CN111235279A - 肿瘤相关成纤维细胞特异性长非编码rna在肺腺癌预后评估中的应用 - Google Patents
肿瘤相关成纤维细胞特异性长非编码rna在肺腺癌预后评估中的应用 Download PDFInfo
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Abstract
本发明公开了肿瘤相关成纤维细胞特异性lncRNA标志物或其组合标志物及其应用,所述lncRNA标志物或其组合标志物包括LINC01614、AC134312.5、LINC01429、LINC02544、AC012213.3或LINC01705中的一种或几种。本发明公开了肿瘤相关成纤维细胞特异性高表达lncRNA(LINC01614、AC134312.5、LINC02544、LINC01705)是一类可靠的与肺腺癌预后相关分子靶标,可以通过检测患者该类基因表达水平用于肺癌预后判断,指导临床制定个体化化疗方案,提高疗效。
Description
技术领域
本发明属于生物技术和治疗领域,涉及肿瘤相关成纤维细胞特异性长非编码RNA在肺腺癌预后评估中的应用。
背景技术
肺癌发病率及死亡率一直稳居所有恶性肿瘤之首,其中超过2/3病理类型为肺腺癌,发病率逐年增加,尤其是在女性,非吸烟患者和年轻人中。影响肺腺癌预后的因素包括肿瘤分期,早期患者通过外科手术可以有效控制肿瘤的复发、转移。而晚期患者常常需要接受化疗、放疗、分子靶向治疗及免疫治疗,其中肿瘤耐药、微环境中免疫细胞耗竭等决定了晚期患者的预后。越来越多研究表明,肿瘤微环境细胞在肿瘤的预后中意义重大,肿瘤的进展依赖于癌细胞与微环境中的成纤维细胞,内皮细胞,免疫细胞和基质成分。肿瘤相关的成纤维细胞(cancer associated fibroblasts, CAFs)是一类多种来源的异质细胞群体,研究表明正常成纤维细胞、间充质细胞、上皮细胞、周细胞、脂肪细胞和内皮细胞都可能转化为CAFs。CAFs作为肿瘤基质的主要成分,可以通过细胞直接接触或分泌细胞因子等方式促进肿瘤细胞的侵袭转移,增强细胞化疗耐药。肺腺癌是富含CAFs的肿瘤之一,CAFs能够调控肿瘤细胞生长、转移,干细胞微环境,免疫抑制和化疗耐药,与肺腺癌预后密切相关。
长链非编码RNA(long non-coding RNA,lncRNA)是一类长度大于200个核苷酸,数量远大于编码蛋白基因的RNA,因为生物学功能丰富而成为近年来研究热点。在肺癌中,已发现肺腺癌转移相关转录因子(metastasis associated lung adenocarcinomatranscript 1, MALAT1)影响非小细胞肺癌患者复发和转移,是重要的预后指标;HOX转录反义RNA(HOX Transcript Antisense RNA, HOTAIR)可通过调控p21促进细胞增殖、介导铂类耐药。此外,外泌体lncRNA也成为了一类具有肿瘤标志物潜能的分子靶标。微环境细胞来源的外泌体lncRNA通过细胞间的交互作用,将不同表型传递给邻近细胞,促进肿瘤发展。
发明内容
发明目的:本发明首先基于美国癌症基因图谱(TCGA)及肿瘤细胞株百科全书(CCLE)数据库的RNA-seq数据,初步筛选出高表达于肺腺癌但在51种肺腺癌细胞株均不表达的lncRNA共195条。将该195条lncRNA在TCGA数据中结合肺腺癌单细胞测序数据中细胞亚群标志物进行相关性分析,获得20条潜在肺癌微环境高表达lncRNA,其中有6条lncRNA与CAFs细胞亚群高-中相关(LINC01614、AC134312.5、LINC01429、LINC02544、AC012213.3、LINC01705)。这6条lncRNA是一组潜在的CAFs来源高表达lncRNA,我们将其命名为肿瘤相关成纤维细胞来源lncRNA(Cancer Associated Fibroblasts Derived LncRNAs 1-6,CAFDL1-6)。
上述6条lncRNA均与CAFs密切相关。获取5例肺腺癌患者新鲜组织,原代培养CAFs细胞及癌旁正常成纤维细胞(normal fibroblasts, NFs),荧光定量PCR显示:6条lncRNA均只表达于CAFs中,显著高于NFs细胞。获取50例肺腺癌患者组织样本,检测6条lncRNA的表达,结果表明其在癌中表达显著高于正常组织。将6条lncRNA与TCGA肺腺癌患者生存时间进行相关性分析,发现LINC01614、AC134312.5、LINC02544、LINC01705表达可以用于肺腺癌预后评估,呈现表达越高,患者预后越差。
本发明为以LINC01614、AC134312.5、LINC02544、LINC01705为基础建立有效的术后预后评估标志物,为开发个体化治疗靶点提供新的途径和理论依据。
技术方案:为了解决上述技术问题,本发明提供了肿瘤相关成纤维细胞特异性lncRNA标志物或其组合标志物,所述lncRNA标志物或其组合标志物包括LINC01614、AC134312.5、LINC01429、LINC02544、AC012213.3或LINC01705中的一种或几种。
其中,所述LINC01614、AC134312.5、LINC01429、LINC02544、AC012213.3和LINC01705的核苷酸序列分别如SEQ ID NO:1 、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示。
在一些具体的实施方式中,本发明提供了用于检测前面所述的lncRNA表达水平的试剂。
在一些具体的实施方式中,所述试剂为荧光定量PCR方法检测lncRNA表达水平的试剂。
具体地,所述试剂可包含反转录试剂盒和荧光定量PCR试剂盒。反转录试剂盒包括聚合酶链式反应所必需的元件。荧光定量PCR试剂盒包含6对特异性针对lncRNA的引物。各引物是具有特异性针对所述基因的核酸序列的核酸,其长度约20bp,扩增产物约200bp。其中,所述LINC01614、AC134312.5、LINC01429、LINC02544、AC012213.3和LINC01705对应的引物对分别为SEQ ID NO:7和SEQ ID NO:8、SEQ ID NO:9和SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示。
此外,所述试剂盒还可包含针对内参基因的核酸序列引物。
其中,在试剂盒的肺癌预后评估中,当肿瘤组织肿瘤相关成纤维细胞特异性lncRNAs (CAFDLs)表达量高于特异性lncRNA标志物对应的cutoff值,则提示所述受试者预后不良可能,否则提示所述受试者的预后良好;具体的,所述特异性lncRNA标志物中LINC01614、AC134312.5、LINC02544、LINC01705对应的cutoff值分别为1.12,5.97,5.80,6.06。
其中,所述荧光定量PCR试剂盒可包含PCR反应管、预混合反应液和无菌水。
在一些具体的实施方式中,本发明的lncRNA在TCGA肺腺癌组织中高表达。
在一些具体的实施方式中,本发明的lncRNA在CCLE数据库中的肺腺癌细胞株及正常肺成纤维细胞株(SALE)中低表达或者不表达。
在一些具体的实施方式中,本发明的lncRNA在肺腺癌单细胞测序数据中与CAFs细胞亚群高-中相关。
在一些具体的实施方式中,本发明的lncRNA在NJLCC非小细胞肺癌队列转录组测序数据中与CAFs相关。
在一些具体的实施方式中,本发明的lncRNA在原代肺腺癌CAFs中高表达,NFs中低表达。
在一些具体的实施方式中,本发明的lncRNA在TCGA数据库肺腺癌组织中与患者预后相关,呈现表达越高,预后越差。基于此,我们提出lncRNA基因表达检测可以应用于肺腺癌预后评估,指导个体化治疗。
在一些具体的实施方式中,本发明将以上肿瘤相关成纤维细胞特异性lncRNA标志物分别进行了肺癌预后评估中的应用。
在一些具体的实施方式中,本发明还将肿瘤相关成纤维细胞特异性lncRNA组合标志物进行了肺癌预后评估中的应用。
在一些具体的实施方式中,本发明还将肿瘤相关成纤维细胞特异性lncRNA标志物或其组合标志物的检测试剂在制备肺癌预后评估的试剂盒中的应用。
本发明还涉及一种用于肺癌预后评估的试剂和/或试剂盒,所述试剂盒包含反转录试剂盒和/或荧光定量PCR试剂盒。
在一些具体的实施方式中,所述荧光定量PCR试剂盒包含特异性针对lncRNA的引物对。
在一些具体的实施方式中,所述试剂盒还可包含针对内参基因的核酸序列引物。
在一些具体的实施方式中,所述荧光定量PCR试剂盒还包含PCR反应管、预混合反应液和无菌水。
有益效果:与现有技术相比,本发明的优点是:本发明公开了肿瘤相关成纤维细胞特异性高表达lncRNA(LINC01614、AC134312.5、LINC02544、LINC01705)是一类可靠的与肺腺癌预后相关分子靶标,可以通过检测患者该类基因表达水平用于肺癌早期诊断或判断预后,指导临床制定个体化化疗方案,提高疗效。
附图说明
图1肺腺癌微环境CAFs细胞来源高表达lncRNA筛选流程;
图2肺腺癌微环境CAFs细胞来源高表达lncRNA筛选流程;图2中代码分别代表:N1-NCIH441,N2-NCIH22,N3-NCIH2228,N4-SKLU1,N5-HCC44,N6-HCC78,N7-RERFLCAD1,N8-NCIH1703,N9-NCIH1395,N10-NCIH1781,N11-MORCRP,N12-NCIH1563,N13-NCIH1358,N14-NCIH1650,N15-NCIH1437,N16-NCIH1792,N17-NCIH1355,N18-RERFLCAD2,N19-NCIH2347,N20-NCIH2087,N21-NCIH2085,N22-NCIH1573,N23-NCIH1651,N24-NCIH1666,N25-NCIH1734,N26-NCIH2342,N27-NCIH322,N28-NCIH2291,N29-NCIH2023,N30-DV90,N31-NCIH1648,N32-NCIH1373
N33-NCIH1623,N34-NCIH1755,N35-NCIH1693,N36-CALU3,N37-NCIH2009,N38-HCC4006,N39-NCIH2405,N40-NCIH2126,N41-HCC827,N42-LXF289,N43-HCC2108,N44-NCIH650,N45-NCIH854,N46-CORL105,N47-HCC2279,N48-HCC1833,N49-NCIH3255,N50-HCC364,N51-HCC827GR5;
图3微环境亚群细胞高-中度相关高表达lncRNA筛选;
图4 NJLCC转录组测序验证6条CAFs来源lncRNA相关性;
图5为CAFs和NFs细胞鉴定;
图6为CAFDLs在CAFs和NFs细胞中的表达
图7为CAFDLs在肺腺癌组织中的表达;
图8为CAFDLs与肺腺癌预后的关系;TCGA数据库提示CAFDLs高表达与肺腺癌不良预后相关;
图9为CAFDLs与肺腺癌预后的关系;qPCR结果提示CAFDLs高表达与肺腺癌不良预后相关;
图10为CAFDL1,CAFDL2,CAFDL4和CAFDL6联合检测与肺腺癌预后的关系;qPCR结果提示联合检测值高表达与肺腺癌不良预后密切相关。
具体实施方式
下面通过具体的实施例对本发明进一步说明,应当指出,对于本领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干变型和改进,这些也应视为属于本发明的保护范围。下述实施例中的实验方法,如无特殊说明,均为常规方法。
实施例1
1、原代细胞培养
选取新鲜肺腺癌肿瘤组织或癌旁正常组织100 mg,PBS清洗3次后,将组织剪碎成1.5~2.0 mm3大小,放入含0.02%EDTA胰酶消化20 min。血清终止消化后,过400目筛网。PBS洗涤,1000 rpm离心5 min,弃上清。重复3次。用含10%胎牛血清,0.37%碳酸氢钠,0.1 mM非必需氨基酸,100μg/ml青链霉素的DMEM培养基置37°C,5%CO2培养箱中培养。每3-4天换液一次,细胞达90%融合时传代。采用分离培养后的第3~6代细胞用于实验。
2、肺腺癌组织样本收集
从江苏省肿瘤医院生物样本库申请获取含预后的肺腺癌组织和配对正常组织各50例,所有样本离体后立即放入液氮冻存,整个操作及保存过程遵循无酶原则。
3、RNA提取
Trizol裂解原代培养细胞组织。利用细胞/组织RNA抽提试剂盒(TIANGEN公司)提取总RNA,具体操作详见说明书。
4、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 2100 Bioanalyzer检测总RNA的浓度、RIN值、28S/18S和片段大小,RNA完整性指数合格(RIN值>7,28S/18S比值>1.5,样品峰图基线平整,18S、28S可见清晰单峰),RNA浓度≥20ng/μl,进行mRNA文库构建。
5、逆转录和荧光定量PCR
将RNA的定量统一后,按照TaKaRa反转录试剂盒(PrimeScript® RT reagent Kitwith gDNA Eraser)的说明书进行第一链cDNA的制备:
1)基因组DNA的除去反应:
2)基因组DNA的除去反应程序:42℃反应2分钟;4℃保存;
3)逆转录反应:
4)程序:37℃反应15分钟;85℃反应5秒钟;4℃反应5分钟;
5)将逆转录所得到的cDNA样品以及剩余的RNA样品,-80℃冰箱内保存备用。
6)荧光定量PCR
10μl Real-Time PCR 体系:
引物序列:
7)PCR扩增程序:95℃,3分钟;
8)PCR结果与计算
以β-actin为内参基因,△Ct值=目的基因Ct值-内参基因Ct值,△△Ct值=癌组织△Ct值-癌旁组织△Ct值。以2-△△Ct表示样本目的基因相对表达量。采用独立样本t检验, P<0.05具有统计学意义。
6、细胞免疫组化染色
将5×104/ml细胞接种到细胞爬片,培养2天后用4%多聚甲醛固定30分钟,PBS洗涤后,封闭30分钟,加入一抗,4℃孵育过夜。PBS洗涤三遍后,加入二抗,室温孵育2小时。PBS洗涤三遍后,DAPI复染细胞核。封片观察。染色结果见图2。
7、TCGA及CCLE数据检索
从TCGA数据网站(https://genome-cancer.ucsc.edu/)下载人肺腺癌RNA-seq数据和预后。其中,来自398个患者的433个样本(包含32个配对的肿瘤正常样本)的RNA-seq数据可用。依据肿瘤与癌旁的正常组织间mRNA表达存在统计学差异FC > 2,p < 0.01原则筛选出823条差异表达lncRNA。从CCLE数据网站(https://portals.broadinstitute.org/ccle/)下载人肺腺癌细胞株和正常肺成纤维细胞株RNA-seq数据,依据TCGA高表达,CCLE不表达原则筛选出195条lncRNA。
8、统计学分析
采用SPSS19.0和GraphpadPrism6.0软件进行数据统计和结果分析,结果以平均值±标准误表示。肿瘤和癌旁两组间比较时用独立样本t检验分析。以P<0.05和P<0.01为差异,具有统计学意义。
9、实验结果:
1)CAFs相关lncRNA筛选
基于美国癌症基因图谱(TCGA)及肿瘤细胞株百科全书(CCLE)数据库的RNA-seq数据,初步筛选出高表达于肺腺癌但51种肿瘤细胞株均不表达的lncRNA共195条(图1&图2)。进一步在TCGA数据中结合肺腺癌单细胞测序各细胞亚群标志物进行相关性分析,获得20条潜在肺腺癌微环境细胞高表达lncRNA,其中有6条lncRNA与CAFs细胞亚群高-中度相关(LINC01614、AC134312.5、LINC01429、LINC02544、AC012213.3、LINC01705)(图3)。在团队已发表的NJLCC队列转录组测序数据中进行验证,发现上述6条lncRNA均与CAFs细胞密切相关(图4),提示这6条lncRNA是一组潜在的CAFs来源高表达lncRNA,因此我们将其命名为肿瘤相关成纤维细胞来源lncRNA(Cancer Associated Fibroblasts Derived LncRNAs 1-6,CAFDL1-6)。
2)原代细胞培养及鉴定
获得5例肺腺癌患者新鲜组织,消化法培养原代CAFs和NFs,可见细胞呈梭形,胞质丰富,胞核位于细胞中央,圆形或椭圆形。细胞生长较快,呈放射状或漩涡状排列。免疫组化染色显示,CAFs细胞表达间质标志蛋白Vimentin及活化的成纤维细胞标志物αSMA,而不表达上皮细胞标志蛋白cytokeratin。NFs细胞αSMA和Cytokeratin蛋白阴性表达,Vimentin蛋白阳性表达(图5)。
3)CAFDL在CAFs及肺腺癌组织中高表达
qPCR检测结果表明,CAFDL1-6在CAFs中高表达,在NF中均为低表达,P<0.05,具有统计学差异(图6)。在50对肺腺癌组织中,CAFDL1-6表达均高于配对的正常癌旁组织,P<0.05(图7)。
4)LINC01614、AC134312.5、LINC02544、LINC01705高表达与患者预后相关
将6条CAFDLs在TCGA肺腺癌数据库中进行生存分析,根据每条lncRNA的FPKM值,计算出最佳cutoff值,其中:CAFDL1:0.46,CAFDL2:0.08,CAFDL4:0.19,CAFDL6:0.06。将FPKM值大于cutoff值的定义为高表达,低于cutoff值的定义为低表达。死亡患者标记为1,否则标记为0。使用K-M法计算高表达及低表达组患者的生存曲线。结果表明CAFDL1,CAFDL2,CAFDL4,CAFDL6四条lncRNA表达越高,肺腺癌患者预后越差,平均生存时间明显短于低表达组。(图8)。
进一步,在50对肺腺癌组织中进行生存验证,其中CAFDL1 cutoff值:1.12,CAFDL2cutoff值:5.97,CAFDL4 cutoff值:5.80,CAFDL6 cutoff值:6.06。结果可见CAFDL1,CAFDL2,CAFDL4,CAFDL6高表达组,患者生存时间较低表达组显著减少(图9)。
最后,将CAFDL1,CAFDL2,CAFDL4,CAFDL6四条lncRNA联合检测用于预后比较。以Score = 2.492×CAFDL1 + 2.408×CAFDL2 + 3.288×CAFDL4 + 2.534×CAFDL6计算每个样本表达值,联合cutoff值为1.59。结果表明,CAFDL1,CAFDL2,CAFDL4,CAFDL6联合检测值表达越高,患者预后越差,统计差异越显著(P<0.001),提示联合检测更有助于肺腺癌患者预后判断(图10)。
序列表
<110> 江苏省肿瘤医院
<120> 肿瘤相关成纤维细胞特异性长非编码RNA在肺腺癌预后评估中的应用
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 648
<212> DNA
<213> LINC01614(CAFDL1)
<400> 1
gggaaaaggg ctgagggggt agaggtataa aaatcagata gctcagcact tcgctccaaa 60
ggagctgaag gagagaggat tccactgcgt gtatgttttc tctttcacgt ctcacttttg 120
ttctctgcca tcaggaaatg cagtagacat ttgtcaccag cagctctttg gagacttcaa 180
tgttccttcc tcccaggatc cgaaagggga cttcagacac ggagaatcta agacaccagg 240
agctcagaag aatggagtct caagacatca ggcaaatacc gaagagatca gagccaagtt 300
ctaaagggca gccatttccc aaatgtcaac caagagcgaa gccaagagaa ttatgatttc 360
ctggctatac aatgacagga aaagcttgat caaagctgga gtgcaagtta aaaagtgtaa 420
ggtactcaag tgctagggtc tgtgtccaag cccaatcaat tgatcattag ttgtttttaa 480
cttcaacgtt tcttttgcaa cagcacctct tccttaaagt agcaatctta gcagactgcc 540
accttacatg atgcatctta ttctccaagg acaatgaaga ctgaactatc gcacattacc 600
taagaaagat gggaattgac atgcacatca caattgtata cacaacag 648
<210> 2
<211> 2066
<212> DNA
<213> AC134312.5(CAFDL2)
<400> 2
ggatgccgga gccctcccag cgagagccgt gtttatttta ctttacatac attttccaac 60
acggagcggc ttgcacacat gcagctctta ggcccgggcc gcacgtctca gaagccccgt 120
gtgcgacttt gaccgccgca cgatcctctg ccgggggagg tgggcccgct gcgctttggg 180
agcacccgcg cccgacactg aggtctcggt gctgtgttcg gcctcttcgt ccctgcgggt 240
cccctctggg agcagaggcg gtcggaaaac cctggggctg aagtgcaggc ttcgggagga 300
cgcgacctgc caagatcagc tcccggcacg tgatgggagc ctggctcacc ttcccccagc 360
gcacgatggg ccgcagcctc cccggtcggc ctggcctgct ggaaaggagc agctctgttt 420
ccagaggctt ctggcgaagc ccacggcctc ccattgttgg ctgatttata aggaaagagg 480
ggaaaggcca agtgtggatg ccattagcat aacctaatcc agaccccatg acaagtccag 540
gatcctgcag ggagagggca tccttgaacg tgaaggactg gctttggaaa cttggcctcc 600
cggaagaaag gtctccgggc ccacccacac ccaccttgtg gacgcccccg cagtcgaata 660
cactccacag gaagacggac cacaaacagc agcagcctcc ggtgtcggcc cagtgatccg 720
ggagctcaga gttgcagcaa gcttaaactt ccttaaaaac aaacctctgg gaagaggaaa 780
acgaggcaag atgctgagaa aaagtcatct cccactctgc ccttctctgg caaatcgaag 840
tgtcctcttg tgggaggcgg tgggcagcga cactcctgtt ggacgtcagt gctctcggga 900
tgggcccggc caccccggag cagaactcac ttagggcctg gtgcccctgt gcccagttct 960
cagaacgtcg gagtgctcag ttctcagaac gccggagtgc cccattcata acctgcagcc 1020
ccgttcatag cctgcagctc ccctccttcc tctcctcccg gtcgtcaggg agcaccccca 1080
gctcccctcc ccactctcaa accctcctcc tccctcccct ctctcatttc cctgtttgca 1140
ggatctcctc ctgcccactc tccagcccag gtcctcccct ctcatctcca attctgcttc 1200
atgggccatg acagcaggca ggccatggcc tgctctcagc agtttgatcg atggactaag 1260
gagatcactc ctatgagagt gaaaaagtca aatgtgcctg agattcggat ggggagctct 1320
caaagctacg gattgcaaga gtcaacaccg cctcaaaagt acaaggccgg ccgggtgcag 1380
tggctcacac ctgtaatccc agcactttgg gagactgagg cgggtggatc acctgaggtc 1440
aggagctcaa gaccagcctg accaacatgg tgaaatcccg tctctattaa aaatacaaaa 1500
aagattagcc gggtgtggtg gtgcatgctt gtagtcccag ctactcggga ggctgaggca 1560
gggggagcac ttgaacccgg gaggtggagg ttgcagtgag ccgagattgc accattgcac 1620
tccagcctgg gcaacagagt gagaccctgc ctcaaaaaaa taaaaaaata aaagtacgag 1680
gcccggttta aatgcaggag gaatctggtg agcgagtttc actctgatgc aacagaaaag 1740
ccaagccaag ccttggctgt gcctggtctt ggccggggag ccacaggcag ggtctgctgg 1800
tttcggtgtc aggggcacat ccccaggacc acgtctgaga tgggaagtgc cttgatggaa 1860
aactgatacc tttttctttc cgagtaactt tagaacatgt cgcttcaaat ataactaata 1920
tcctgatttc agtggacttg catggaagga aacgcaagtt catttgccag tggggggtga 1980
ggactgggag agtggggagc agggtgggga agaggggagc attccacaag ccgcaagagc 2040
ctggacacct ccagccttca ataaat 2066
<210> 3
<211> 2326
<212> DNA
<213> LINC01429(CAFDL3)
<400> 3
tttctgctgg ctggatgggg atgtggagga gaggaccctt ggaccatggg gatgggagca 60
acgtgccagg catggcagcg caatagaggg tggaacatct tttgaagagc tcagaccctg 120
gacccctgat cttaatgagc tttgcagcct tcagtgacac aacctggatt taaactcatg 180
gatgtcggcc tctgctctgc gttgaattgt atcctcccaa aaaggagatg ttggagctct 240
gagacccagc acctcagaat gtgtttctac ttggaaatag ggtctttacc aagaccctat 300
tccaatagga ctggtgacct gtgggagaaa tgtggacaca gagacagaca gacatagagg 360
gaagatgacg gcagagtcac agggagaaga cggtcatcta cgagccaagg agagaggcct 420
ggcacagatc cttccctcac agcacccaga gagccttcag acaatacatc tgtgctgtgt 480
aagccgctcc gtctgtggct ctctgttaca gcagccccag aaaacaaata gagactctta 540
attgccaagc catgttcctc ccatagtacg atttctcatc agatagtcta tcttcgctgc 600
taaaacaggt atttctggat ggtagaggtc atgtgtagct tgcatattta gtatgaggta 660
tgtgaaagtc acctcataga tattaaatta tttggctaca gctttgagct tggcctctct 720
cttaaggatt accttgccat agggacagga agccatcgtg atataaaggc tgaagtttct 780
gtaagatttt gatttttaat ctaacatgat attaacaact gtgcccgttg ggcgtgatgg 840
ctcacaccta caatcccagc actttgggag gccaaggcgg aggatcactt gaggtcagga 900
gtttgagacc agcctggcca acatggtgaa accccgtctc taccaaaaat acaaaaatta 960
gccgggcttg ggggtgcatg cctgtaatcc cagctacttg ggaggcgaag gcaggagaat 1020
tgcttgaacc caggaggtgg aggttgcagt gagccaagac tgtaccactg cgctccagcc 1080
tcggcaacag agtgagactc tgtctcaaaa cagacaaaca aacaaaataa taactgtacc 1140
tatacaaggt ccccaagata attaaaaggc tcacttgagc agagtaactg aggagagtct 1200
tttggggttc tttgtttttt ttttttagag acagtctcac tctgtcgccc agtctagagt 1260
gcagaggcgc aatcttggct cactgcaacc tccgcctccc aggttcaaga gatcctcctg 1320
cctcggcctc tcaaagtgct gggactacag gcacatgcca ccatgtccgg ctgaggaatg 1380
tttaataaaa ggggtattta ccaagatata agcaaggtat agagaaatca gcagggttga 1440
tgaagcaccc cggggctacc aacaaccaaa agcccttccc tctccaaggc ctccaaaggc 1500
agcaggaggg agtggttatc agaaaatggc aagatccata ggaaaaaggc caccccatgg 1560
gagctgtggg tttcagtaga gggacacagc cactgccaga ccatggccag ccagggagtt 1620
aatagcccag ccccaccttg gccctcccat ctcccactcc tccttattgg ctgaacccag 1680
caagaaacca gatgtcacag aaccaggaga tgtggtcccc actgcccggc ctcttgggca 1740
cagaggaggg aagagaaaat ggagagtgga tctggagggg caaatgaagt attaatattc 1800
tgtacactca gaatgtccac agagtgaaca ctggcttgcc ggctgacccc atttcttgaa 1860
aatcatccta tccctgcagg ttatttatgt tgttaccctt ttgcaagaac cacataaaat 1920
attttacatg cttttaaaac tcataaatgt aatgaagcca agatgtttca gcaaatgtgc 1980
tcaatgggca aatccttcca aagatctgct ctcagctgac gtcaggacaa aaggcacatc 2040
tcacgacctg cagagcgtct gacgttaaag ccaataaatt catccccctg acccacatgg 2100
cttccagaat gtacaagctg tcagtggatc ttaaaaatgt atctattcct tcattcactt 2160
cctgccgttc gtctcttgca agtatttact gctctttaaa aactgaactc caccagctgg 2220
aaatgcatta gtgcagggag atggctgtac tttgttccgg caggaattgt gcatgcaact 2280
tccaatctga cttttctgag acattaataa aaacacatgc gtcctg 2326
<210> 4
<211> 672
<212> DNA
<213> LINC02544(CAFDL4)
<400> 4
ataccgttct tctggtagtg aacaagtctc acaagagctg atggttttat gaagggtttc 60
tacatttgca tcttcctcat tttctcttgc cgccgccaca taagaagtgc ctttcacctc 120
tcatcatgat tctgtggcct ccccagccat gtggaacttt tttctcttgc tgctaccatg 180
taagaagtgc ctttcacctc gtgccatgat tatgagactt ccccagccat gtggaactgt 240
taagtccaat taaacatctt tttcttctta gtcccaggtt gttctcattc gtggctggat 300
cacaccagca agaaggattt taaaggacaa ctccttgggg ccgtccaaca ctgaaaccgc 360
ctgaccgctg ctgtctttgt gcttgcagaa aatcacggtg ggcacaaaat tttggacacg 420
atttcgagag cgtgagagct actgttcacg aatccagggc cagcctcgtg tctgtcttcc 480
tttaaagcac ctggcttggt acctaaaatg ttcctttttt tttttttttt gaaattcaat 540
aacttcagaa caggcgttga tgtagacaga tctccatctt tttggttttt tgagtgagtt 600
tttgctctgt aggctgaatt ctttggaatt tagttctgaa atcaccctat tgttgagtat 660
tcgtttccta gc 672
<210> 5
<211> 777
<212> DNA
<213> AC012213.3(CAFDL5)
<400> 5
attttaatat attatgaact catgtattta aacatatttg atgttttaaa ccattgcagt 60
tctctttatt gatgcttaaa ttgtgtcatc tttaacagtg agaaccaatt caaatgttgg 120
ctcttggatc cttttgagtt gtttttcata gctgccttgt tttccagtat gacaaaatgt 180
tccatgctca tattgtacat ttcctgcccc ataggtgaaa tctcccattt cttcaaggat 240
ttttagttcc ttttagtagg aaatgggagt gtagagatca cattctgggc attactggat 300
tggtcattgt ttctgggcct tttcggtaaa caaagctaga ttttgttgct gttttgtagc 360
tttatttaag ataaagtact ttaggagttc atactgatac ttccaattca agcccagaat 420
ttacttaact tcactaatct tacatgtata ctgccttctt ggattttttt ctgatcacct 480
cagccagaag tgatcactct tcctttgaac cacatcaccc acttcaacac ttttatagta 540
atcaacacat taacttttga tttattgtta tttctatata tgtctgatat tctctaccaa 600
atggtaagga atttgaggat agaatccatc ctttgttacc ctttgttaaa taaagataaa 660
attcctaaag tgctgatata tttcctgaca tataaatacc actttctctt tgtattcctt 720
taaaaatgga accatattat acatgtacta aaaattaata aatgagtgtt aaatgaa 777
<210> 6
<211> 873
<212> DNA
<213> LINC01705(CAFDL6)
<400> 6
ctctttgcct gctgcaatcc atgtaagatg tgacttgttc ctccttgcct tctgccatga 60
ttgtgaggcc tccacagcca tgcggaactg ttgaaataca cactacctga agctggataa 120
ctcatgaact ctgttgacaa ttatattctt tgtcttttta attaactcat ttggagttgg 180
tgtcctgagg acctaagcct aagaatcttc aagatggaag agtagagtga acaatgaatg 240
agccagaggg ctcttgtcct ctgtgtgcag ctctgggcag ccagcaggtg gatatgggac 300
ttggcctgct ggaggggccc tggcaagagg agctggcagg agcagatggc aaagttcact 360
cactctggga gacactggct tccctgttgt gaggacactc aagcagccct attcagaggt 420
ccatgtggta aggtacgaag gcctcctgtc aattctagtg agaaacctag gtttcttgct 480
aacagctatg tgagtgagcc atcttggaag cagattctcc caccccagat gagccttcgg 540
atgactggag ttctggctga catcttgact gcaatctcat cagagacctc aagccaaaat 600
taccccactt ccaaattcct gaccctacag aaactgtgtg agataatata tgcttattgt 660
ttaagccact aagttgttct tttggtgggg gggatgggtg atttgttatg caacaacaga 720
aaactaatac agaaattata cctgccatta tcaaagaggc ctccagtgag ggttgatggt 780
atccttgaat atttagggaa actaagcatg ttaagtgtaa atacagaaac tgtctttgtc 840
aaaactaaat aaagcaaggg aaatggaact tga 873
<210> 7
<211> 20
<212> DNA
<213> CAFDL1-F(Artificial Sequence)
<400> 7
cctcacctgg ctatggctac 20
<210> 8
<211> 20
<212> DNA
<213> CAFDL1-R(Artificial Sequence)
<400> 8
gagctcccaa gggccaaata 20
<210> 9
<211> 20
<212> DNA
<213> CAFDL2-F(Artificial Sequence)
<400> 9
tgcatggaag gaaacgcaag 20
<210> 10
<211> 20
<212> DNA
<213> CAFDL2-R(Artificial Sequence)
<400> 10
attgaaggct ggaggtgtcc 20
<210> 11
<211> 20
<212> DNA
<213> CAFDL3-F(Artificial Sequence)
<400> 11
acccacatgg cttccagaat 20
<210> 12
<211> 20
<212> DNA
<213> CAFDL3-R(Artificial Sequence)
<400> 12
acagccatct ccctgcacta 20
<210> 13
<211> 20
<212> DNA
<213> CAFDL4-F(Artificial Sequence)
<400> 13
caacactgaa accgcctgac 20
<210> 14
<211> 20
<212> DNA
<213> CAFDL4-R(Artificial Sequence)
<400> 14
aaggaagaca gacacgaggc 20
<210> 15
<211> 21
<212> DNA
<213> CAFDL5-F(Artificial Sequence)
<400> 15
accacatcac ccacttcaac a 21
<210> 16
<211> 22
<212> DNA
<213> CAFDL5-R(Artificial Sequence)
<400> 16
acaaagggta acaaaggatg ga 22
<210> 17
<211> 20
<212> DNA
<213> CAFDL6-F(Artificial Sequence)
<400> 17
tgggaaaggt caggctacca 20
<210> 18
<211> 20
<212> DNA
<213> CAFDL6-R(Artificial Sequence)
<400> 18
aggccaaaac catgtggagt 20
<210> 19
<211> 23
<212> DNA
<213> β-actin-F(Artificial Sequence)
<400> 19
gtcattccaa atatgagatg cgt 23
<210> 20
<211> 20
<212> DNA
<213> β-actin-R(Artificial Sequence)
<400> 20
gctatcacct cccctgtgtg 20
Claims (10)
1.肿瘤相关成纤维细胞特异性lncRNA标志物或其组合标志物,其特征在于,所述lncRNA标志物或其组合标志物包括LINC01614、AC134312.5、LINC01429、LINC02544、AC012213.3或LINC01705中的一种或几种。
2.权利要求1所述的肿瘤相关成纤维细胞特异性lncRNA标志物或其组合标志物的检测试剂在制备肺癌预后评估的试剂盒中的应用。
3.根据权利要求2所述的应用,其特征在于,所述LINC01614、AC134312.5、LINC01429、LINC02544、AC012213.3和LINC01705的核苷酸序列分别如SEQ ID NO:1、SEQ ID NO:2、SEQID NO:3、SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示。
4.一种用于肺癌预后评估的试剂或试剂盒,其特征在于,所述试剂或试剂盒包括权利要求1所述的lncRNA标志物或其组合标志物。
5.根据权利要求4所述的试剂或试剂盒,其特征在于,所述试剂盒包含反转录试剂盒或荧光定量PCR试剂盒。
6.根据权利要求4所述的试剂或试剂盒,其特征在于,所述荧光定量PCR试剂盒包含特异性针对权利要求1所述的lncRNA标志物的引物对。
7.根据权利要求4所述的试剂或试剂盒,其特征在于,所述LINC01614、AC134312.5、LINC01429、LINC02544、AC012213.3和LINC01705对应的引物对分别为SEQ ID NO:7和SEQID NO:8、SEQ ID NO:9和SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示。
8.根据权利要求4所述的试剂或试剂盒,其特征在于,所述试剂盒还可包含针对内参基因的核酸序列引物。
9.根据权利要求4~8任一项所述的试剂或试剂盒,其特征在于,在肺癌预后评估中,当肿瘤组织肿瘤相关成纤维细胞特异性lncRNAs表达量高于cutoff值,则提示受试者预后不良,否则提示受试者的预后良好。
10.根据权利要求9所述的试剂或试剂盒,其特征在于,所述特异性lncRNA标志物中LINC01614、AC134312.5、LINC02544、LINC01705对应的cutoff值分别为1.12,5.97,5.80,6.06。
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CN113699240A (zh) * | 2020-10-10 | 2021-11-26 | 广西医科大学 | Nrk在肺癌治疗和预后诊断中的医药用途 |
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