CN111154882A - 长链非编码rna linc01107在制备白血病诊断试剂盒上的应用 - Google Patents
长链非编码rna linc01107在制备白血病诊断试剂盒上的应用 Download PDFInfo
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Abstract
本发明提供了一种长链非编码RNA LINC01107在制备急性髓性白血病诊断试剂盒上的应用。本发明通过实验发现相较于正常人，患有急性髓性白血病的患者外周血中长链非编码RNA LINC01107的表达明显上调，而完全缓解的患者外周血中长链非编码RNA LINC01107的表达量相较于急性髓性白血病患者显著下调，针对该结果本发明设计了一种检测长链非编码RNA LINC01107表达量的试剂盒，用于急性髓性白血病患者的早期诊断和预后诊断。
Description
技术领域
本发明属于生物医药技术领域，尤其涉及长链非编码RNA LINC01107在制备白血病诊断试剂盒上的应用。
背景技术
白血病是在造血干细胞水平异常转化的一类克隆性恶性血液病。白血病细胞由于分化停滞、增殖失控、凋亡受阻等机制在骨髓和其他造血组织中大量的克隆增生，并且浸润其他组织和器官。白血病的死亡率在男性恶性肿瘤中居第六位，在女性恶性肿瘤中居第八位。按照患者的起病缓急，白血病可以分为急性白血病和慢性白血病。近年来，随着环境污染的不断加剧，急性髓性白血病的发病率明显升高。因此，实现急性髓性白血病患者的快速诊断对于实现患者的早发现，早治疗具有重要的意义。
长链非编码RNA是缺乏开放阅读框，长度约为200nt-100000bp的一种不具有编码蛋白质能力的RNA。目前认为，长链非编码RNA已被证明参与基因表达调控的几乎每一个层面，涉及转录调控、细胞内物质运输和染色体重塑，并且参与细胞分化、生长发育、应激反应以及疾病发生发展等多种生物学进程。目前已有研究显示，长链非编码RNA不仅参与调节机体的生理过程，在疾病治疗前后对某些异常表达的长链非编码RNA表达水平的检测也成为目前肿瘤诊断及预后的重要部分，同时，长链非编码RNA在肿瘤的发生发展中发挥着重要作用。目前，长链非编码在急性髓性白血病中的研究还处于起始阶段，发明新的急性髓性白血病相关长链非编码RNA对于白血病的临床诊断、靶向治疗均具有重要的意义。
目前，关于LINC01107在急性髓性白血病中的作用尚未见报道。
发明内容
针对上述现有技术，本发明的目的在于提供一种通过荧光定量PCR技术在正常组，白血病组以及完全恢复组检测LINC01107的差异表达情况，以此设计检测试剂盒为急性髓性白血病患者的早期诊断和预后诊断提供支持。
为实现上述目的，本发明提供了如下技术方案：
本发明提供了一种长链非编码RNA，所述长链非编码RNA为LINC01107；LINC01107在GeneBank中的ID号为151171，转录本为NR_037809；LINC01107的序列如SEQ ID NO.1所示。
此外，本发明提供了长链非编码 RNA LINC01107在制备急性髓性白血病诊断试剂盒中的应用。
优选地，所述试剂盒包括检测长链非编码 RNA LINC01107表达量的试剂。
优选地，所述试剂包括检测长链非编码 RNA LINC01107的引物对。
优选地，所述引物对的序列如SEQ ID NO.2和SEQ ID NO.3所示。
除此之外，本发明提供了一种用于急性髓性白血病诊断的荧光定量检测试剂盒，所述试剂盒包括权利要求5所述的引物对。
优选地，所述试剂盒还包括管家基因GAPDH引物对；所述GAPDH引物对的序列如SEQID NO.4和SEQ ID NO.5所示。
优选地，所述试剂盒还包括逆转录试剂、SYBR Green荧光染料、PCR缓冲液、DEPC水。
本发明的有益效果在于
本发明首次证明了长链非编码 RNA LINC01107与急性髓性白血病的相关性，并且提供了一种荧光定量PCR检测试剂盒用于白血病患者的早期诊断和预后诊断，相较于传统检测方法，本发明具有灵敏度高，对患者创伤小的优点。
附图说明
图1 LINC01107在正常组，白血病组和完全恢复组中的差异表达情况。
图2 正常组和白血病组的ROC曲线。
图3 白血病组和完全恢复组的ROC曲线。
图4 LINC01107在单个核细胞、U937细胞、HL-60细胞中的表达情况。
图5 Ara-C（阿糖胞苷）处理HL-60细胞后LINC01107的表达情况。
具体实施方式
实施例1
检测LINC01107在正常组，白血病组和完全恢复组中的差异表达情况
1.材料
30例急性髓性白血病患者外周血样本，30例正常人外周血样本，10例完全恢复外周血样本来自于北京大学肿瘤医院。所有样本均通过医院伦理委员会审查和批准，且所有样本来源均签署知情同意书。
所有白血病患者均通过病理诊断确认，且患者未患有其他肿瘤，并且未采取放疗或化疗等。
2. RNA提取
（1）采用Ficoll法分离单个核细胞；
（2）加入1ml Trizol充分吹打混匀，室温静置5min；
（3）加入200μl氯仿，震荡离心管，充分混匀后，室温放置10min；
（4）置于离心机中，12000rpm/min 离心15min；
（5）将上层水相转移至另一离心管中，加入等体积预冷异丙醇，上下颠倒混匀，混匀后冰上静置10min；
（6）置于离心机中，4℃，12000rpm离心10分钟；
（7）小心去除上清，加入1ml 75%乙醇悬浮沉淀；
（8）置于离心机中，4℃，12000rpm离心5分钟；
（9）小心去除乙醇液体，室温下放置5min充分干燥沉淀，加入DEPC水溶解沉淀；
（10）使用紫外分光光度计测量RNA的纯度和浓度。
3.荧光定量PCR检测
参照Takara试剂盒
（1）去除基因组DNA
反应体系：5×gDNA Eraser Buffer 2.0μl，gDNA Eraser 1μl，Total RNA 1μg，RNaseFree dH2O up to 10μl；
反应条件42℃ 2min，4℃。
（2）逆转录反应
反应体系：步骤（1）反应液 10μl，PrimeScript RT Enzyme Mix I 1.0μl ，RT PrimerMix 1.0μl，5×PrimeScript Buffer 2 4.0μl，RNase Free dH2O 4.0μl。
反应条件：37℃ 15分钟；85℃ 5s；4℃。
（3）PCR检测
反应体系：SYBR Green Premix Ex Taq（2×） 10μl，正向引物0.4μl，反向引物 0.4μl，cDNA模板 2μl，ddH2O 7.2μl。
反应条件：95℃ 5min；95℃ 10s，60℃ 40s，72℃ 30s,35个循环；75℃，30s。
LINC01107
正向引物序列5’-TGCTTGTGAGATTCCACGCA-3’
反向引物序列5’-GGGTCTTCTGATGATGTGCTCA-3’
GAPDH
正向引物序列5’-AATGGGCAGCCGTTAGGAAA-3’
反向引物序列5’-ATCTAGGAAAAGCATCACCCGG-3’
（4）结果统计
A： LINC01107在正常组，白血病组，完全缓解组的表达量差异
使用2-△△Ct处理得到的数据，计算LINC01107的表达水平。结果如图1所示，从图1可以看出，相较于正常组，白血病组（5.49±0.48）的LINC01107表达量显著上调，差异具有统计学意义。
相较于正常组，完全缓解组（1.25±0.07）的LINC01107表达量有部分上调，但差异无统计学意义。
相较于白血病组，完全缓解组的LINC01107表达量显著降低，差异具有统计学意义。
综上所述，通过检测LINC01107的表达量，可以用于白血病患者的早期诊断以及预后诊断。
B：LINC01107对于白血病的诊断价值
正常组和白血病组的ROC曲线如图2所示，AUC值为 0.9633，Std. Error 0.01979，95%confidence interval 0.9245-1.002，P ＜0.0001,表明检测LINC01107表达量对于急性髓性白血病患者的早期诊断具有一定的价值。
白血病组和完全恢复组的ROC曲线如图3所示，AUC值为0.9533，Std. Error 0.0,3159，95% confidence interval 0.8914-1.015，P ＜0. 0001,表明检测LINC01107表达量对于急性髓性白血病患者的预后诊断也具有一定的价值。
实施例2
检测LINC01107在单个核细胞、U937细胞、HL-60细胞中的表达情况，每组设置3个重复。
实验方法同实施例1
实验结果
实验结果如图4所示，相较于单个核细胞，人急性骨髓白血病细胞U937和HL-60细胞中的LINC01107的表达量显著上调，差异具有统计学意义。其中，HL-60细胞中LINC01107的表达量最高（8.23±0.37），因此，选择其进行后续的实验。
实施例3
Ara-C（阿糖胞苷）处理HL-60细胞后LINC01107的表达情况
采用 500nM 的 Ara-C 处理 HL-60 细胞株，48h后提取RNA，检测LINC01107的表达情况，每组设置3个重复。
实验方法同实施例1。
实验结果如图5所示，可以看出，Ara-C处理HL-60细胞后，细胞中的LINC01107表达量显著下调，差异具有统计学意义，进一步证明了检测LINC01107表达量可以用于急性髓性白血病患者的早期诊断和预后诊断。
实施例4 用于急性髓性白血病诊断的LINC01107荧光定量检测试剂盒
试剂盒包括如下组分：LINC01107引物对，GAPDH引物对，SYBR Green PCR反应体系；
LINC01107引物对正向引物序列5’-TGCTTGTGAGATTCCACGCA-3’；反向引物序列5’-GGGTCTTCTGATGATGTGCTCA-3’。
GAPDH引物对正向引物序列5’-AATGGGCAGCCGTTAGGAAA-3’；反向引物序列5’-ATCTAGGAAAAGCATCACCCGG-3’。
SYBR Green PCR反应体系为SYBR Green荧光染料、dNTPs和PCR缓冲液。
反应条件：95℃ 5min；95℃ 10s，60℃ 40s，72℃ 30s,35个循环；75℃，30s。
本发明的实施例仅用于解释本发明，并不意味着限制本发明的保护范围。需要注意的是，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，还可以对本发明进行若干改进。因此在不背离权利要求及等同范围所限定的一般概念下，本发明并不限于特定的细节和这里示出与描述的图例。
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<110> 山东殷氏干细胞有限公司
<120> 长链非编码RNA LINC01107在制备白血病诊断试剂盒上的应用
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2420
<212> DNA
<213> 人源(Human)
<400> 1
tacttctgct ttccttgggt ttgtttgtct ttcatttcaa gtgatttctt aatttccttt 60
aacagttttt aaactgtgga attattttct cagtatagtc caagctacat ctaataagct 120
ctagcatgta actttttctt ttttgttctt ttctaaatat tgtgtcactg ctgatttggt 180
ttttctcttt tatccatgag atgactgttt taagttccca ggaagcgagt cttcttgttc 240
caatttattc ctgaattttt attttgactg tcattacact gtagttagga gatgtgacct 300
ctactgtctc cagcatttag aatgtattgc ggatccaggg acacagcaag tcaggtgggc 360
tctgtcgcaa ggatggttac aacatagact acttccatgt cagtcttcca ttatttgcat 420
actcgagacc ctgatgtgtt ggctaatttg ccaagaaacc cccgcagcag actgcgtccg 480
cagggcaggg ctgagtagca ggggcagcac ctctcacagg ggcagggccg cttgcccagc 540
cttgaggaag ggggacccag acattgcctt tctcaatact gagcccctaa acaggatttc 600
tggtctgggg tctgggacta ggctatctgg acagtcaaac tcttcccact gcgccatcag 660
ccccactagg aaagaaagag gagaaattag ggctttgctt taaagagttc aacggtcaga 720
atatgtcccc cgcaaacgga catgctggaa tcctcatccc caagtgaggg cggggcctca 780
cattaggagg tgatgaggtc acagaggcag aaacctcacg aacgggatca ggctcatcaa 840
agaggcctga gagacgacac ctaccagctg gaaggcggtt ccgcaccaga caccgaagct 900
gctgggcctt cgacctcgga cctccgtctt ccagaactgt gagccacaca ttcctgatgc 960
tcctaagcca cgccgtgtac ggtattttgt tacggtagcc caaatggacc aagataggag 1020
ctaaaggtgc ttgtgagatt ccacgcatca tactagaggt gtgagcactg cacctaaggg 1080
tgcctcggat catccctgag atgcaggcac tgcaccagga ggtgtcttgg atcattcctg 1140
aggtgtgagc acatcatcag aagacccttt tgtccatggg gtcccattct caccttctgg 1200
atacacatca ctctcggggc cgccattcta cctgcctcat ctgtgcgcgg cagtctggac 1260
ctagaagctc tgtcccctct gctgcccagt ggagtcgagt gcctggcacc caacacccag 1320
acagggtggc caccaacctg gttgagggga ctgtccccaa gatgatcaca gaacatcagg 1380
cagggctcct gagccagcgc cccccccaag cctgcctcgc tcccagtacg gccggaagag 1440
tagctgaggc caacccctga agcagccaag gcgtcctcta tgcccagaag tgccagggat 1500
cgggggctgc ccagttccag acagggtcat cacactccga ccttcagagg ggccccccgc 1560
ctgccagctc cagacagggc catcacactc ccgatccaca gagggccccc ccacgcctcc 1620
cagctcctga cggaagcgcc tgtccagctt caccgacctc catgccgctg cctggggtct 1680
tgctcgaacg caagagaaca cgggtctcgt tgagggctcc tctgccccgc atctcctgct 1740
cctggacacc caggcctgcc ctcaccactg ctgctggaca ccgggcaggg gtcctggcca 1800
ctcttctgga aggccagtct gacgttgcag gcgcagccac ttcactgttt gtccttcacg 1860
gtatgccaga ctccctcctc cctgcaagag cctctcccag ggcccagtcc tcccccgaga 1920
ggtgctgatg agagtggagt atggaaccat ctgccagcca accccaggtg gcctctcggg 1980
ggtgccctgc cccacagggc catgcacggt gtggccctcc ccggtgctgt cacgtctgaa 2040
tgtggccctg cagacttgcc cccactgggc tggagcacat cccctggaat ctgatcccag 2100
gagctccggg agcagctcgc acacagctcc atggatgccc ctcacagtgg gagagacagg 2160
aaaggccagc tccgagtctc acacctcact cttcaagctg ggaaaatgat gctgttacct 2220
tgggaggtga gaccctccag atgggaacct cgacgctggg agctggtgtg ttcccggaat 2280
tcgggggaat atttcattcc ctgcgtatgg tgtctggact ttcagatccc tcctcccagg 2340
cacccaggca tcatttcaca accccatgct cagcaagccc cagagaaaac tcctcaataa 2400
actacgagtg ttaatacccg 2420
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tgcttgtgag attccacgca 20
<210> 3
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gggtcttctg atgatgtgct ca 22
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
aatgggcagc cgttaggaaa 20
<210> 5
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atctaggaaa agcatcaccc gg 22
Claims (8)
1.一种长链非编码RNA，其特征在于，所述长链非编码RNA为LINC01107。
2.长链非编码 RNA LINC01107在制备急性髓性白血病诊断试剂盒中的应用。
3.根据权利要求2所述的应用，其特征在于，所述试剂盒包括检测长链非编码 RNALINC01107表达量的试剂。
4.根据权利要求3所述的应用，其特征在于，所述试剂包括检测长链非编码 RNALINC01107的引物对。
5.根据权利要求4所述的应用，其特征在于，所述引物对的序列如SEQ ID NO.2和SEQID NO.3所示。
6.一种用于急性髓性白血病诊断的LINC01107荧光定量检测试剂盒，其特征在于，所述试剂盒包括权利要求5所述的引物对。
7.根据权利要求6所述的试剂盒，其特征在于，所述试剂盒还包括管家基因GAPDH引物对；所述GAPDH引物对的序列如SEQ ID NO.4和SEQ ID NO.5所示。
8.根据权利要求6所述的试剂盒，其特征在于，所述试剂盒还包括逆转录试剂、SYBRGreen荧光染料、PCR缓冲液、DEPC水。
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| CN111118013A (zh) * | 2020-03-09 | 2020-05-08 | 山东殷氏干细胞有限公司 | 白血病生物标志物linc02033及其应用 |
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| CN112877439A (zh) * | 2021-04-12 | 2021-06-01 | 广东医科大学 | 长链非编码rna enst00000522339在all白血病中的应用 |
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| CN113151466A (zh) * | 2021-04-12 | 2021-07-23 | 广东医科大学 | 长链非编码RNA uc002ubt.1在ALL白血病中的应用 |
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Cited By (6)
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| CN111118013A (zh) * | 2020-03-09 | 2020-05-08 | 山东殷氏干细胞有限公司 | 白血病生物标志物linc02033及其应用 |
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| CN112877439A (zh) * | 2021-04-12 | 2021-06-01 | 广东医科大学 | 长链非编码rna enst00000522339在all白血病中的应用 |
| CN112961923A (zh) * | 2021-04-12 | 2021-06-15 | 广东医科大学 | 长链非编码rna tcons00026679在all白血病中的应用 |
| CN113151466A (zh) * | 2021-04-12 | 2021-07-23 | 广东医科大学 | 长链非编码RNA uc002ubt.1在ALL白血病中的应用 |
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