CN116574808A - 一种肺癌生物标志物及其检测系统和试剂盒 - Google Patents
一种肺癌生物标志物及其检测系统和试剂盒 Download PDFInfo
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Abstract
本发明公开了一种肺癌生物标志物及其检测系统和试剂盒。所述生物标志物为SPLUNC1和HIF‑1α联合。本发明的肺腺癌标志物的检测系统包括检测模块和分析模块;检测模块用于检测样本中所述生物标志物的含量;分析模块用于接受并分析检测模块获得的数据之间的相关性。本发明的肺腺癌检测试剂盒包括检测试剂,检测试剂包括用于检测所述生物标志物的试剂。本发明联合应用SPLUNC1和HIF‑1α不仅可以作为诊断肺腺癌的生物学指标,也可以提高肺腺癌患者预后评估的预测价值,并发现低氧环境下HIF‑1α直接与SPLUNC1启动子结合,调节其转录,从而引起SPLUNC1蛋白表达增多。
Description
技术领域
本发明涉及生物技术领域,特别涉及一种肺癌生物标志物及其检测系统和试剂盒。
背景技术
肺癌是全世界癌症死亡的首要原因。根据世界卫生组织国际癌症研究机构的最新统计,目前全球范围内几乎四分之一的癌症死亡是由肺癌引起的,给人们的健康和生活带来严重影响。肺癌大致分为小细胞肺癌和非小细胞肺癌(Non–small cell lung cancers,NSCLC),腺癌在NSCLC中的发病率最高,占到40%。在临床上初诊时很多肺腺癌的原发病灶虽然很小,但已出现转移的现象。这就是临床上常说的“小腺癌,大转移”。据报道,转移前诊断和治疗明确的肺癌患者的5年生存率达到50-70%,而远处转移患者的5年生存率下降到不到5%。因此,提高早期肺癌的诊断和控制转移是降低肺癌死亡率的有效办法。世界范围内的肺腺癌发生率持续增加,因此迫切需要有效的生物标志物以诊断和/或治疗肺腺癌。
在2000年何志巍教授克隆出一个在成人鼻咽组织中高表达而在成人鼻咽低分化磷状细胞癌组织中低表达的新基因,命名为YH1。同年国外两位教授分别在胚胎期小鼠和人的上腭、鼻咽上皮和肺部也克隆出了此基因,为了强调该基因在这几个部位的特异性,将其命名为PLUNC,它编码的蛋白为SPLUNC1蛋白。目前有研究表明SPLUNC1可作为肺癌诊断的生物标志物,并且具有特异性强,灵敏度高的特点。而且它还能促进肺癌的生长与转移,但是SPLUNC1参与肺癌转移的分子机制还不明确。
HIF是由氧敏感的HIF-α亚基和结构型表达的HIF-β亚基组成的异二聚体蛋白,其中HIF-α可分为HIF-1α、HIF-2α和HIF-3α。HIF-1活性主要通过氧依赖的蛋白质稳定性变化和HIF-1α的反式激活来调节。在常氧条件下,抑制HIF-1α的脯氨酸羟化酶(PHD2)羟化HIF-1α氧依赖降解(ODD)区域的Pro402和Pro564。这种羟化通过含有von Hippel-Lindau(VHL)的E3泛素连接酶来触发泛素化,从而导致HIF-1α蛋白的快速降解。此外,天冬酰胺羟化酶(FIH-1)羟基化天冬氨酸残基N803,在HIF-1α的C末端反式激活区域,导致其反式激活活性被抑制。缺氧条件下,PHD2和FIH-1无法利用氧原子对HIF-1α亚基进行羟化作用,导致HIF-1α蛋白稳定表达并与HIF-1β亚基形成二聚体在细胞核内结合辅因子p300,结合于下游靶基因启动子区的HRE,激活下游靶基因的基因转录,从而协调细胞对低氧压力的适应性反应。
至今为止被证实的HIF-1α的靶基因有很多,参与肿瘤发生发展的各个方面。肿瘤代谢重编程方面,HIF-1α可调控葡萄糖转运蛋白家族,如GLUT1,GLUT3,以及糖酵解过程中大量催化葡萄糖最终变为乳酸的酶,如HK1、HK2、LDHA、PGK1和GAPDH等,同时HIF-1α通过上调PDK1的活性抑制线粒体氧化;促血管淋巴管生成方面,HIF-1α可活化下游一系列调控血管和淋巴管生成的基因,如VEGF、SDF1、PGF、PDGFB和ANGPT家族等;促进细胞侵袭转移方面,HIF-1α可调控基质金属蛋白酶MMP2和MMP9以及LOX家族(LOXL2,LOXL4)降解和重塑细胞外基质,上调血光生成素样蛋白(ANGPTL4)促进肿瘤细胞溢出血管和在转移灶形成集落;在促进肿瘤发生内皮细胞间质转化方面,HIF-1α可活化EMT相关基因如SNAI1、SNAI2、TCF3、BPIFA1和ZEB2等。近年来越来越多的研究发现HIF-1α也参与肿瘤干细胞调控的过程,研究者发现HIF-1α可调控端粒酶(TERT),干细胞多功能因子NANOG和OCT4以及阻止细胞衰老的因子如磷酸甘油酸变位酶(PGM)。
综上,目前亟需新的肺癌尤其是肺腺癌的诊断、预后评估和治疗的有效靶点。
发明内容
针对现有技术中的缺陷,本发明提出了一种肺癌生物标志物及其检测系统和试剂盒。本发明发现联合应用SPLUNC1联合HIF-1α不仅可以作为诊断肺腺癌的指标,也可以提高肺腺癌患者预后评估的预测价值。并在分子水平发现低氧环境下HIF-1α直接与SPLUNC1启动子结合,调节其转录,从而引起SPLUNC1蛋白表达增多。
本发明提供一种肺癌生物标志物,所述生物标志物为SPLUNC1和HIF-1α联合。
进一步的,所述生物标志物用于肺腺癌识别或肺腺癌预后评估。
进一步的,所述生物标志物在肺腺癌个体中的表达量升高。
进一步的,HIF-1α直接与SPLUNC1启动子结合,调节其转录,从而引起SPLUNC1蛋白表达量升高。
本发明还提供一种肺腺癌标志物的检测系统,包括检测模块和分析模块;所述检测模块用于检测样本中所述生物标志物的含量;所述分析模块用于接受并分析检测模块获得的数据之间的相关性。
进一步的,所述检测模块的含量检测为mRNA表达水平和/或蛋白表达水平。
进一步的,所述样本包括但不限于肺癌组织。
进一步的,所述分析模块用来分析所述生物标志物的含量变化关系。
本发明还提供一种肺腺癌的检测试剂盒,所述试剂盒包括检测试剂,所述检测试剂包括用于检测所述生物标志物的试剂。
本发明还提供所述的生物标志物或所述的检测系统或所述的试剂盒在以下任一项中的应用:
(1)制备诊断肺腺癌的产品;
(2)肺腺癌发病机制的研究;
(3)制备用于肺腺癌预后效果评估的产品。
综上,与现有技术相比,本发明达到了以下技术效果:
1.本发明首次发现缺氧环境下可以诱导SPLUNC1的表达增多,并且初步阐明了HIF-1α对SPLUNC的转录调控机制,发现了介导缺氧微环境驱动肺癌进展的一条新通路—HIF-1α/SPLUNC1。
2.本发明首次发现肺腺癌组织中SPLUNC1和HIF-1α的蛋白表达水平具有高度正相关性,联合两者不仅能提高肺腺癌诊断的敏感性,还可以提高肺腺癌患者预后评估的预测价值。
3.本发明为开发联合检测SPLUNC1和HIF-1α的试剂盒或抗体试纸提高肺腺癌诊断和评估预后中的价值提供实验依据,并指导临床医生对肺腺癌预后预期良好的患者尽早开展治疗并密切随访,以期提高肺腺癌患者的总体生存率,最终为肺腺癌的诊治带来临床获益。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1为本发明的技术路线。
图2为本发明实施例1中Real-time PCR和Western blot检测缺氧(1%O2)条件下肺癌细胞株中SPLUNC1的表达水平。A、B、C、D的左图分别为肺癌细胞株H292、H827、H1299和A549在缺氧培养0h、6h、12h、24h和48h时SPLUNC1的mRNA表达水平。A、B、C、D的右图分别为肺癌细胞株H292、H827、H1299和A549中HIF-1α、SPLUNC1和β-actin蛋白表达水平随缺氧培养时间的变化。*p<0.05。
图3为本发明实施例1中CoCl2模拟缺氧环境下,SPLUNC1在肺癌细胞株中的表达情况。A、B、C、D的左图分别为在不同浓度CoCl2处理条件下,肺癌细胞株H292、H827、H1299和A549中SPLUNC1的mRNA表达情况;A、B、C、D的右图分别为Western blot检测肺癌细胞株H292、H827、H1299和A549在不同浓度CoCl2处理后HIF-1α和SPLUNC1的表达情况。*P<0.05。
图4为本发明实施例1中肺癌细胞经CoCl2(100μM)处理24h后,终止处理,Western印迹法检测H292、H827和A549细胞中SPLUNC1的蛋白表达水平,直至48h。
图5为本发明实施例1中采用Western blot检测肺癌细胞株中干扰HIF-1α表达后SPLUNC1的蛋白表达情况。用siRNA手段瞬时肺癌细胞株H292和H1299,下调HIF-1α的表达,SPLUNC1的蛋白表达水平均明显下调。
图6为本发明实施例1中肺癌细胞株中SPLUNC1和HIF-1α在细胞内的定位。A,H292;B,H827;C,H1299;D,A549。
图7为本发明实施例2中免疫组织化学方法检测SPLUNC1蛋白在肺癌组织和癌旁肺组织中的表达水平。结果表明,SPLUNC1蛋白在癌旁和肺癌上皮细胞胞浆中均有表达。SPLUNC1在肺癌组织中的表达明显高于正常肺组织。****P<0.0001。
图8为本发明实施例2中免疫组织化学法观察肺癌组织中SPLUNC1表达的细胞定位。
图9为本发明实施例2中肺癌细胞系中SPLUNC1表达的细胞内定位在细胞浆。A,H292;B,H827;C,H1299。
图10为本发明实施例2中SPLUNC1表达水平与肺癌不良预后之间的关系。在80例肺癌患者中检测SPLUNC1的表达水平,按照SPLUNC1高表达(n=32)和SPLUNC1低表达分组(n=48),Kaplan-Meier法两组总体生存率之间的差异,***P<0.001。
图11为本发明实施例3中免疫组织化学方法检测HIF-1α蛋白在肺癌组织和癌旁肺组织中的表达水平。结果表明,HIF-1α蛋白在癌旁和肺癌上皮细胞胞浆中均有表达。HIF-1α在肺癌组织中的表达明显高于正常肺组织,****P<0.0001。
图12为本发明实施例3中HIF-1α表达水平与肺癌不良预后之间的关系。在80例肺癌患者中检测HIF-1α的表达水平,按照HIF-1α高表达(n=26)和HIF-1α低表达分组(n=54),Kaplan-Meier法两组总体生存率之间的差异,***P<0.001。
图13为本发明实施例3中80例肺腺癌患者组织中的HIF-1α表达水平与SPLUNC1蛋白的表达密切相关。(A)同一患者同一视野组织切片中SPLUNC1和HIF-1α蛋白染色情况。(B)SPLUNC1和HIF-1α蛋白表达情况的相关性分析。(C)SPLUNC1和HIF-1α联合用于肺腺癌诊断的ROC分析。
图14为本发明实施例3中联合应用SPLUNC1与HIF-1α可以提高肺腺癌患者预后的评估价值。
图15为本发明实施例1中BPIFA1是HIF-1α的直接靶点结果。(A)分析BPIFA1-2000至0区域的启动子序列,发现1个HIF-1α结合位点;(B)具有潜在或突变的HIF-1α结合位点的BPIFA1启动子结构图;(C,D,E)构建包含HRE野生型和突变型的BPIFA1启动子区域报告基因质粒,并与pcDNA3.1-HIF-1α质粒共转染细胞24h后检测荧光素酶活性的变化(*P<0.05;**P<0.01;***P<0.001)。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分的实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例,都应当属于本发明保护的范围。
本发明技术路线如下(参见图1):
1.缺氧环境下HIF-1α调控SPLUNC1在肺癌细胞系中的表达
(1)采用化学诱导剂及低氧培养箱模拟缺氧环境,在肺癌细胞系中分别采用qRT-PCR及Western blot法检测HIF-1α和SPLUNC1的表达水平,分析两者之间在缺氧状态下是否具有相关性。
(2)生物信息学分析SPLUNC1启动子中是否有HIF-1α的结合元件,并利用过表达或RNAi等方法改变HIF-1α的表达水平分析SPLUNC1的变化趋势。
(3)克隆SPLUNC1启动子及其突变体,双荧光素酶报告基因检测系统分析HIF-1α调控SPLUNC1启动子的能力及其具体结合位点。
2.SPLUNC1和HIF-1α在人肺腺癌组织中的表达及其临床意义
利用含80对癌与癌旁的肺腺癌组织芯片,采用IHC法检测SPLUNC1和HIF-1α的表达水平;以此分析SPLUNC1和HIF-1α在人肺腺癌组织中的表达情况及其在肺腺癌诊断和预后评估中的临床意义。
本发明经过一系列的实验,发现肺腺癌细胞株和肺腺癌组织中SPLUNC1和HIF-1α的蛋白表达水平具有高度正相关性,联合两者不仅能提高肺腺癌诊断的敏感性,还可以提高肺腺癌患者预后评估的预测价值,结果如下:
(1)采用化学诱导剂及低氧培养箱模拟缺氧环境,在肺癌细胞系中分别采用qRT-PCR及Western blot法检测HIF-1α和SPLUNC1的表达水平,结果表明缺氧状态下可以诱导SPLUNC1在mRNA水平和蛋白水平的增多,且其变化趋势与HIF-1α的表达趋势是一致的。利用RNAi等方法改变HIF-1α的表达水平分析SPLUNC1的变化趋势说明SPLUNC1的变化与HIF-1α有关而与HIF-2α无关。
(2)生物信息学分析SPLUNC1启动子中发现1个特异性的缺氧应答原件(Hypoxia-Responsive Element,HRE)位点(5’-TGACGTGCAA-3’),HRE(-468,-458)。利用双荧光素酶报告基因检测系统分析HIF-1α调控SPLUNC1启动子的能力及其具体结合位点。实验结果显示,转染包含HRE(-436,-432)启动子导致荧光强度明显增强、启动子活性明显增强,提示HRE是HIF-1α调控SPLUNC1启动子区域的结合位点。
(3)组织芯片结果说明肺腺癌组织中SPLUNC1蛋白高表达,癌旁组织中低表达,SPLUNC1蛋白可以作为区分肺腺癌组织与癌旁组织的生物标志物,可以用以肺腺癌的诊断。SPLUNC1蛋白表达高低与肺腺癌患者的肿瘤大小、T分期、淋巴结转移和病理分级密切相关,且SPLUNC1高表达组的肺腺癌患者的整体生存期显著低于SPLUNC1低表达组。进一步说明SPLUNC1蛋白可以作为肺腺癌预后不良的生物标志物。
(4)组织芯片结果说明肺腺癌组织中HIF-1α蛋白高表达,癌旁组织中低表达,HIF-1α蛋白可以作为区分肺腺癌组织与癌旁组织的生物标志物,可以用以肺腺癌的诊断。HIF-1α蛋白表达高低与肺腺癌患者的肿瘤大小、病理分级、淋巴结转移和远处转移密切相关,且HIF-1α高表达组的肺腺癌病人总体生存期降低,说明HIF-1α蛋白可以作为肺腺癌预后不良的生物标志物。
(5)根据SPLUNC1和HIF-1α蛋白在肺腺癌组织中的共表达情况,进行两者联合的ROC分析,结果发现曲线下面积为0.812,比SPLUNC1和HIF-1α两者单独的ROC曲线下面积都有不同程度的提高,说明联合SPLUNC1和HIF-1α两种蛋白可以作为诊断肺腺癌的联合指标,可以提高诊断效率。
(6)高表达SPLUNC1和HIF-1α的肺腺癌患者的整体生存期明显低于两者同时低表达组;同时高表达SPLUNC1和HIF-1α的肺腺癌患者与单独SPLUNC1或者HIF-1α高表达的患者相比,整体生存率也显著降低,提示联合应用SPLUNC1与HIF-1α可以提高肺腺癌患者预后的评估价值。
具体参见以下实施例。
实施例1缺氧环境下HIF-1α调控SPLUNC1在肺癌细胞系中的表达
(一)(1%O2)缺氧环境下,肺癌细胞中SPLUNC1表达增多
缺氧(1%O2)条件下培养肺癌细胞株,选取0h、6h、12h、24h和48h共5个时间点,以缺氧环境的标志性调控因子HIF-1α的表达变化为阳性对照,以β-actin为内参检测SPLUNC1的表达水平变化。如图2所示,缺氧处理48h后,四株肺癌细胞株中BPIFA1 mRNA和SPLUNC1蛋白水平的表达均明显增加。并且HIF-1α与SPLUNC1表达水平会随着缺氧处理时间的增加而增加,两者的表达趋势是一致的。说明缺氧环境下可以诱导SPLUNC1在mRNA水平和蛋白水平的增多。
(二)诱导剂CoCl2模拟的缺氧环境下,肺癌细胞中SPLUNC1表达增多
采用化学诱导剂CoCl2模拟缺氧环境。分别采用0、100、150、200、250和300(μM)浓度的CoCl2处理肺癌细胞株H292、H827、H1299和A549,培养24h后用real-time PCR和Western blot的方法分别检测肺癌细胞株中HIF-1α和SPLUNC1在mRNA和蛋白水平表达量的变化。如图3所示,肺癌细胞株中SPLUNC1在mRNA水平和蛋白水平的变化趋势均随CoCl2浓度的提高表达呈阶梯状上升趋势,且表达水平在250μM组到达高峰,且其变化趋势与HIF-1α的表达趋势是一致的。本实验的研究结果与缺氧(1%O2)条件处理下SPLUNC1表达的变化趋势基本一致。说明缺氧可以诱导SPLUNC1在mRNA水平和蛋白水平的增多,这些变化可能与HIF-1α有关。
(三)缺氧诱导的SPLUNC1表达依赖于HIF-1α
CoCl2(100μM)预处理H292、H827和A549等肺癌细胞株24h后撤离,继续培养12h、24h和48h,WB法检测各个时间点SPLUNC1、HIF-1α和HIF-2α蛋白水平的表达情况。结果如图4所示,CoCl2(100μM)处理各种肺癌细胞株后SPLUNC1蛋白表达水平显著有所增加。此外,在终止CoCl2(100μM)处理后,各种肺癌细胞株中SPLUNC1蛋白水平随着时间的推移而持续增加,并且SPLUNC1蛋白变化的趋势与HIF-1α的变化趋势一致而与HIF-2α的变化趋势无关。基于以上结果,推测缺氧环境下SPLUNC1表达增多,可能与HIF-1α的调控有关而与HIF-2α无关。
接下来,利用HIF-1α敲低质粒进一步研究SPLUNC1是否受到HIF-1α的调控。分别在肺癌细胞株H292和H1299中通过siRNA抑制HIF-1α的表达,结果如图5所示,发现SPLUNC1的蛋白表达水平均随之下降,进一步说明SPLUNC1的表达可能受到HIF-1α的调控。
(四)HIF-1α和SPLUNC1在肺癌细胞中存在共定位
分别在肺癌细胞株H292、H827、H1299和A549中用免疫荧光的方法检测HIF-1α和SPLUNC1在细胞中的定位情况,结果如图6所示,发现HIF-1α在四种肺癌细胞株中主要表达在细胞核,而SPLUNC1在四种细胞株中除了表达在细胞核外还表达在胞浆和胞膜,尤其在H299、HCC827和H292中非常明显。说明HIF-1α可能在细胞核中调控了SPLUNC1的转录从而促进其蛋白表达增多。
(五)HIF-1α直接与SPLUNC1启动子结合,调节其转录
前面的研究表明,HIF-1α在基因和蛋白水平上可调节SPLUNC1的表达,接下来,本实施例进一步探讨了HIF-1α是如何调控SPLUNC1表达的。对SPLUNC1启动子区域-2000~+1区域的序列进行分析,发现1个特异性的缺氧应答原件(Hypoxia-Responsive Element,HRE)位点(5’-TGACGTGCAA-3’),HRE(-468,-458)。为了确定SPLUNC1是否是HIF-1α的直接靶点,将759bp片段的SPLUNC1启动子序列插入荧光素酶报告基因质粒pGL4.16-Basic中,构建pGL4.16-SPLUNC1报告基因质粒。将pcDNA3.0-HA-HIF-1α过表达质粒、报告基因质粒和pRL-TK质粒按照相应比例混合,共转染HEK293T细胞,孵育24h,然后利用荧光素酶检测试剂盒(Promega)检测荧光素酶的活性。实验结果如图15所示,转染包含HRE(-436,-432)启动子导致荧光强度明显增强、启动子活性明显增强,提示HRE是HIF-1α调控SPLUNC1启动子区域的结合位点。
实施例2肺腺癌组织中SPLUNC1蛋白高表达,且其与肿瘤的临床预后密切相关
(一)SPLUNC1蛋白在肺腺癌组织中高表达
将肺腺癌组织芯片(上海卓立公司,Cat#ZL-LUC1601)进行SPLUNC1蛋白的免疫组化染色,并利用Visiopharm分析软件分析阳性细胞着色部位,阳性细胞数、阳性面积及染色强度,以及计算阳性面积和染色强度的乘积即积分光密度值(Integrate opticaldensity,IOD)。根据IOD值进行后续的统计学分析。
和癌旁组织相比,SPLUNC1蛋白在肺腺癌组织中明显高表达(图7A),在肺腺癌组织中SPLUNC1蛋白表达的IOD为6.95*107±2.56*107,而癌旁组织中的SPLUNC1蛋白表达的IOD为5.55*107±2.89*107,肺腺癌组织中SPLUNC1蛋白的表达水平显著高于癌旁组织,且差异具有统计学意义(图7B,P<0.0001)。根据SPLUNC1蛋白在肺腺癌组织中的表达情况,进行ROC曲线分析结果显示AUC值为0.8089(95%CI:0.7403to 0.8762,P<0.0001)(图7C)。这些结果说明SPLUNC1蛋白可以作为区分肺腺癌组织与癌旁组织的生物标志物,可以用以肺腺癌的诊断。
(二)SPLUNC1的亚细胞定位
基于肺腺癌组织芯片中SPLUNC1蛋白免疫组织化学染色结果,观察SPLUNC1蛋白表达的细胞内定位,结果发现SPLUNC1主要富集在肿瘤细胞的胞浆,少量在胞膜,肿瘤间质表达量很弱(图8)。
为进一步证实SPLUNC1蛋白在肺癌细胞内的定位情况,选取肺癌细胞系H292、H827和H1299,通过免疫细胞化学的方法检测SPLUNC1蛋白在各肺癌细胞系的定位情况。结果发现,H292、H827和H1299细胞系中的SPLUNC1蛋白主要表达在细胞浆,少量在胞膜(图9)。
(三)肺腺癌临床样本中,SPLUNC1蛋白的表达与患者临床病理特征的关系
免疫组织化学染色的方法观察80例肺腺癌组织中SPLUNC1的表达情况,统计结果显示肺腺癌组织芯粒中SPLUNC1高表达组有32例,低表达组有48例。进一步分析SPLUNC1的表达情况与肺腺癌临床病理特征之间的相关性,结果显示SPLUNC1高表达与年龄、性别和远处转移无显著相关性(P>0.05),而SPLUNC1高表达强度与肿瘤大小(P=0.0079)、T分期(P=0.0141)、病理分级(P=0.0367)和淋巴结转移(P=0.0037)密切相关(如表1所示)。总之,这些结果表明SPLUNC1蛋白表达高低与肺腺癌患者的肿瘤大小、T分期、淋巴结转移和病理分级密切相关,进一步说明SPLUNC1蛋白可以作为肺腺癌预后不良的生物标志物。
表1SPLUNC1蛋白表达水平与肺癌患者临床病理特征的关系
(四)SPLUNC1的表达与肺癌预后的关系
对80例肺腺癌患者组织中的SPLUNC1表达水平与患者整体生存率(OverrallSurvival,OS)进行相关性分析发现,SPLUNC1高表达组的肺腺癌患者的整体生存期(Mediansurvival=36.5months)显著低于SPLUNC1低表达组,提示SPLUNC1的高水平表达可引起肺腺癌患者的预后不良(图10,P<0.05),说明SPLUNC1高表达是肺癌患者预后不良的危险因素。实施例3HIF-1α蛋白在肺腺癌组织中高表达,且与SPLUNC1蛋白表达密切相关
(一)HIF-1α蛋白在肺腺癌组织中高表达
利用免疫组织化学方法检测肺腺癌组织芯片(上海卓立公司,Cat#ZL-LUC1601)中HIF-1α蛋白的表达情况,结果显示HIF-1α蛋白在肺腺癌和癌旁组织细胞胞浆中均有表达,和癌旁组织相比,HIF-1α蛋白在肺腺癌组织中明显高表达(图11A),在肺腺癌组织中HIF-1α蛋白表达的IOD为8.98*107±3.93*107,而癌旁组织中的HIF-1α蛋白表达的IOD为5.72*107±3.80*107,肺腺癌组织中HIF-1α蛋白的表达水平显著高于癌旁组织,且差异具有统计学意义(图11B,P<0.0001)。根据HIF-1α蛋白在肺腺癌组织中的表达情况,进行ROC曲线分析发现AUC值为0.7433(95%CI:0.6650to 0.8216,P<0.0001)(图11C),说明HIF-1α蛋白可以作为肺腺癌的诊断生物标志物。
(二)肺腺癌临床样本中,HIF-1α蛋白的表达与患者临床病理特征的关系
免疫组织化学染色的方法观察80例肺腺癌组织中HIF-1α的表达情况,统计结果显示肺腺癌组织芯粒中HIF-1α高表达组有26例,低表达组有54例。本实施例进一步分析HIF-1α的表达情况与肺癌临床病理特征之间的相关性,结果显示HIF-1α高表达与年龄、性别和T分期无显著相关性(P>0.05),而HIF-1α高表达强度与肿瘤大小(P=0.0161)、病理分级(P=0.0056)、淋巴结转移(P=0.009)和远处转移(P=0.0202)密切相关(如表2所示)。总之,这些结果表明HIF-1α蛋白表达高低与肺腺癌患者的肿瘤大小、病理分级、淋巴结转移和远处转移密切相关,进一步说明HIF-1α蛋白可以作为肺腺癌预后不良的生物标志物。
表2HIF-1α蛋白表达水平与肺癌患者临床病理特征的关系
(三)HIF-1α的表达与肺腺癌患者预后的关系
对80例肺腺癌患者组织中的HIF-1α表达水平与患者整体生存率(OverrallSurvival,OS)进行相关性分析后发现,HIF-1α高表达组的肺腺癌病人总体生存期降低(Median survival=27months),提示HIF-1α高表达的肺腺癌患者预后较差(图12,***P<0.001)。
(四)HIF-1α蛋白和SPLUNC1蛋白的表达水平在肺腺癌组织中密切相关
对80例肺腺癌患者的组织样本(2张相同货号芯片)通过免疫组织化学染色的方法分别观察SPLUNC1和HIF-1α的蛋白表达情况,发现同一患者组织样本中同一视野下两者的表达趋势基本一致的(图13A所示)。进一步采用Pearson法分析两者表达的相关性,结果显示SPLUNC1与HIF-1α在肺腺癌组织中的蛋白表达水平呈现显著正相关(R=0.8146,P<0.001,图13A和13B)。
根据SPLUNC1和HIF-1α蛋白在肺腺癌组织中的共表达情况,进行两者联合的ROC分析,结果发现曲线下面积为0.812,(95%CI:0.744to 0.879,P<0.0001图13C),比SPLUNC1和HIF-1α两者单独的ROC曲线下面积都有不同程度的提高,说明联合SPLUNC1和HIF-1α两种蛋白可以作为诊断肺腺癌的联合指标,可以提高诊断效率。
(五)联合应用SPLUNC1与HIF-1α可以提高肺腺癌患者预后的评估价值
此外,本实施例发现同时高表达SPLUNC1和HIF-1α的肺腺癌患者的整体生存期明显低于两者同时低表达组(图14A);同时高表达SPLUNC1和HIF-1α的肺腺癌患者与单独SPLUNC1或者HIF-1α高表达的患者相比,整体生存率也显著降低,提示联合应用SPLUNC1与HIF-1α可以提高肺腺癌患者预后的评估价值(图14B)。
综合以上实施例,本发明发现缺氧环境下HIF-1α直接与SPLUNC1启动子结合,调节其转录,从而引起SPLUNC1蛋白表达增多。并且SPLUNC1的高水平表达提示肺腺癌瘤体比较大、病理级别低和容易发生淋巴结转移。且SPLUNC1高表达组的肺腺癌患者的整体生存期较SPLUNC1低表达的患者更低,提示SPLUNC1高表达可能是肺腺癌患者预后不良的危险因素。
通过上述实施例已经证明HIF-1α可以靶向调控SPLUNC1,而且HIF-1α已被证实是肺腺癌不良预后的独立预测因子,因此拟进一步探究两者联合应用在提高肺腺癌患者预后评估中的价值。首先本实施例通过连续切片的肺腺癌组织免疫组织化学染色结果发现SPLUNC1与HIF-1α在同一患者组织样本中同一视野下的表达趋势是一致的(图13A所示),相对于癌旁组织,癌组织中的两者的表达量都上调;且Pearson法分析结果显示两者在肺腺癌组织中的表达呈显著正相关(R=0.8146,P<0.001,图13B)。根据SPLUNC1和HIF-1α蛋白在肺腺癌组织中的共表达情况,进行两者联合的ROC分析,结果发现AUC为0.812,(图13C,P<0.0001),比SPLUNC1和HIF-1α两者单独的AUC都有不同程度的提高,说明联合SPLUNC1和HIF-1α两种蛋白可以作为诊断肺腺癌的联合指标。进一步分析发现,同时高表达SPLUNC1和HIF-1α的肺腺癌患者的整体生存期明显低于同时低表达SPLUNC1和HIF-1α的肺腺癌患者(图14A);对比单一指标高表达的患者,SPLUNC1和HIF-1α同时高表达的肺腺癌患者整体生存预后情况最差,提示联合应用SPLUNC1与HIF-1α可以提高肺腺癌患者预后评估的预测价值(图14B)。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种肺癌生物标志物,其特征在于,所述生物标志物为SPLUNC1和HIF-1α联合。
2.根据权利要求1所述的生物标志物,其特征在于,所述生物标志物用于肺腺癌识别或肺腺癌预后评估。
3.根据权利要求1或2所述的生物标志物,其特征在于,所述生物标志物在肺腺癌个体中的表达量升高。
4.根据权利要求1或2所述的生物标志物,其特征在于,HIF-1α直接与SPLUNC1启动子结合,调节其转录,从而引起SPLUNC1蛋白表达量升高。
5.一种肺腺癌标志物的检测系统,其特征在于,包括检测模块和分析模块;所述检测模块用于检测样本中权利要求1所述生物标志物的含量;所述分析模块用于接受并分析检测模块获得的数据之间的相关性。
6.根据权利要求5所述的检测系统,其特征在于,所述检测模块的含量检测为mRNA表达水平和/或蛋白表达水平。
7.根据权利要求5所述的检测系统,其特征在于,所述样本包括但不限于肺癌组织。
8.根据权利要求5所述的检测系统,其特征在于,所述分析模块用来分析所述生物标志物的含量变化关系。
9.一种肺腺癌的检测试剂盒,其特征在于,所述试剂盒包括检测试剂,所述检测试剂包括用于检测权利要求1所述生物标志物的试剂。
10.权利要求1-4任一项所述的生物标志物或权利要求5-8任一项所述的检测系统或权利要求9所述的试剂盒在以下任一项中的应用:
(1)制备诊断肺腺癌的产品;
(2)肺腺癌发病机制的研究;
(3)制备用于肺腺癌预后效果评估的产品。
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