CN109609645B - 检测IncRNA LNC_004208表达量的试剂在制备胶质瘤诊断试剂中的应用 - Google Patents
检测IncRNA LNC_004208表达量的试剂在制备胶质瘤诊断试剂中的应用 Download PDFInfo
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Abstract
本发明公开了一种检测长链非编码RNALNC_004208表达量的试剂在制备脑胶质瘤诊断试剂中的应用。本发明通过研究发现,LNC_004208在高级别胶质瘤组织中高表达,且与胶质瘤分子标记IDH1突变相关。说明LNC_004208是与脑胶质瘤诊断相关的分子标记,可用于制备脑胶质瘤诊断的制剂,为脑胶质瘤患者的诊断提供强有力的分子生物学依据,具有深远的临床意义和重要的推广应用前景。
Description
技术领域
本发明属于肿瘤分子生物学技术领域,具体涉及一种检测IncRNA LNC_004208表达量的试剂在制备胶质瘤诊断试剂中的应用。
背景技术
脑胶质瘤是中枢神经系统最常见的恶性肿瘤,约占原发性恶性脑肿瘤的80%,根据欧洲神经肿瘤协会(EANO)统计,全世界脑胶质瘤每年发生率约为6/100,000。脑胶质瘤的治疗方法是世界性的难题,特别是恶性胶质母细胞瘤,即便采取最优治疗方案,患者预后仍旧十分不理想,平均生存期仅13.3-15个月。替莫唑胺(Temozolomide,TMZ)是近二十年来出现的一种治疗脑胶质瘤效果较好的新药,经美国FDA批准、美国国立综合癌症网络指南推荐已成为治疗脑胶质瘤的首选化疗药物。TMZ属于新型咪唑嗪类口服抗肿瘤药,易于透过血瘤屏障,其活性产物MTIC通过N3、N7、O6位鸟嘌呤和O3位的腺嘌呤DNA烷基化,发挥细胞毒性作用。由于肿瘤细胞内在的因素或者获得性抗性的出现,脑胶质瘤患者对TMZ治疗逐渐产生了抵抗,肿瘤治疗疗效降低。
O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)通过转移DNA上异常的O6甲基,从而抑制TMZ介导的DNA损伤,导致胶质瘤细胞TMZ抵抗。最新研究表明,除了MGMT之外,肿瘤细胞表皮生长因子(epidermal growth factor receptor,EGFR)等多种信号通路的异常均参与了TMZ抵抗的发生,然而其具体机制仍未阐明。因此,深入探讨TMZ抵抗发生的分子机制,并采取有效的措施进行干预,将为提高脑胶质瘤的化疗疗效提供重要的新思路。
近年来研究发现,不同类别的脑胶质瘤组织中lncRNAs表达谱具有显著差异,脑胶质瘤的发病及预后与lncRNAs的表达和功能失调密切相关。分子病理学在精准医学时代的肿瘤研究与诊治中日益发挥着极其重要的作用。随着肿瘤发生发展机制研究及针对性药物开发与临床应用的飞速进展,肿瘤相关分子病理检测也从组织或者细胞学标本,扩展至液体活检;采用的分析手段也更为灵敏、多层面及高通量,并逐步向智能化迈进。精准分子分型已成为肿瘤诊断、治疗及全程管理过程中不可或缺的环节。特别是跨越肿瘤发生部位、以基因型作为直接靶标的治疗模式的探索与实践,给分子病理学提出了新的要求。本发明以lncRNA LNC_004208为靶标的脑胶质瘤诊断、预后以及治疗新策略,为研究lncRNAs在脑胶质瘤TMZ抵抗中的分子机制提供新的实验证据。
发明内容
本发明的目的是提供一种检测IncRNA LNC_004208表达量的试剂在制备胶质瘤诊断试剂中的应用,该IncRNA LNC_004208的序列如SEQ NO:1所示。
所述的检测IncRNA LNC_004208表达量的试剂用于检测脑胶质瘤组织。
所述的检测IncRNA LNC_004208表达量的试剂用于制备诊断高级别脑胶质瘤的试剂。
所述的检测IncRNA LNC_004208的试剂包括实时荧光定量检测试剂。
扩增lncRNA LNC_004208的引物包括以下两对或一对:
LNC_004208(1)-正向引物5’-gccaatccaaaccgaaagcc-3’,见SEQ NO:2;
LNC_004208(1)-反向引物5’-agactcagaccccaggctac-3’,见SEQ NO:3;
LNC_004208(2)-正向引物5’-tttgctacatcccagctcca-3’,见SEQ NO:4;
LNC_004208(2)-反向引物5’-tccccctttcccttgtaggt-3’,见SEQ NO:5。
检测试剂中用于对照的β-actin
正向引物:5’-catgtacgttgctatccaggc-3’,见SEQ NO:6。
反向引物:5’-ctccttaatgtcacgcacgat-3’,见SEQ NO:7。
试剂中还含有:
(1)从脑胶质瘤组织中抽提总RNA所用试剂,包括稳定溶液、试剂、三氯甲烷、异丙醇、无酶水;
(2)以总RNA为模板将RNA LNC_004208逆转录为cDNA所用试剂,包括逆转录缓冲液、三磷酸碱基脱氧核普酸、RNA酶抑制剂、MMLV逆转录酶以及随机引物;
(3)将cDNA实时定量PCR所用试剂,包括实时荧光定量SYBR染料、无酶水。
所述的胶质瘤诊断试剂,包括检测IncRNA LNC_004208表达量的试剂与胶质瘤分子标记IDH1突变检测试剂。
本发明通过研究发现,LNC_004208在高级别胶质瘤组织中高表达,且与胶质瘤分子标记IDH1突变相关。说明LNC_004208是与脑胶质瘤诊断相关的分子标记,可用于制备脑胶质瘤诊断的制剂,为脑胶质瘤患者的诊断提供强有力的分子生物学依据,具有深远的临床意义和重要的推广应用前景。
附图说明
图1为转录组测序技术和lncRNA测序技术分析TMZ敏感/抗性细胞表达谱;
图2为筛选胶质瘤TMZ抵抗细胞中差异表达的lncRNA;
图3为筛选并验证胶质瘤TMZ抵抗细胞中差异表达的lncRNA;
图4为胶质瘤分级与lncRNA表达的相关性;
图5为胶质瘤分子诊断标记物与lncRNA表达的相关性。
具体实施方式
以下结合具体实施方式进一步说明本发明,而非限制本发明。
实施例1转录组测序技术和lncRNA测序技术分析TMZ敏感/抗性细胞表达谱
采用转录组测序技术和lncRNA测序技术(Illumina HiSeqTM 2000),选取脑胶质瘤T98G/U-118MG细胞系和TMZ抵抗的T98G-R/U-118MG-R细胞系进行表达谱分析。在进行筛选之前,我们首先使用Cuffmerge软件,对各样品拼接得到的转录本进行合并,并去掉其中链方向不确定的转录本,得到本次测序完整的转录组信息。筛选过程分以下5个步骤(图1A):
step1:转录本exon个数筛选:过滤转录组拼接结果中大量低表达量,低可信度的单外显子转录本,选择exon个数>=2的转录本(如无特殊要求,植物会保留exon个数=1的转录本);
step2:转录本长度筛选:选择转录本长度>200bp的转录本;
step3:转录本已知注释筛选:通过Cuffcompare软件,筛除与数据库注释exon区域有重叠的转录本,并将数据库中,与本次拼接转录本exon区域有重叠的lncRNA作为数据库注释lncRNA纳入到后续分析;
step4:转录本表达量筛选:通过Cuffquant计算每条转录本的表达量,选择FPKM>=0.5的转录本(对于单外显子转录本筛选阈值为2);
step5:编码潜能筛选:分为CNCI,CPC,PFAM,phyloCSF(当物种为非哺乳动物时,不进行phyloCSF分析)。
并通过下列软件进行分析(图1B):
CNCI:通过CNCI(Coding-Non-Coding Index)软件进行的。CNCI能够根据相邻三核苷酸的频谱有效地区分编码和非编码的序列。CNCI可以有效地对不完整的转录本和反义转录本对进行预测;
CPC:通过CPC(Coding Potential Calculator,编码潜能计算)软件进行的。将转录本与已知蛋白数据库做BLAST比对,CPC依据转录本各个编码框的生物学序列特征,通过支持向量机的分类器来评估转录本的编码潜能。十折交叉验证表明CPC能够精确地区分出具有编码能力的转录本;
PFAM:通过pfamscan软件进行的。Pfam-A数据库记录了大部分已知蛋白的、高质量的结构域,而Pfam-B数据库则更加全面的覆盖了结构域家族,是对Pfam-A的补充;我们将转录本各个编码框上的蛋白序列与Pfam-A和Pfam-B数据库做hmmscan的同源搜索,能比对到这两个数据库的序列、即具有某个结构域的转录本,被认为具有编码能力,而比不到的转录本极有可能是非编码的转录本;
phyloCSF(只针对部分哺乳动物进行此项分析):通过PhyloCSF软件进行的。密码子替换频率(Codon Substitution Freuqencies,CSF)是指某密码子替换在多序列比对中的出现的频率,编码和非编码区的密码子替换频率比值是一个有效区分一段序列能否编码蛋白的方法;PhyloCSF能够结合系统进化树,在转录本的多物种的基因组比对结果上,通过编码区和非编码区的密码子替换频率对转录本的编码潜能进行打分。phyloCSF利用多物种间的全基因组序列比对文件定义一段基因组区域是否有编码潜能。通过文献查询,我们发现不同的物种间phyloCSF阙值不尽相同,故首先随机选择本项目研究物种一定数目的已知coding和lncRNA基因进行阙值分析,再筛选候选转录本分析结果。此分析仅限于哺乳动物。
最终分析结果共鉴定出5166个lncRNA(图1C-F)。利用cuffdiff软件进行差异表达分析,筛选到94个差异表达的lncRNA(图2)。
实施例2筛选并验证胶质瘤TMZ抵抗细胞中差异表达的lncRNA
(1)细胞总RNA提取(TRIzol法)
将细胞接种于6孔板中,置于37℃、5%CO2培养箱中孵育。待细胞完全贴壁长满后,向每孔中加入1000μl TRIzol。静置至完全溶解后,转移至1.5ml EP管中。加入0.2ml氯仿,剧烈振荡15秒,室温放置3min。于4℃、12000rpm离心15min,将上清液转移到新EP管中。加入0.5ml异丙醇,室温放置10min。4℃、12000rpm离心10min后,在EP管底部出现胶状沉淀。弃去上清液,用1ml75℅乙醇洗涤RNA沉淀,并于4℃、7500rpm离心5min再次弃去上清液。空气干燥5min中后,加入25μl DEPC水,用枪头吸打几次使其充分溶解。放置于-80℃保存。
(2)逆转录合成cDNA
①取5.0μg RNA,将下列反应成分加至EP管中
加入完成后轻轻摇晃混匀,在65℃下孵育5分钟,然后在冰上孵育1分钟。
②在EP管中按顺序添加下列物质(cDNA合成混合物)
轻轻混匀,瞬时离心后收集。PCR仪逆转录程序:在25℃10min,50℃50min。在85℃5min进行终止反应,冰上冷却。将合成的cDNA放置于-20℃保存。
(3)Real time-PCR
PCR引物序列如下:
LNC_004208(1)-正向引物5’-GCCAATCCAAACCGAAAGCC-3’
LNC_004208(1)-反向引物5’-AGACTCAGACCCCAGGCTAC-3’
LNC_004208(2)-正向引物5’-TTTGCTACATCCCAGCTCCA-3’
LNC_004208(2)-反向引物5’-TCCCCCTTTCCCTTGTAGGT-3’。
进一步通过Real-Time PCR验证差异表达lncRNA的表达水平,证实lncRNA XLOC_024808在T98G-R、U-118MG-R细胞系均显著高表达,结果见附图3。
实施例3胶质瘤分级与lncRNA表达的相关性
脑胶质瘤的等级划分是根据星形细胞瘤按其恶性程度进一步分级,通用WHO分级是根据非典型性、核分裂指数、内皮细胞增殖和坏死程度分为4级:1级、一般为良性以毛细胞型星形细胞瘤为主,占胶质瘤5%左右是可以治愈的;2级、为一般的星形细胞瘤或星形-少突细胞瘤,占胶质瘤的30~40%左右。3级、为间型星形细胞瘤,占胶质瘤的15~25%左右,一般由2级演变而来。4级、为胶质母细胞瘤,占胶质瘤的1/3左右。脑胶质瘤按肿瘤细胞在病理学上的恶性程度,可以进一步分类。低级别胶质瘤(WHO 1-2级),为分化良好的胶质瘤;虽然这类肿瘤在生物上并不属于良性肿瘤,但是患者的预后相对而言,还是不错。高级别胶质瘤(WHO 3-4级),为低分化胶质瘤;这类肿瘤为恶性肿瘤,患者生存较差预后。胶质瘤由于恶性程度不同,其所产生症状的速度也不同。例如,低级别胶质瘤患者的病史往往在几个月甚至上年,而高级别胶质瘤患者的病史往往在几个星期至几个月。在104例胶质瘤患者中,我们通过分组研究发现,LNC_004208在高级别胶质瘤组织中高表达(图4A),然而与分期无关(图4B),与患者胶质瘤是初诊或复发无关(图4C)。
实施例4胶质瘤分子诊断标记物与lncRNA表达的相关性
IDH突变是肿瘤早期发生的现象。目前认为IDH突变是低级别胶质瘤和继发性胶质母细胞瘤的重要标记物。2008年文献首次报道IDH1突变,而且IDH1突变的胶质瘤患者具有较好的预后。其中精氨酸突变为组氨酸(R132H)是IDH1最常见的类型,约占80%~90%。研究者发现,在胶质母细胞瘤中IDH1突变主要发生在继发性胶母细胞瘤,原发性胶母细胞瘤的IDH1几乎都是野生型的。在104例胶质瘤患者中,我们通过分组研究发现,在IDH1野生型的患者中,lncRNA XLOC_024808表达量显著高于突变型患者(P=0.0366)(图5)。
上述结果提示lncRNA XLOC_024808可与其他分子诊断标记物联合诊断胶质瘤,作为分子病理学依据,判定其预后,尤其是在一些难以鉴别的颅内肿瘤。
序列表
<110> 中南大学湘雅医院
<120> 检测IncRNA LNC_004208表达量的试剂在制备胶质瘤诊断试剂中的应用
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1236
<212> DNA
<213> 智人(Homo sapiens)
<400> 1
gtctggccat cggtagaggg atgcagccac taccaaccac agtatggcag agaagtagca 60
gagaagaaat aaaagaaatc ccctctcact ctccaattct gttgttgcct ccatgcgcca 120
atccaaaccg aaagccagaa ggaagaagag tgaggagttt gctacatccc agctccacat 180
cactggaaat gggatttgag gtaatgacta ggagtttaac ctgtattccc tgcagttaga 240
aggacagtat tggtagcctg gggtctgagt ctagactaag aaatgaacat gcagcaggct 300
gtagtggttt tgtggcagca aaatcatgac ctgaagtttg gtgggaagct gttcttccac 360
agtttattgg gaaaaagact tgtaatttcc attgaccaga ccttcataga gaggcagcat 420
catctctctc ccaatccctt catccaaccg caccagctaa ttagaggacc tgattagcta 480
gaaggagaga aggagtaaaa gaattagggg aggaaaaaga ggggaagcca atcagcttcc 540
tttcttttgt tgtttgtcca cagtaggacc tacaagggaa agggggatat gcctgaaatc 600
aaggttagat tctttgatga tgacatagga ctggacattt taggggctga attgagactc 660
tagtaaatga atgaacagga attattaatt tataataaat cgtgaggcta cccagtttac 720
ctgaacaggg aaaaggggaa gaaatatctc cactcatcaa ctgtaaaaga caatgagaag 780
caaaactaga atcactttat gactacatcc catgagtcat gtttgttccc attatagtta 840
tattggcttg acctgatgat ttctgagagg aagtgttaac atcccctatt atggttatgt 900
ctttacctac tagtccttat atagttcact gggtttgctt tagcatatgt taaactgtta 960
cagctaggag aataaagact cctaactgtt ttataagttc ctagtaaaag ggactttgtg 1020
tcataataaa ataggcctct ttacccaaat aattgatctt cacattgaat tctatttttt 1080
aattatatca ctgttgccac agccattttt aaaataaatt ttactagata ttttttttcc 1140
atcctacatt tttaaacagt gtatcatttt gtttgggatg tgtctcttaa aaataccatt 1200
tagtaacttg ggttgtctat ttgttcttat tctggt 1236
<210> 2
<211> 20
<212> DNA
<213> 未知(Unknown)
<400> 2
gccaatccaa accgaaagcc 20
<210> 3
<211> 20
<212> DNA
<213> 未知(Unknown)
<400> 3
agactcagac cccaggctac 20
<210> 4
<211> 20
<212> DNA
<213> 未知(Unknown)
<400> 4
tttgctacat cccagctcca 20
<210> 5
<211> 20
<212> DNA
<213> 未知(Unknown)
<400> 5
tccccctttc ccttgtaggt 20
<210> 6
<211> 21
<212> DNA
<213> 未知(Unknown)
<400> 6
catgtacgtt gctatccagg c 21
<210> 7
<211> 21
<212> DNA
<213> 未知(Unknown)
<400> 7
ctccttaatg tcacgcacga t 21
Claims (5)
1.检测IncRNA LNC_004208表达量的试剂在制备鉴别高级别和低级别脑胶质瘤试剂中的应用,该IncRNA LNC_004208的序列如SEQ NO:1所示。
2.根据权利要求1所述的应用,其特征在于,所述的检测IncRNA LNC_004208表达量的试剂用于检测脑胶质瘤组织。
3.根据权利要求1所述的应用,其特征在于,所述的检测IncRNA LNC_004208的试剂包括实时荧光定量检测试剂。
4.根据权利要求3所述的应用,其特征在于,扩增lncRNA LNC_004208的引物:
LNC_004208(2)-正向引物 5’-TTTGCTACATCCCAGCTCCA-3’
LNC_004208(2)-反向引物 5’-TCCCCCTTTCCCTTGTAGGT-3’。
5.根据权利要求3所述的应用,其特征在于,检测试剂中用于对照的β-actin正向引物为5’-CATGTACGTTGCTATCCAGGC-3’,反向引物5’-CTCCTTAATGTCACGCACGAT-3’。
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Publication number | Priority date | Publication date | Assignee | Title |
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CN103966339A (zh) * | 2014-05-26 | 2014-08-06 | 中南大学 | 长链非编码rna crnde的应用方法 |
CN105506155A (zh) * | 2016-01-29 | 2016-04-20 | 中南大学 | 检测长链非编码rna loc284454表达量的试剂的应用 |
CN108504737A (zh) * | 2018-04-11 | 2018-09-07 | 中南大学湘雅医院 | 一种长链非编码rna tralr的应用 |
CN108531484A (zh) * | 2018-04-11 | 2018-09-14 | 中南大学湘雅医院 | 抑制长链非编码rna tralr制剂的应用 |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103966338A (zh) * | 2014-05-26 | 2014-08-06 | 中南大学 | 长链非编码rna rmst的应用方法 |
CN103966339A (zh) * | 2014-05-26 | 2014-08-06 | 中南大学 | 长链非编码rna crnde的应用方法 |
CN105506155A (zh) * | 2016-01-29 | 2016-04-20 | 中南大学 | 检测长链非编码rna loc284454表达量的试剂的应用 |
CN108504737A (zh) * | 2018-04-11 | 2018-09-07 | 中南大学湘雅医院 | 一种长链非编码rna tralr的应用 |
CN108531484A (zh) * | 2018-04-11 | 2018-09-14 | 中南大学湘雅医院 | 抑制长链非编码rna tralr制剂的应用 |
Non-Patent Citations (1)
Title |
---|
An Insight into the Increasing Role of LncRNAs in the Pathogenesis of Gliomas;Yuanliang Yan等;《Frontiers in Molecular Neuroscience》;20170228;第10卷;Article 53 * |
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