CN112094911A - Nrk在肺癌治疗和预后诊断中的医药用途 - Google Patents
Nrk在肺癌治疗和预后诊断中的医药用途 Download PDFInfo
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Abstract
本发明公开了NRK在肺癌治疗和预后诊断中的医药用途。具体是NRK在制备诊断肺癌预后的产品中的应用，在制备治疗肺癌的药品中的应用；以及NRK中和抗体在制备诊断肺癌的产品中的应用，在制备治疗肺癌的药品中的应用。本发明利用RNA‑seq和综合生物信息学方法对来自LUADⅠ、Ⅱ、Ⅲ期组织的CAFs和NFs进行分析，发现NRK在肺癌肿瘤相关成纤维细胞中特异表达，它可作为肺癌的临床诊断生物标记物，将能提供一个更好的诊断预后指标；LUAD相关CAFs中NRK显著升高，可将NRK作为LUAD联合治疗的靶点。
Description
技术领域
本发明涉及生物医药技术领域，具体而言，涉及NRK在肺癌治疗和预后诊断中的医药用途。
背景技术
肺癌是世界范围内癌症死亡的主要原因。肺癌分为小细胞肺癌(SCLC)和非小细胞肺癌(NSCLC)，其中NSCLC占所有肺癌的85％。NSCLC主要分为腺癌(LUAD)、鳞状细胞癌(LUSC)和大细胞癌，LUAD是NSCLC的主要亚型，约占NSCLC的50％。尽管手术、化疗、靶向治疗和免疫治疗等临床治疗方法的迅速发展已经显著改善了包括LUAD在内的肺癌患者的预后，但由于耐药或反应不良，这些患者的预后仍然很差，5年平均生存率仅为15％。此外，大多数LUAD患者被诊断为晚期转移，随后迅速复发，这也限制了患者生存率。因此，迫切需要为LUAD治疗寻找新的治疗靶点和准确地预测患者预后，从而改善患者的预后。
尽管癌症药物开发传统上侧重于以癌症为中心，但最近重点已转向肿瘤微环境(TME)以寻求新的治疗和预防策略，因为TME可能会促使肿瘤复发和治疗耐药。在TME的所有基质细胞群中，肿瘤相关成纤维细胞(CAFs)是最丰富的，并且与癌症的发生和发展密切相关。有研究发现，CAFs可通过细胞间通讯调节癌细胞和其他基质细胞的生物学特性，通过释放细胞因子或趋化因子，重塑细胞外基质(ECM)或基底膜，调节信号通路和抗肿瘤免疫反应。因此，靶向CAFs可能通过重塑TME，减少免疫抑制发生而非直接杀伤癌细胞提供克服癌症的一条有效途径。事实上，多项临床前研究表明，CAFs可以被选为癌症治疗的新兴靶点，包括抗肿瘤免疫疗法。例如，含有FAP-α基因的重组腺病毒载体感染的树突状细胞可以诱导保护性抗肿瘤免疫，并延长免疫小鼠的总生存时间，提示FAP-α有可能成为靶向CAF和开发免疫原性肿瘤疫苗的潜在靶点。除了CAFs的直接消耗之外，维生素D和维生素A对CAFs的重编程，将促肿瘤CAFs的激活状态重置为静止状态，这在胰腺导管腺癌和结肠癌被关注。此外，CAFs分泌的成纤维细胞生长因子9(FGF9)促进了LUAD细胞的生长，在体内抑制成纤维细胞生长因子受体(FGFRs)导致肿瘤大小和数量显著减少，尽管还不足以完全消除肿瘤，但这为联合策略治疗肺癌提供了证据。值得注意的是，除了癌细胞外，靶向CAFs可能会增强抗肿瘤效果。例如，将奥沙利铂与小分子二肽基肽酶抑制剂PT-100(通过靶向成纤维细胞激活蛋白(FAP)来抑制CAFs相结合，可以改善化疗反应，减少免疫促肿瘤细胞的招募和小鼠结肠癌异种移植模型中的血管生成。然而，CAFs是一个异质的细胞群体，在实体恶性肿瘤中既起到肿瘤支持作用，又起到肿瘤抑制作用，这被认为是开发新的CAF靶向诊断和治疗的主要障碍。
NRK(NIK相关激酶)是由x染色体中的NRK基因编码的蛋白激酶，属于生发中心激酶(GCK)亚家族，参与13种激活MAPK级联。其首先从小鼠中克隆NRK，并在小鼠胚胎发生过程中在骨骼肌中初步发现。这与大多数成纤维细胞的胚胎起源是一致的。NRK也有可能在人脑中表达。NRK(也称为NESK)是促进cofilin(一种必需的肌动蛋白调节蛋白)磷酸化和诱导肌动蛋白聚合的关键。此外，据报道，它通过调节AKT磷酸化来调节滋养层细胞的增殖和胎盘的发育。而且，NRK是银屑病患者真皮间充质干细胞(DMSCs)的高表达基因之一，调节外周血源性单核细胞(PBMCs)向DMSCs的迁移。最近研究发现，NRK在人血管中有很强的表达，并与基质金属蛋白酶(MMPs)和趋化因子(如MMP8、MMP12、CCL8、CXCL9)的诱导有关，表明NRK可能是动脉粥样硬化中的抗炎因子。除上述功能外，NRK与三阴性乳腺癌(TNBC)患者的生存呈正相关。怀孕期间NRK缺乏导致小鼠乳腺癌的触发。然而，NRK在其他组织或细胞、其底物中的表达以及调节其激酶活性和调节信号级联的机制尚未阐明。目前，也尚未发现NRK在肺癌治疗和预后诊断中的相关医药用途。
发明内容
本发明的目的在于提供NRK在肺癌治疗和预后诊断中的医药用途。
一方面，本发明提供了NRK在制备诊断肺癌预后的产品中的应用。
具体的，所述产品以NRK作为诊断标记物。
更具体的，所述NRK的高表达预示肺腺癌患者预后不良。
具体的，所述产品为实时荧光定量PCR检测试剂盒或诊断试纸。
更具体的，所述实时荧光定量PCR检测试剂盒中含有检测用引物，其具体序列为：
正向引物，5′-CAGCAGGTTCGGTCACTGATGTAG-3′；
反向引物，5′-CAGCAGGTTCGGTCACTGATGTAG-3′。
另一方面，本发明还提供了NRK在制备治疗肺癌的药品中的应用。
具体的，所述药品以NRK作为治疗靶标。
另一方面，本发明还提供了NRK中和抗体在制备诊断肺癌的产品中的应用。
具体的，所述产品是诊断试纸或试剂盒，所述产品的诊断样品是肺分泌物或肺组织。
具体的，所述产品以NRK中和抗体作为诊断标记物。
另一方面，本发明还提供了NRK中和抗体在制备治疗肺癌的药品中的应用。
本发明利用RNA-seq和综合生物信息学方法对来自LUADⅠ、Ⅱ、Ⅲ期组织的CAFs和NFs进行分析，发现NRK在肺癌肿瘤相关成纤维细胞中特异表达，它可作为肺癌的临床诊断生物标记物，将能提供一个更好的诊断预后指标；LUAD相关CAFs中NRK显著升高，可将NRK作为LUAD联合治疗的靶点。
附图说明
图1是CAFs和NFs的形态图。
图2是CAFs、NFs和A549的表达。
图3是IF实验和WB实验中CAFs、NFs和A549的表达。
图4是CAFs和NFs的转录分析图。
图5是DEGs火山图，其中红点(上方浅色灰点)代表650个上调基因，蓝点(下方深色黑点)代表1499个下调基因。
图6是log2(Fc)热图。
图7-图12是基因本体论(GO)分析，其中图7-图9分别为上调的DEGs的BP(生物学过程)、MF(分子功能)和CC(细胞成分)，图10-图12分别为下调的DEGs的BP、MF和CC。
图13是上调DEGs的KEGG分析。
图14是下调DEGs的KEGG分析。
图15是RNA-Seq数据、LncRNA微阵列和ArrayExpress的公共数据集(E-MTAB-6149和E-MTAB-6653)中获得的重叠DEG，其中|Fc|>2、P值<0.05。
图16是十个重叠DEGs基因的柱形图。
图17是NRK在CAFs、NFs、BEAS-2B、A549和H1299中的表达水平。
图18是NRK蛋白在CAFs、NFs、BEAS-2B、A549和H1299的Western Blot表达结果。
图19是NRK在CAF的胞核和胞浆、NFs的胞核和胞浆、BEAS-2B的胞核和胞浆、A549的胞核和胞浆和H1299的胞核和胞浆中的表达。
图20是NRK表达热图。
图21是NRK总生存图。
具体实施方式
为了能够更清楚地理解本发明的上述目的、特征和优点，下面结合附图和具体实施方式对本发明技术方案作进一步非限制性的详细描述。
一、材料和方法
肺腺癌组织及其对应的非恶性肺组织(距离肿瘤至少5cm)前瞻性的收集自在广西医科大学附属肿瘤医院被诊断为原发性的肺腺癌患者。选择标准包括：1)病理诊断为LUAD；2)患者在手术前没有接受化疗或放疗；3)患者无其他类型肿瘤史；4)患者有完整的医疗记录。缺乏AJCC分期信息、缺乏组织学信息以及术后30天内死亡或因其他疾病原因死亡的患者被排除，不纳入分析。研究用人体组织经过广西医科大学涉及人体受试者研究的伦理审查委员会审查和批准。
二、细胞的分离、鉴定和培养
CAFs细胞的分离、鉴定和培养
将CAFs和正常纤维细胞(normal fibroblasts)NFs分离。新鲜组织用PBS洗涤后分别切成1～2mm3，然后在25cm2的组织培养瓶中加入1.5ml DMEM培养基(Gibco，美国)、10％FBS(Gibco，美国)和1％青霉素-链霉素(Solarbio，中国北京)，将5～6块组织块分布在25cm2的组织培养瓶中。24h后取出培养基，在组织培养瓶中加入2ml添加10％胎牛血清的新培养基。大约七天后，当成纤维细胞出现突起时，再次更换培养基。细胞融合达到50％后，去除组织，在含10％胎牛血清的DMEM培养基中培养成纤维细胞。
LUAD细胞系的培养
正常人肺支气管上皮细胞系BEAS-2B取自中国科学院昆明分院细胞库。人LUAD细胞系A549和H1299由中国科学院干细胞库提供。BEAS-2B在特定的BEGM(Lonza co.，美国)、A549和H1299在含10％胎牛血清和1％青霉素-链霉素的RIPM-1640(Gibco，美国)培养基上培养。
三、RNA提取
用TRIZOL试剂(Invitgen，美国)提取CAFs和NFs的总RNA。用Agilent BioAnalyzer2100(Agilent Technologies，美国)、NanoDrop ND-1000分光光度计(NanoDropTechnologies，美国)和Qubit 2.0(Invitgen，美国)评估RNA的数量和质量。所有样品的RNA完整性数(RIN)均大于9.0。
四、文库制备和测序
使用NEBNext UltraTM RNA Library Prep Kit for Illumina(New EnglandBiolabs(NEB)，美国)生成测序文库，并将索引代码添加到每个样品的属性序列中。用聚T寡核苷酸连接的磁珠从总RNA中纯化mRNA。在NEBNext第一链合成反应缓冲液(5X)中，用二价阳离子在高温下进行裂解。利用随机六聚体引物和M-MuLV逆转录酶合成第一链cDNA。然后用DNA聚合酶I和核糖核酸酶H进行第二链cDNA合成，DNA片段3’端腺苷基化后，连接具有发夹环结构的NEBNext接头，为杂交做准备。为了选择长度优先于240bp的cDNA片段，用AMPureXP系统(Beckman Coulter，美国)纯化文库片段。然后使用3μl用户酶(New EnglandBiolabs(NEB)，美国)，与大小选择的接头连接的cDNA37℃作用15min，然后在95℃下5min，然后使用Phusion High-Fidelity DNA聚合酶、通用PCR引物和Index(X)引物PCR。然后用Phusion高保真DNA聚合酶、通用PCR引物和Index(X)引物进行PCR扩增。最后对PCR产物进行纯化(AMPure XP系统)，并在Agilent BioAnalyst 2100系统上对文库质量进行评价。
五、使用Deseq2进行CAFs的生物信息学分析和差异表达分析。
Deseq2发现调整后P值<0.05和|倍数变化|>2的基因被归类为差异表达基因。然后用每千碱基外显子片段数(FPKM)估计基因表达水平。基因本体论(GO)分析由GO SEQ R软件包完成，京都基因和基因组百科全书(KEGG)途径分析基于KEGG数据库(http://www.genome.jp/kegg/)。根据RNAseq数据，LncRNA微阵列和ArrayExpress(https://www.ebi.ac.uk/arrayexpress/)的公共数据集(E-MTAB6149和E-MTAB6653)选择|折叠变化|>2且调整后P值<0.0 5的DEG作为覆盖DGE。
六、应用RT-qPCR验证RNA-Seq数据。
RT-qPCR验证了10个重叠DEG在CAFs、NFs、BEAS-2B和LUAD细胞(A549、H1299)中的mRNA表达水平。使用RNA分离试剂盒(Axygen，美国)从细胞中提取总RNA。用PrimeScriptTMRT Master Mix Kit(日本高原市)逆转录合成。用Power SYBR Green PCRMaster Mix(Invitgen，美国)和ABI 7500实时PCR系统(美国应用生物系统公司)进行RT-qPCR。用2-ΔΔCT法测定这些DEGS的相对表达量。表1列出了RT-qPCR检测中使用的特定引物。
表1 RT-qPCR引物序列
七、生存分析
在CCLE数据库(https://portals.broadinstitute.org/ccle/about)中检索“NRK”，并下载NRK在肺癌细胞系中的表达数据，验证NRK在CCLE和Kapan-Meier绘图仪上的表达情况。共选择192个细胞系。PC-14细胞系在国际细胞系鉴定委员会、交叉污染或错误鉴定细胞系数据库(http://iclac.org/databases/cross-contaminations/)，中被报道为污染或鉴定错误，因此被排除在本研究之外。共收集191个细胞系进行下一步分析。此外，利用HEMI(heatmap Illustrator，version1.0；http://hemi.biocuckoo.org/)创建了基于肺癌细胞系中nrk表达的热图。这191个细胞株表现出不同程度的NRK高(红)或低(蓝)表达。在Kapan-Meier绘图仪(https://kmplot.com/analysis/)上进行生存分析。
八、Western Blot(WB)检测
用RIPA缓冲液(Beyotime，中国，上海)从细胞中提取蛋白质，并用BCA蛋白检测试剂盒(Beyotime，中国，上海)定量。用10％SDS-聚丙烯酰胺凝胶分解20微克变性蛋白，并转移到PVDF膜(MilliPore；Merck KGaA，Darmstadt，德国)。然后在5％脱脂牛奶中封闭膜，并与一抗在4℃孵育过夜。使用以下一抗：兔抗α-SMA单抗(1:1000；细胞信号技术公司，美国)、兔抗α-GAPDH单抗(1:1000；Abcam，英国坎布)；兔抗Vimentin单抗(1：1000；细胞信号技术公司，美国)；抗E-钙粘附素单抗(1:1000；细胞信号技术公司，美国)；兔抗NRK(1:500；Invitgen；Thermo Fisher Science，美国)；兔抗GAPDH mAb(1:5000；细胞信号技术公司，美国)。所有膜在PBST中清洗3次，然后在室温下与抗兔IgG二抗(DyLightTM800 4XPEG偶联物，Cst，美国)孵育1h。然后使用红外荧光成像系统奥德赛(LI-COR，美国)对膜带进行成像。
九、免疫荧光(IF)检测
细胞用PBS清洗3次，室温下用4％多聚甲醛(Solarbio,中国北京)固定1h，再用PBS轻轻清洗3次。然后用5％的山羊血清(Solarbio，中国北京)阻断细胞30min。封闭后的细胞分别与兔抗α-SMA单抗(1:200；美国细胞信号技术公司)、兔抗FAP-α(1:200；英国坎布)、兔抗NRK(1:25；Origene，美国)抗NRK孵育，再与二抗(1:200；Abcam，英国)室温孵育1h。免疫标记细胞用25μl DAPI(Solarbio，中国北京)室温复染8min。细胞样本在共聚焦显微镜下观察(Leica Microsystems CMS GmbH/Model DMi8，德国)。
十、实验结果
1、组织标本的临床特征
共纳入13例LUAD患者样本进行RT-qPCR验证，所有LUAD患者在诊断时均处于I期、II期或III期。患者的临床特点见表2。
表2肿瘤组织和癌旁组织用于分离CAFs和NFs的患者的临床特点
2、CAFs的鉴定
如图1所示，CAFs的特征表现为典型的纺锤形、扁平和成纤维细胞样的形态，类似于NFs。随机选择一对CAFs和NFs，通过WB和IF分析鉴定激活的成纤维细胞的特异性生物标志物。如图2所示，CAFs强表达α-SMA和FAP-α，而NFs和A549弱表达这两个活化成纤维细胞的生物标志物。此外，CAFs和NFs均强表达间充质标志物Vimentin，但不表达上皮标志物E-cadherin。然而，LUAD细胞系A549强表达E-cadherin，低表达Vimentin(图2)。IF试验中这些标记物的表达与WB试验中的表达一致(图3)。结果表明，成功地分离了CAFs和NFs，没有上皮细胞或癌细胞的污染。
3、从RNA-Seq中鉴定DEGS
CAFs和NFs的转录分析显示总共检测到35,259个基因。主成分分析(PCA)结果表明，CAFs与NFs分别聚为一类，PC1聚类率高达45.9％(图4)，表明这两类具有不同的生物学特性，适合于下一步的差异性分析。DEGS上调的标准为差异倍数(Fc)>2，调整后P值<0.05；下调DEGs的标准为FC<-2，调整后P值<0.05。总共鉴定出1799个DEGs，包括650个上调的DEGs和1499个下调的DEGs。上调和下调的DEGs的分布由火山图显示(图5)。上调的DEG用红色表示，下调的DEGs用蓝色表示。与NFs(图6)相比，log2(Fc)的热图在CAFs中显示出不同的表达模式。
4、CAFs中DEG的GO分析与非CAF中DEG的功能比较
为确定DEG在CAFs中的功能。与NFs相比，GO分析对潜在功能进行了分类(调整后的P值<0.05)。BP类有188个种类，MF类有83个种类，CC类有40个种类。如图7-12所示，分别显示了BP、MF、CC类别中上调和下调DEGs的前15个显著丰富的集合。在BP类别中，大多数上调的DEGS集中在骨骼系统的发育(GO：0001501)，也与细胞器分裂(GO：0048285)和核分裂(GO：0000280)有关(图7)。此外，上调的DEGs还与MF类(图8)的细胞外基质结构成分(GO：0005201)、G蛋白偶联胺受体活性(GO：0008227)和单链ATP酶活性(GO：0043142)以及CC类(图9)的染色体区域(GO：0098687)、细胞外基质(GO：0031012)和含胶原的细胞外基质(GO：0062023)相关。同时，下调的DEGs在BP类(图10)富含细胞外结构(GO：0043062)、血管内径正调控(GO：0097755)、肌肉系统突起(GO：0003012)，在MF类(图11)富含受体调节活性(GO：0030545)、受体配体活性(GO：0048018)、细胞外基质成分(GO：0005201)，在CC类(图12)富含细胞外基质(GO：0031012)、含胶原的细胞外基质(GO：0062023)、神经元胞体(GO：0043025)。综上所述，我们的结果表明上调和下调的DEG主要参与ECM功能
CAFs中的KEGG通路分析发现，上调的DEGs最常参与与糖酵解/糖异生、戊糖磷酸途径、半乳糖代谢、果糖和甘露糖代谢、抗坏血酸和醛酸代谢相关的5条信号通路(图13)。另一方面，与DEGs表达下调相关的8条信号通路分别为糖酵解/糖异生、半乳糖代谢、脂肪酸生物合成、抗坏血酸和醛酸代谢、戊糖和葡萄糖醛酸相互转换、脂肪酸伸长、戊糖磷酸途径和果糖和甘露糖代谢(图14)。因此，代谢途径尤其是糖酵解/糖异生途径丰富了DEGs，提示CAFs可能主要通过代谢途径调控LUAD的发生发展。
5、用RT-qPCR验证重叠DEGS
从RNA-Seq数据、LncRNA微阵列和ArrayExpress的公共数据集(E-MTAB-6149和E-MTAB-6653)(图15，表3)中获得10个|Fc|>2和调整P值<0.05的重叠DEG，包括6个上调的DEG和4个下调的DEGs(图15，表3)，其中6个是上调的DEGs，4个下调的DEG是来自ArrayExpress(图15，表3)的公共数据集(E-MTAB-6149和E-MTAB-6653)。与RNA-seq数据一致，6个重叠DEGs(ZNF93、ZNF827、NRK、DPYSL4、HES4、LYPD6B)在CAFs中显著上调，而FCGBP和CFI在CAF中显著下调。相反，在RNA-seq低表达的CAF中，SETBP1和HMCN1表达升高。如图16所示，NRK被确认为十个重叠DEGs中最显著的上调基因。
表3 CAFs与NFs相比，CAFs中有10个交集差异DEGs
6、NRK在LUAD患者CAF中的表达水平及其与预后的关系
CAFs中NRK的mRNA水平显著高于相应的NFs、正常肺上皮细胞BEAS-2B和两个LUAD细胞系A549和H1299(图17)。WB结果表明，NRK蛋白在CAF中强表达，而在NFs、BEAS-2B、A549和H1299中弱表达(图18)。此外，结果表明，NRK蛋白主要在CAF的胞核和胞浆中表达，而在NFs、BEAS-2B、A549和H1299中几乎不表达(图19)。另外，我们还选择了CCLE数据库中表达NRK的191个肺癌细胞株。每株细胞均有相应的NRK表达值。191个肺癌细胞株的NRK表达热图显示大部分条带为蓝色，表明NRK在肺癌中低表达(图20)。仅32株细胞株NRK高表达，其中LUAD细胞株13株，LUSC细胞株1株，大细胞癌细胞株1株，小细胞肺癌细胞株3株，其他亚型肺癌细胞株14株。而NRK低表达的细胞株有159个，包括42个LUAD细胞系，6个LUSC细胞系，3个大细胞癌细胞系，30个小细胞肺癌细胞系，78个其他亚型肺癌细胞系(如下表，图20)。此外，NRK的mRNA在LUAD患者中的高表达预示着较短的总生存期(logrank P＝0.0093)(图21)。结果表明，NRK的mRNA在大多数LUAD细胞系中弱表达或不表达，但在LUAD的TME内的CAFs中更好地表达，并与LUAD患者的低生存率相关。
综合以上结果，利用RNA-seq和综合生物信息学方法对来自LUADⅠ、Ⅱ、Ⅲ期组织的CAFs和NFs进行分析，发现NRK在肺癌肿瘤相关成纤维细胞中特异表达，它可作为肺癌的临床诊断生物标记物，将能提供一个更好的诊断预后指标；LUAD相关CAFs中NRK显著升高，可将NRK作为LUAD联合治疗的靶点。
需要指出的是，上述较佳实施例仅为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施，并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围之内。
序列表
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<120> NRK在肺癌治疗和预后诊断中的医药用途
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Claims (10)
1.NRK在制备诊断肺癌预后的产品中的应用。
2.根据权利要求1所述的应用，其特征在于：所述产品以NRK作为诊断标记物。
3.根据权利要求2所述的应用，其特征在于：所述NRK的高表达预示肺腺癌患者预后不良。
4.根据权利要求1所述的应用，其特征在于：所述产品为实时荧光定量PCR检测试剂盒或诊断试纸。
5.NRK在制备治疗肺癌的药品中的应用。
6.根据权利要求5所述的应用，其特征在于：所述药品以NRK作为治疗靶标。
7.NRK中和抗体在制备诊断肺癌的产品中的应用。
8.根据权利要求7所述的应用，其特征在于：所述产品是诊断试纸或试剂盒，所述产品的诊断样品是肺分泌物或肺组织。
9.根据权利要求7所述的应用，其特征在于：所述产品以NRK中和抗体作为诊断标记物。
10.NRK中和抗体在制备治疗肺癌的药品中的应用。
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