐药性小细胞肺癌药物。
10.如权利要求8或9任一项所述的药物,其特征在于,所述药物还包含小细胞肺癌化疗药物,所述化疗药物为顺铂。
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