CN113862359B - Fam13a基因转录本在评估胶质母细胞瘤患者临床预后中的应用 - Google Patents
Fam13a基因转录本在评估胶质母细胞瘤患者临床预后中的应用 Download PDFInfo
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Abstract
本发明公开了FAM13A基因转录本在评估胶质母细胞瘤患者临床预后中的应用。本发明通过对胶质母细胞瘤细胞系进行检测，发现RBMX表达水平和胶质母细胞瘤患者临床预后相关，而FAM13A基因编码的两条转录本FAM13A‑004及FAM13A‑001的表达与RBMX表达水平及临床预后相关。表明FAM13A‑004及FAM13A‑001转录本检测可用来评估胶质母细胞瘤患者临床预后。本发明的提出为指导胶质母细胞瘤患者的个体化治疗，优化治疗方案以达到最大的治疗效益提供了有效的技术手段，有助于提高脑胶质母细胞瘤患者的总体生存期，推动胶质母细胞瘤精准治疗方案的进展。
Description
技术领域
本发明涉及FAM13A基因转录本在评估胶质母细胞瘤患者临床预后中的应用。特别涉及FAM13A基因的两个转录本FAM13A-004及FAM13A-001在评估胶质母细胞瘤患者临床预后中的应用。本发明属于医药技术领域。
背景技术
胶质母细胞瘤(Glioblastoma，GBM)是中枢神经系统最常见的肿瘤之一，其侵袭性强，恶性程度高，而预后较差。胶质母细胞瘤治疗以手术切除为主，并结合放化疗等多种治疗手段，但治疗效果欠佳。胶质母细胞瘤为起源于神经上皮的肿瘤，不同类型的胶质母细胞瘤在病理组织学形态、侵袭进展及临床预后结局有着很大的差异，而根据不同基因的表达谱特征来区分不同胶质母细胞瘤亚型为目前国际上较为公认的、有意义的肿瘤分型。结合不同胶质母细胞瘤亚型并对患者的临床预后进行预测，可以指导患者的个体化治疗，优化治疗方案以达到最大的效益。
与其他肿瘤类似，胶质母细胞瘤存在上皮间质转化(Epithelial to mesenchymaltransition，EMT)，间质样生长的肿瘤(间质型，Mesenchymal)有较高的恶性行为倾向，并过度表达N-cadherin、β-catenin等EMT间质标记物，参与肿瘤的侵袭迁移和放化疗耐受等生物学活动。
RNA选择性转录(AT，Alternative transcription)增加了RNA和编码蛋白的多样性，并参与了肿瘤的EMT、细胞增殖及凋亡等过程。本发明通过对TCGA、 GSE16011等数据库胶质母细胞瘤样本的亚型注释，联合多平台结果及预后数据最终发现转录因子RBMX(RNAbinding motif protein X-linked，亦称作HNRNPG) 在Mesenchymal型胶质母细胞瘤中低表达，RBMX高表达的胶质母细胞瘤患者生存时间更长。RBMX基因属于RBMY基因家族，该基因是Y染色体RBMY基因的一个活跃的X染色体同源体，广泛表达。RBMX编码的RNA结合蛋白在转录过程中参与特异性基因转录调控和mRNAs前体的选择性转录，FAM13A基因编码的两个转录本FAM13A-004和FAM13A-001的表达量及预后与RBMX表达量相关性相反。本发明发明人发现RBMX介导的FAM13A基因转录本的选择性转录，产生具有不同作用机制的功能蛋白，调控脑胶质母细胞瘤上皮间质转化的过程。
因此，FAM13A-004和FAM13A-001的PCR引物及原位杂交探针可用于评估胶质母细胞瘤患者临床预后，从而指导患者的个体化治疗，优化治疗方案以达到最大的治疗效益，有助于提高脑胶质母细胞瘤患者的总体生存期，推动胶质母细胞瘤精准治疗方案的进展。
发明内容
本发明的目的之一在于提供FAM13A基因的两个转录本FAM13A-004及 FAM13A-001在评估胶质母细胞瘤患者临床预后中的应用。
本发明的目的之二在于提供一种用于评估胶质母细胞瘤患者临床预后的试剂盒及其检测方法。
为了达到上述目的，本发明采用了以下技术手段：
本发明通过对胶质母细胞瘤细胞系进行检测，发现RBMX表达水平和胶质母细胞瘤患者临床预后相关。接着通过生物信息学技术发现FAM13A基因编码的两条转录本FAM13A-004及FAM13A-001的表达与RBMX表达水平及临床预后相关，且两个转录本与RBMX表达量相关性(FAM13A-004，r＝0.43； FAM13A-001，r＝-0.33)和对应的临床预后(FAM13A-004，HR＝0.80，P＝0.04； FAM13A-001，HR＝1.67，P＝5.05E-04)相反。RBMX选择性转录FAM13A转录本调控上皮向间质转化促进脑胶质母细胞瘤恶性进展。因此，通过对FAM13A-004 及FAM13A-001转录本的表达水平进行检测可用来评估胶质母细胞瘤患者的临床预后。
在上述研究的基础上，本发明提出了FAM13A基因转录本在制备评估胶质母细胞瘤患者临床预后的试剂或试剂盒中的应用，所述的FAM13A基因转录本为 FAM13A-004以及FAM13A-001其中之一或其组合，其中，FAM13A-004的序列如SEQ ID NO.1所示，FAM13A-001的序列如SEQ ID NO.2所示，高表达 FAM13A-004的胶质母细胞瘤患者预后较好，高表达FAM13A-001的胶质母细胞瘤患者预后较差。
进一步的，本发明还提出了用于检测FAM13A基因转录本的试剂在制备评估胶质母细胞瘤患者临床预后的试剂或试剂盒中的应用，所述的FAM13A基因转录本为FAM13A-004以及FAM13A-001其中之一或其组合，其中，FAM13A-004的序列如SEQ ID NO.1所示，FAM13A-001的序列如SEQ ID NO.2所示，高表达 FAM13A-004的胶质母细胞瘤患者预后较好，高表达FAM13A-001的胶质母细胞瘤患者预后较差。
其中，优选的，所述的试剂为检测FAM13A-004及FAM13A-001转录本的 qRT-PCR特异性引物及/或原位杂交探针。
其中，优选的，检测FAM13A-004转录本的qRT-PCR特异性引物为：
正向引物：TCGCCCTCAAGTTTGTTCCT
反向引物：AGCAAGTTTGCCCTGCCTAA
检测FAM13A-001转录本的qRT-PCR特异性引物为：
正向引物：GAGCAGAAGGAGTAGGGTGC
反向引物：CAGGGAAGCAGCTGAACCA。
其中，优选的，检测FAM13A-004及FAM13A-001转录本的原位杂交探针的序列分别如SEQ ID NO.5以及SEQ ID NO.6所示。
更进一步的，本发明还提出了一种用于评估胶质母细胞瘤患者临床预后的试剂盒，所述的试剂盒中包括检测FAM13A-004及FAM13A-001转录本的qRT-PCR 特异性引物及/或原位杂交探针。
其中，优选的，检测FAM13A-004转录本的qRT-PCR特异性引物为：
正向引物：TCGCCCTCAAGTTTGTTCCT
反向引物：AGCAAGTTTGCCCTGCCTAA
检测FAM13A-001转录本的qRT-PCR特异性引物为：
正向引物：GAGCAGAAGGAGTAGGGTGC
反向引物：CAGGGAAGCAGCTGAACCA。
其中，优选的，检测FAM13A-004及FAM13A-001转录本的原位杂交探针的序列分别如SEQ ID NO.5以及SEQ ID NO.6所示。
相较于现有技术，本发明的有益效果是：
本发明通过对胶质母细胞瘤细胞系进行检测，发现RBMX表达水平和胶质母细胞瘤患者临床预后相关，而FAM13A基因编码的两条转录本FAM13A-004 及FAM13A-001的表达与RBMX表达水平及临床预后相关。表明FAM13A-004 及FAM13A-001转录本检测可用来评估胶质母细胞瘤患者临床预后。本发明的提出为指导胶质母细胞瘤患者的个体化治疗，优化治疗方案以达到最大的治疗效益提供了有效的技术手段，有助于提高脑胶质母细胞瘤患者的总体生存期，推动胶质母细胞瘤精准治疗方案的进展。
附图说明
图1为胶质母细胞瘤中RBMX影响患者预后及肿瘤恶性进展；
其中：(A-B)Mesenchymal和Proneural亚型胶质母细胞瘤中存在差异表达基因，其中RBMX是该差异表达基因中参与调控蛋白与核酸结合；(C)RBMX 表达水平与胶质母细胞瘤患者预后的关系；(D-E)RBMX过表达测序结果及人源 HG7和HG11原代胶质母细胞瘤细胞系的RBMX敲低和过表达细胞系构建验证。
图2为RBMX调控胶质母细胞瘤侵袭能力和调控肿瘤恶性增殖；
其中：(A)RBMX调控AKT信号通路的激活水平；(B)Transwell细胞侵袭实验检测过表达和敲低RBMX对胶质瘤细胞侵袭能力的影响；(C)小动物体内研究验证敲低和过表达RBMX调控肿瘤生长大小和小鼠预后状况；
图3为RBMX调控脑胶质瘤EMT进展；
其中：(A-B)过表达和敲低RBMX对EMT标记物蛋白水平的影响；(C) 体内实验验证过表达和敲低RBMX对EMT标记物蛋白水平的影响；
图4为RBMX调节FAM13A-001和FAM13A-004转录本的表达水平；
其中：(A)与RBMX表达水平相关的转录本及FAM13A-001与FAM13A-004 转录本表达水平对胶质瘤患者预后的影响；(B)RBMX表达水平对FAM13A-001 和FAM13A-004转录本表达的影响。
图5为FAM13A-004及FAM13A-001转录本原位杂交检测试剂盒在脑胶质瘤患者组织中的应用；
图6为利用转录本原位杂交检测试剂盒，分别在过表达或敲低RBMX的原代肿瘤细胞小动物肿瘤模型中检测FAM13A-004及FAM13A-001转录本的应用。
具体实施方式
下面通过实验并结合实施例对本发明做进一步说明，应该理解的是，这些实施例仅用于例证的目的，决不限制本发明的保护范围。
实施例1 RBMX表达水平评估胶质母细胞瘤亚型和预后
1、通过数据库筛选与胶质母细胞瘤预后相关的转录调控因子
TCGA(The Cancer Genome Atlas)的GBM转录组分型为目前最常用的分型系统，其中包含了前神经元型(Proneural)和间质型(Mesenchymal)，两种亚型间联系紧密，常常涉及GBM的EMT进展。本发明通过对来自TCGA数据库和 GSE16011数据库中的1000余例GBM样本进行亚型注释，在Proneural型GBM 和Mesenchymal型GBM之间筛选了404个转录因子基因表达差异，然后联合 TCGA-U133A、Agilent、HiSeq和GSE16011多平台结果及临床预后数据，发现 RBMX在Mesenchymal型GBM中低表达，在Proneural型GBM中高表达，且 RBMX高表达的胶质母细胞瘤患者生存时间更长(图1)。
2、构建过表达和敲低RBMX的细胞系
通过对胶质母细胞瘤原代细胞HG7及HG11，分别使用过表达和敲低RBMX 慢病毒(上海吉凯基因公司)转染细胞，分别稳定培养出过表达和敲低RBMX 的细胞系，按照以下方法检测RBMX蛋白和AKT信号通路的表达水平变化。
(1)提取组织或细胞蛋白，收集胶质母细胞瘤细胞至具有1％蛋白酶抑制剂的RIPA缓冲液(Solarbio)中；
(2)用分光光度计(NanoDrop)测定蛋白质浓度；
(3)电泳：所有样品均经过十二烷基硫酸钠聚丙烯酰胺凝胶(EpiZymeScientific)电泳，电泳条件为恒压100V，待染料前沿移行到距离凝胶底部2-3mm 时方可停止(120min)；
(4)电转膜仪转膜：将凝胶转移到PVDF膜上(Millipore，USA)，转膜条件为恒流250mA，2h(时间可根据目的蛋白分子量适当调整)；
(5)封闭：将膜在5％脱脂牛奶-TBST溶液室温封闭1h；
(6)加入一抗(兔抗人RBMX抗体)，在抗体覆盖PVDF膜后，放入4℃冰箱中孵育过夜；
(7)回收一抗，PBST洗膜3次，10min/次；
(8)二抗孵育：与辣根过氧化物酶标记的二抗(羊抗兔IgG抗体)室温孵育1h；
(9)洗膜：PBST洗膜2次，PBS洗膜一次，10min/次；
(10)显色：在ChemiDocTM MP Imaging System(BioRad)中用SuperEnhanced 化学发光检测试剂(Applygen Technologies Inc)检测蛋白条带。
结果提示，成功构建了RBMX敲低和过表达细胞系，并且影响AKT信号通路，结果见图2A。
3、Transwall实验检测RBMX敲低和过表达胶质瘤细胞的侵袭能力
使用transwell技术检测RBMX敲低和过表达细胞系中细胞增殖和侵袭性的变化，具体方法如下：
(1)铺基质胶：4℃条件下将Matrigel胶用无血清的细胞培养基按照1：8 比例稀释，取100μl均匀涂抹于上室的聚碳酸酯膜表面，37℃放置0.5-1h；
(2)细胞培养：取对数生长期的待测细胞，并用PBS洗涤，接着用无血清培养基悬浮细胞，调整细胞密度为1-10×105/ml；
(3)接种细胞：24孔板下室加入500μL含10％FBS的培养基，用镊子将 Transwell小室置于24孔板内，取细胞悬液200μL加入上室，放入培养箱中培养 12-48h；
(4)细胞固定：取出小室，吸走培养基，用棉签轻轻擦拭Matrigel和上室内的细胞。取新的24孔板加入4％多聚甲醛600μL，固定20-30min；
(5)细胞染色及计数：弃固定液，0.2％结晶紫染色10min，PBS洗3遍，用棉签轻轻擦拭小室的上侧，将非特异性结合于小室上表面的染料擦掉。适当风干后，显微镜下观察细胞并计数。
结果提示，RBMX低表达的LN229细胞系，细胞增殖和侵袭能力增强，结果见图2B。
4、敲低和过表达RBMX胶质母细胞瘤裸鼠模型的肿瘤生长
根据所构建的敲低或过表达RBMX的LN229细胞系，选用4周龄雌性 BALB/c裸鼠，随机分为4组，每组10只。将3×105个LN229细胞使用立体定向穿刺技术注入脑内。分别于10天、20天、30天使用荧光成像技术拍照，观察肿瘤生长大小及小鼠生存时间，结果见图2C。
实施例2 RBMX表达与EMT标记物的相关性检测
RBMX在Mesenchymal型胶质母细胞瘤中低表达，在Proneural型胶质母细胞瘤中高表达。RBMX作为一种转录因子，参与EMT进程。使用免疫印迹(Western blot，WB)、免疫荧光技术和免疫组化技术检测敲低和过表达RBMX细胞系中 EMT相关标记物E-cadherin，Claudin1，N-cadherin，Slug和β-catenin的表达情况。
1、免疫荧光技术
(1)将5×103个细胞均匀铺在24孔板的细胞爬片上，过夜，保证细胞贴壁生长；
(2)利用4％多聚甲醛将细胞固定，使用含0.5％Triton的PBS溶液透膜，利用5％胎牛血清(BSA)封闭细胞爬片；
(3)将E-cadherin，Claudin1，N-cadherin，Slug和β-catenin的特异性抗体 (一抗)加入细胞爬片上，每张爬片加入200μL，将细胞爬片置于4℃冰箱中孵育过夜；
(4)根据一抗种属来源，选择合适的荧光标记二抗，加入到细胞爬片上，于室温孵育1h；
(5)加入DAPI染色剂，对细胞核进行染色，于室温避光状态下孵育20min；
(6)于荧光显微镜下观察细胞爬片，检测E-cadherin，Claudin1，N-cadherin，Slug和β-catenin蛋白的细胞定位以及表达水平。
2、免疫组化技术
(1)将胶质瘤组织制成蜡块，在轮转式切片机上制作0.3mm的石蜡切片，于70℃烤箱内烘烤4h至过夜，确保石蜡充分融化；
(2)将石蜡切片置于二甲苯溶液中透明，并在100％、90％、85％、80％、 75％、70％的乙醇溶液中梯度水化；
(3)将处理后的组织切片置于柠檬酸修复液中，95℃加热，维持8min，进行抗原修复，静置冷却到室温；
(4)在处理后的组织切片上滴加3％的过氧化氢溶液，室温静置10min，去除内源性过氧化物酶；
(5)在PBST浸润清洗组织切片后，滴加E-cadherin，Claudin1，N-cadherin，Slug和β-catenin对应的一抗，于湿盒中4℃冰箱中孵育过夜；
(6)PBS溶液清洗一抗后，滴加对应种属的辣根过氧化物酶标记的二抗，室温静置30min；
(7)PBS溶液清洗二抗后，滴加DAB显色液进行显色，在显微镜下观察显色过程；
(8)显色后，自来水冲洗5min终止显色，并利用苏木素染色20s，自来水冲洗5min终止染色；
(9)利用70％-100％乙醇溶液梯度脱水、二甲苯溶液透明处理，使用树胶封片，于70℃烤箱内烤干过夜，室温保存组织切片，以备后续检测观察。
结果提示，在RBMX高表达的胶质母细胞系LN229及原代细胞HG7中， E-cadherin和Claudin1表达明显升高，N-cadherin，Slug和β-catenin表达明显下降；在RBMX低表达的胶质母细胞系LN229及原代细胞HG7中，E-cadherin和 Claudin1表达明显下降，N-cadherin，Slug和β-catenin表达明显升高，结果见图 3。
实施例3使用qPCR技术和琼脂糖凝胶电泳技术检测转录本FAM13A-004 及FAM13A-001表达水平
RBMX编码的RNA结合蛋白在转录前和转录后过程中起着多种调节作用，与组织特异性基因转录调控和前mRNAs的选择性转录有关。通过分析TCGA转录本测序数据发现RBMX表达与7000余条转录本表达水平相关(r>0.3或<-0.3)，其中，我们发现FAM13A基因编码的两个转录本与RBMX表达量相关性相反 (FAM13A-004，r＝0.43)及FAM13A-001，r＝-0.33)，高表达FAM13A-004的胶质母细胞瘤患者预后较好(HR＝0.80，P＝0.043)，高表达FAM13A-001的胶质母细胞瘤患者预后较差(HR＝1.67，P＝0.044)，结果见图4A。FAM13A-004转录本包含一个RAS同源GTP酶激活蛋白(RhoGAP，Ras homologous GTPase-activating protein)结构域，影响GTPase活性，下调RhoA信号通路，抑制EMT进展，抑制GBM恶性增殖。而FAM13A-001转录本包含两个coiled coil domain(CCDs)结构域，参与蛋白质间互作，通过结合RANGAP1蛋白，促进RhoA信号通路，促进EMT进展，促进GBM发生发展。
1、FAM13A-004、FAM13A-001的序列
利用Ensemble数据库查询FAM13A(ENSG00000138640)的全长序列，全长包括385444个碱基，存在23个转录本。其中FAM13A-004包含5个外显子、2762个碱基，序列如SEQID NO.1所示。FAM13A-0001包含17个外显子、4945 个碱基。序列如SEQ ID NO.2所示。
2、FAM13A-004、FAM13A-001特异性序列
通过仔细比对FAM13A所有转录本的外显子序列所在区段位置，发现 FAM13A的两个转录本FAM13A-004和FAM13A-001均存在一段独有的特异性序列。FAM13A-004的特异性序列位于3’末端最后一个外显子区域内(1665个碱基) 序列如SEQ ID NO.3所示。FAM13A-001的特异性序列位于5’起始端第一个外显子区域内(103个碱基)，序列如SEQ ID NO.4所示。
3、设计引物
使用NCBI的Primer Blast网站设计FAM13A-004及FAM13A-001的上游及下游引物，由优宁维公司负责引物合成。利用qRT-PCR技术检测FAM13A-004 及FAM13A-001表达量，在RNA层面检测二者的存在及表达量。
FAM13A-004：
Sequence(5'->3') Template strand
Forward primer TCGCCCTCAAGTTTGTTCCT
Reverse primer AGCAAGTTTGCCCTGCCTAA
FAM13A-001：
Sequence(5'->3') Template strand
Forward primer GAGCAGAAGGAGTAGGGTGC
Reverse primer CAGGGAAGCAGCTGAACCA
4、利用qPCR技术，检测FAM13A-004和FAM13A-001的表达水平。
(1)RNA提取：
①每个组织样品均取100mg，研磨成粉末
②按照从Sigma公司购买的Trizol，提取RNA
(2)逆转录反应
每个样品取相同量RNA，用随机引物进行逆转录，反应体系如下。
(3)PCR扩增反应
①按下表配制RCR反应体系，选择GAPDH作内参基因，根据下表配制20μl/ 孔PCR反应体系
②准备96孔PCR反应板，每例样本重复三次，充分离心避免气泡产生
③反应条件
④数据分析：观测扩增曲线图，设置基线和阈值，以GAPDH为内源性对照，应用相对CT值的方法计算所检测FAM13A-004和FAM13A-001的相对表达量。
5、琼脂糖凝胶电泳
(1)制备1％的琼脂糖凝胶，取适量的琼脂糖凝胶溶于TBE缓冲溶液中，加热、摇匀至琼脂糖完全溶解，按照琼脂糖溶液体积加入适量GelRed试剂，并将琼脂糖溶液倒入模具中，制备琼脂糖凝胶；
(2)将PCR产物按照顺序加入对应的琼脂糖凝胶的泳道中，以110V恒压在TBE溶液中进行电泳；
(3)观察电泳结果，在合适的时间终止电泳，将琼脂糖凝胶转移至凝胶成像系统中，在紫外线照射下观察对应产物的相对表达程度。
结果显示，FAM13A-004在RBMX敲低细胞系中表达下降，在RBMX过表达细胞系中表达升高；FAM13A-001在RBMX过表达细胞系中表达升高，在 RBMX敲低细胞系中表达下降。RBMX的表达与FAM13A-004表达成负相关，与FAM13A-001表达成正相关。结果见图4B。
实施例4使用原位杂交探针检测转录本FAM13A-004及FAM13A-001表达水平
根据上述FAM13A-004和FAM13A-001独有的特异性序列，委托ACD公司根据上述序列设计合成寡核苷酸原位杂交探针(靶向FAM13A-004的探针序列如 SEQ ID NO.5所示，靶向FAM13A-001的探针序列如SEQ ID NO.6所示)。该产品包含了BaseScopeTMDuplex试剂盒(预处理试剂盒，检测试剂盒和洗脱液)， FAM13A-004和FAM13A-001对应的探针及阳性和阴性对照探针，Vector Labs封片剂和ImmEdgeTMHydrophobic阻水笔。FAM13A-004和FAM13A-001 Basescope 探针可以使用Basescope试剂盒实现石蜡组织切片染色。
结果显示，在RBMX表达水平较高的脑胶质瘤患者组织中(用于提取原代细胞HG7的脑胶质瘤组织)，FAM13A-004相对FAM13A-001表达水平较高； RBMX表达水平较低的脑胶质瘤患者组织中(用于提取原代细胞HG11的脑胶质瘤组织)，FAM13A-004相对FAM13A-001表达水平较低，结果见图5。
在RBMX过表达细胞中，FAM13A-004表达量升高；在RBMX敲低细胞中， FAM13A-001表达量升高，结果见图6。
这些结果提示，FAM13A基因编码的两条转录本与RBMX表达水平和预后相关。因此，FAM13A基因编码的两条转录本(FAM13A-004，FAM13A-001) 检测可以应用于评估胶质母细胞瘤患者临床预后。
序列表
<110> 哈尔滨医科大学
<120> FAM13A基因转录本在评估胶质母细胞瘤患者临床预后中的应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2762
<212> DNA
<213> Homo sapiens
<400> 1
atgtgggttc agcggaaaga gaagcaaaac cactcttcct aaaatgttag aagctgctct 60
tcgcttacct tggggccttt gcattgggag ctgtttttca catcaaagaa tatgtgctga 120
atggaatttt agtattttgc tgtcgtttta atattttcgt ctggtcttcc tcagttcttc 180
cagacgcttt ctgagagaat gggggcagga gctctagcca tctgtcaaag taaagcagcg 240
gttcggctga aagaagacat gaaaaagata gtggcagtgc cattaaatga acagaaggat 300
tttacctatc agaagttatt tggagtcagt ctccaagaac ttgaacggca ggggctcacc 360
gagaatggca ttccagcagt agtgtggaat atagtggaat atttgacgca gcatggactt 420
acccaagaag gtctttttag ggtgaatggt aacgtgaagg tggtggaaca acttcgactg 480
aagttcgaga gtggagtgcc cgtggagctc gggaaggacg gtgatgtctg ctcagcagcc 540
agtctgttga agctgtttct gagggagctg cctgacagtc tgatcacctc agcgttgcag 600
cctcgattca ttcaactctt tcaggatggc agaaatgatg ttcaggagag tagcttaaga 660
gacttaataa aagagctgcc agacacccac tactgcctcc tcaagtacct ttgccagttc 720
ttgacaaaag tagccaagca tcatgtgcag aatcgcatga atgttcacaa tctcgccact 780
gtatttgggc caaattgctt tcatgtgcca cctgggcttg aaggcatgaa ggaacaggac 840
ctgtgcaaca agataatggc taaaattcta gaaaattaca ataccctgtt tgaagtagag 900
tatacagaaa atgatcatct gagatgtgaa aacctggcta ggcttatcat agtaaaagta 960
agcaacttgg tttttaattt tcagtattgc tataattttg gacagaagat tttatttaat 1020
tctttctcat aaaaattcca tatggatata accctcagat tattattcct gtgtatagtg 1080
aatccttctt atgaaatcta ttattccaaa tgcttattaa attgaaatat agccttctaa 1140
aattcaagaa taatagcaat tttatgtttt cttgttgcta taaagtcatc tcaaaaacac 1200
ttgcatgagc ccagtgctct gatcaactgg ccgcaccacc tttactacag aagcctgatg 1260
ttacagcagg aataagtgca tatggctgtg atcaggagtg ctaggttact aactagattg 1320
caaattcctg aagacaatat tactgtgtct ttttgtctct tttgtgtctg gcacaatttg 1380
aagcccaacc tcagattcta agtcccatat attagttttt ggtaacaatc atcagtaaag 1440
gagaatattt taaaaaccta taaaggagtc cttgacaata ctatctaaat ctttttatac 1500
attgataatt ttataatata ccctgtatat attaggtaaa tgcctgtagg tctccaaaga 1560
cctagaattg agaatcagag ggtaaacatc caaacaaatc ccctagatgt gggaaaataa 1620
ggaagttatc ttatttcgtc gtcatttata ttgaggtgaa tcatgatgga gctggtatga 1680
gatttcctca ggaggtttct tgaagcttat caggtttaca gaccataaca tactctttgc 1740
tgattcatat agcaatgaat gataaaatca gaggcacttg gtttgggcac ttaaaggaat 1800
gttttcatct cttctcccag ttgaggccat gactttgaag aaaggttaaa atggtttgag 1860
tatcaagtag catcctacaa aaggatctaa aactagattt tctagtttgc tcagttaaaa 1920
tgataaaatg agataattgg agactatcag ttgtaaatct gagttagaaa taacacggta 1980
gctgaaaaaa atgtcagtga ttcacaagaa aaaataagaa aacaacatta cttcagtttt 2040
gcctcagaaa agttaaatga ttttttaaat agaaaaaaat cgccctcaag tttgttcctc 2100
tagtgcctat ggtactttga aaaggatctc tttttaaaat gttaaaaggt aattaacttt 2160
gtttcagcag catctctcaa tcacatatac cccataaata tgtacaacta ttaaaggacc 2220
tataatgata attaaaaata aaatatttta aaaatctttt ataaacaatt tttaacactc 2280
ataccttttt actagggcag ataaaataat aaggtgaacc tgaagtataa tggaaataga 2340
taaatataat cttaggcagg gcaaacttgc tgagataaag ctaattgctg gaatatgcca 2400
atgggaagtt gatatggttt ggatgtttgt cccctccaaa tctcatattg aaacgcagtc 2460
cctggtgttg gagctggggc ctggtgggag gtgtttggat catgagggcg gatccttcgt 2520
gaatgactta gtgccatccc cttggtgatg agtgagttct tgctgtgagt tcacaggaga 2580
cctgcttgtt taaaagtgtg tggcacttcc ccccctcact cttgctccca ctctcgccat 2640
gtgatatgcc tgctcccctt cttcttctgc cgtgattgtg agcttcctga ggcctcatta 2700
gaggcagatg ttctgtatgg cctgctgaac tgtgagtcaa ttaaacctct ttttgaaata 2760
aa 2762
<210> 2
<211> 4945
<212> DNA
<213> Homo sapiens
<400> 2
attgaggagc agaaggagta gggtgcgggg gaggaggagg agcgccttta gtgctgcagc 60
agctgctgct ctgattggcc cggtggttca gctgcttccc tggaacaaaa ggtcaaagtg 120
gactgcagtg taaatgtaga gaagcagccg ataaaatagc attgcctgaa gaagtttgga 180
ggctgagagc agcagtagac tggccaactg cagagcaagt tgtttctcca gccgtgcggt 240
gcagcctcat gcccccaacc cagcttagcc actgtaagaa gacgttcact gtacagacga 300
ccaaacttgc cgtggaagag acagttgtga gattcccttg caaatttaca tacgagaatg 360
gcttgtgaaa tcatgcctct gcaaagttca caggaagatg aaagacctct gtcacctttc 420
tatttgagtg ctcatgtacc ccaagtcagc aatgtgtctg caaccggaga actcttagaa 480
agaaccatcc gatcagctgt agaacaacat ctttttgatg ttaataactc tggaggtcaa 540
agttcagagg actcagaatc tggaacacta tcagcatctt ctgccacatc tgccagacag 600
cgccgccgcc agtccaagga gcaggatgaa gttcgacatg ggagagacaa gggacttatc 660
aacaaagaaa atactccttc tgggttcaac caccttgatg attgtatttt gaatactcag 720
gaagtcgaaa aggtacacaa aaatactttt ggttgtgctg gagaaaggag caagcctaaa 780
cgtcagaaat ccagtactaa actttctgag cttcatgaca atcaggacgg tcttgtgaat 840
atggaaagtc tcaattccac acgatctcat gagagaactg gacctgatga ttttgaatgg 900
atgtctgatg aaaggaaagg aaatgaaaaa gatggtggac acactcagca ttttgagagc 960
cccacaatga agatccagga gcatcccagc ctatctgaca ccaaacagca gagaaatcaa 1020
gatgccggtg accaggagga gagctttgtc tccgaagtgc cccagtcgga cctgactgca 1080
ttgtgtgatg aaaagaactg ggaagagcct atccctgctt tctcctcctg gcagcgggag 1140
aacagtgact ctgatgaagc ccacctctcg ccgcaggctg ggcgcctgat ccgtcagctg 1200
ctggacgaag acagcgaccc catgctctct cctcggttct acgcttatgg gcagagcagg 1260
caatacctgg atgacacaga agtgcctcct tccccaccaa actcccattc tttcatgagg 1320
cggcgaagct cctctctggg gtcctatgat gatgagcaag aggacctgac acctgcccag 1380
ctcacacgaa ggattcagag ccttaaaaag aagatccgga agtttgaaga tagattcgaa 1440
gaagagaaga agtacagacc ttcccacagt gacaaagcag ccaatccgga ggttctgaaa 1500
tggacaaatg accttgccaa attccggaga caacttaaag aatcaaaact aaagatatct 1560
gaagaggacc taactcccag gatgcggcag cgaagcaaca cactccccaa gagttttggt 1620
tcccaacttg agaaagaaga tgagaagaag caagagctgg tggataaagc aataaagccc 1680
agtgttgaag ccacattgga atctattcag aggaagctcc aggagaagcg agcggaaagc 1740
agccgccctg aggacattaa ggatatgacc aaagaccaga ttgctaatga gaaagtggct 1800
ctgcagaaag ctctgttata ttatgaaagc attcatggac ggccggtaac aaagaacgaa 1860
cggcaggtga tgaagccact atacgacagg taccggctgg tcaaacagat cctctcccga 1920
gctaacacca tacccatcat tgaagaagag gaggggtcag aagacgatag caatgtgaag 1980
ccagacttca tggtcactct gaaaaccgat ttcagtgcac gatgctttct ggaccaattc 2040
gaagatgacg ctgatggatt tatttcccca atggatgata aaataccatc aaaatgcagc 2100
caggacacag ggctttcaaa tctccatgct gcctcaatac ctgaactcct ggaacacctc 2160
caggaaatga gagaagaaaa gaaaaggatt cgaaagaaac ttcgggattt tgaagacaac 2220
tttttcagac agaatggaag aaatgtccag aaggaagacc gcactcctat ggctgaagaa 2280
tacagtgaat ataagcacat aaaggcgaaa ctgaggctcc tggaggtgct catcagcaag 2340
agagacactg attccaagtc catgtgaggg gcatggccaa gcacaggggg ctggcagctg 2400
cggtgagagt ttactgtccc cagagaaagt gcagctctgg aaggcagcct tggggctggc 2460
cctgcaaagc atgcagccct tctgcctcta gaccatttgg catcggctcc tgtttccatt 2520
gcctgcctta gaaactggct ggaagaagac aatgtgacct gacttaggca ttttgtaatt 2580
ggaaagtcaa gactgcagta tgtgcacatg cgcacgcgca tgcacgcaca cacacacaca 2640
gtagtggagc tttcctaaca ctagcagaga ttaatcacta cattagacaa cactcatcta 2700
cagagaatat acactgttct tccctggata actgagaaac aagagaccat tctctgtcta 2760
actgtgataa aaacaagctc aggactttat tctatagagc aaacttgctg tggagggcca 2820
tgctctcctt ggacccagtt aactgcaaac gtgcattgga gccctatttg ctgccgctgc 2880
cattctagtg acctttccac agagctgcgc cttcctcacg tgtgtgaaag gttttcccct 2940
tcagccctca ggtagatgga agctgcatct gcccacgatg gcagtgcagt catcatcttc 3000
aggatgtttc ttcaggactt cctcagctga caaggaattt tggtccctgc ctaggaccgg 3060
gtcatctgca gaggacagag agatggtaag cagctgtatg aatgctgatt ttaaaaccag 3120
gtcatgggag aagagcctgg agattctttc ctgaacactg actgcactta ccagtctgat 3180
tttatcgtca aacaccaagc caggctagca tgctcatggc aatctgtttg gggctgtttt 3240
gttgtggcac tagccaaaca taaaggggct taagtcagcc tgcatacaga ggatcgggga 3300
gagaaggggc ctgtgttctc agcctcctga gtacttacca gagtttaatt tttttaaaaa 3360
aaatctgcac taaaatcccc aaactgacag gtaaatgtag ccctcagagc tcagcccaag 3420
gcagaatcta aatcacacta ttttcgagat catgtataaa aagaaaaaaa agaagtcatg 3480
ctgtgtggcc aattataatt tttttcaaag actttgtcac aaaactgtct atattagaca 3540
ttttggaggg accaggaaat gtaagacacc aaatcctcca tctcttcagt gtgcctgatg 3600
tcacctcatg atttgctgtt acttttttaa ctcctgcgcc aaggacagtg ggttctgtgt 3660
ccacctttgt gctttgcgag gccgagccca ggcatctgct cgcctgccac ggctgaccag 3720
agaaggtgct tcaggagctc tgccttagac gacgtgttac agtatgaaca cacagcagag 3780
gcaccctcgt atgttttgaa agttgccttc tgaaagggca cagttttaag gaaaagaaaa 3840
agaatgtaaa actatactga cccgttttca gttttaaagg gtcgtgagaa actggctggt 3900
ccaatgggat ttacagcaac attttccatt gctgaagtga ggtagcagct ctcttctgtc 3960
agctgaatgt taaggatggg gaaaaagaat gcctttaagt ttgctcttaa tcgtatggaa 4020
gcttgagcta tgtgttggaa gtgccctggt tttaatccat acacaaagac ggtacataat 4080
cctacaggtt taaatgtaca taaaaatata gtttggaatt ctttgctcta ctgtttacat 4140
tgcagattgc tataatttca aggagtgaga ttataaataa aatgatgcac tttaggatgt 4200
ttcctatttt tgaaatctga acatgaatca ttcacatgac caaaaattgt gtttttttaa 4260
aaatacatgt ctagtctgtc ctttaatagc tctcttaaat aagctatgat attaatcaga 4320
tcattaccag ttagctttta aagcacattt gtttaagact atgtttttgg aaaaatacgc 4380
tacagaattt ttttttaagc tacaaataaa tgagatgcta ctaattgttt tggaatctgt 4440
tgtttctgcc aaaggtaaat taactaaaga tttattcagg aatccccatt tgaatttgta 4500
tgattcaata aaagaaaaca ccaagtaagt tatataaaat aaattgtgta tgagatgttg 4560
tgttttcctt tgtaatttcc actaactaac taactaactt atattcttca tggaatggag 4620
cccagaagaa atgagaggaa gcccttttca cactagatct tatttgaaga aatgtttgtt 4680
agtcagtcag tcagtggttt ctggctctgc cgagggagat gtgttcccca gcaaccattt 4740
ctgcagccca gaatctcaag gcactagagg cggtgtctta attaattggc ttcacaaaga 4800
caaaatgctc tggactggga tttttccttt gctgtgttgg gaatatgtgt ttattaatta 4860
gcacatgcca acaaaataaa tgtcaagagt tatttcataa gtgtaagtaa acttaagaat 4920
taaagagtgc agacttataa ttttc 4945
<210> 3
<211> 1665
<212> DNA
<213> Homo sapiens
<400> 3
gtaagcaact tggtttttaa ttttcagtat tgctataatt ttggacagaa gattttattt 60
aattctttct cataaaaatt ccatatggat ataaccctca gattattatt cctgtgtata 120
gtgaatcctt cttatgaaat ctattattcc aaatgcttat taaattgaaa tatagccttc 180
taaaattcaa gaataatagc aattttatgt tttcttgttg ctataaagtc atctcaaaaa 240
cacttgcatg agcccagtgc tctgatcaac tggccgcacc acctttacta cagaagcctg 300
atgttacagc aggaataagt gcatatggct gtgatcagga gtgctaggtt actaactaga 360
ttgcaaattc ctgaagacaa tattactgtg tctttttgtc tcttttgtgt ctggcacaat 420
ttgaagccca acctcagatt ctaagtccca tatattagtt tttggtaaca atcatcagta 480
aaggagaata ttttaaaaac ctataaagga gtccttgaca atactatcta aatcttttta 540
tacattgata attttataat ataccctgta tatattaggt aaatgcctgt aggtctccaa 600
agacctagaa ttgagaatca gagggtaaac atccaaacaa atcccctaga tgtgggaaaa 660
taaggaagtt atcttatttc gtcgtcattt atattgaggt gaatcatgat ggagctggta 720
tgagatttcc tcaggaggtt tcttgaagct tatcaggttt acagaccata acatactctt 780
tgctgattca tatagcaatg aatgataaaa tcagaggcac ttggtttggg cacttaaagg 840
aatgttttca tctcttctcc cagttgaggc catgactttg aagaaaggtt aaaatggttt 900
gagtatcaag tagcatccta caaaaggatc taaaactaga ttttctagtt tgctcagtta 960
aaatgataaa atgagataat tggagactat cagttgtaaa tctgagttag aaataacacg 1020
gtagctgaaa aaaatgtcag tgattcacaa gaaaaaataa gaaaacaaca ttacttcagt 1080
tttgcctcag aaaagttaaa tgatttttta aatagaaaaa aatcgccctc aagtttgttc 1140
ctctagtgcc tatggtactt tgaaaaggat ctctttttaa aatgttaaaa ggtaattaac 1200
tttgtttcag cagcatctct caatcacata taccccataa atatgtacaa ctattaaagg 1260
acctataatg ataattaaaa ataaaatatt ttaaaaatct tttataaaca atttttaaca 1320
ctcatacctt tttactaggg cagataaaat aataaggtga acctgaagta taatggaaat 1380
agataaatat aatcttaggc agggcaaact tgctgagata aagctaattg ctggaatatg 1440
ccaatgggaa gttgatatgg tttggatgtt tgtcccctcc aaatctcata ttgaaacgca 1500
gtccctggtg ttggagctgg ggcctggtgg gaggtgtttg gatcatgagg gcggatcctt 1560
cgtgaatgac ttagtgccat ccccttggtg atgagtgagt tcttgctgtg agttcacagg 1620
agacctgctt gtttaaaagt gtgtggcact tccccccctc actct 1665
<210> 4
<211> 103
<212> DNA
<213> Homo sapiens
<400> 4
attgaggagc agaaggagta gggtgcgggg gaggaggagg agcgccttta gtgctgcagc 60
agctgctgct ctgattggcc cggtggttca gctgcttccc tgg 103
<210> 5
<211> 165
<212> DNA
<213> artificial sequence
<400> 5
tcaatttaat aagcatttgg aataatagat ttcataagaa ggattcacta tacacaggaa 60
taataatctg agggttatat ccatatggaa tttttatgag aaagaattaa ataaaatctt 120
ctgtccaaaa ttatagcaat actgaaaatt aaaaaccaag ttgct 165
<210> 6
<211> 39
<212> DNA
<213> artificial sequence
<400> 6
gccaatcaga gcagcagctg ctgcagcact aaaggcgct 39
Claims (4)
1.用于检测FAM13A基因转录本的试剂在制备评估胶质母细胞瘤患者临床预后的试剂或试剂盒中的应用，所述的FAM13A基因转录本为FAM13A-004以及FAM13A-001其中之一或其组合，其中，FAM13A-004的序列如SEQ ID NO.1所示，FAM13A-001的序列如SEQ ID NO.2所示，高表达FAM13A-004的胶质母细胞瘤患者比低表达FAM13A-004的胶质母细胞瘤患者预后更好，高表达FAM13A-001的胶质母细胞瘤患者比低表达FAM13A-001的胶质母细胞瘤患者预后更差。
2.如权利要求1所述的应用，其特征在于，所述的试剂为检测FAM13A-004及FAM13A-001转录本的qRT-PCR特异性引物及/或原位杂交探针。
3.如权利要求2所述的应用，其特征在于，检测FAM13A-004转录本的qRT-PCR特异性引物为：
正向引物：TCGCCCTCAAGTTTGTTCCT
反向引物：AGCAAGTTTGCCCTGCCTAA
检测FAM13A-001转录本的qRT-PCR特异性引物为：
正向引物：GAGCAGAAGGAGTAGGGTGC
反向引物：CAGGGAAGCAGCTGAACCA。
4.如权利要求2所述的应用，其特征在于，检测FAM13A-004及FAM13A-001转录本的原位杂交探针的序列分别如SEQ ID NO.5以及SEQ ID NO.6所示。
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