CN114075603A - 一种确定AJUBA基因启动子CpG岛差异甲基化位点的方法 - Google Patents

一种确定AJUBA基因启动子CpG岛差异甲基化位点的方法 Download PDF

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CN114075603A
CN114075603A CN202010878080.6A CN202010878080A CN114075603A CN 114075603 A CN114075603 A CN 114075603A CN 202010878080 A CN202010878080 A CN 202010878080A CN 114075603 A CN114075603 A CN 114075603A
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闫池
郭永军
马杰
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Abstract

本发明采用亚硫酸盐扩增子测序技术，通过特异性引物对1和特异性引物对2对AJUBA基因启动子CpG岛在非小细胞肺癌和正常组织中的差异甲基化位点进行分析，同时采用引物对3通过Sanger测序法分析该启动子的突变，结果发现在非小细胞肺癌肿瘤和正常组织间的差异甲基化位点，同时实验结果表明AJUBA启动子的单核苷酸变异与肿瘤患者的不良预后相关，因此AJUBA基因可以作为潜在的肺癌预后的分子标志物。

Description

一种确定AJUBA基因启动子CpG岛差异甲基化位点的方法
技术领域
本发明涉及采用亚硫酸盐扩增子测序的方法确定抑癌基因AJUBA启动子CpG岛差异甲基化位点，该方法跟传统的焦磷酸测序法、甲基化特异PCR法和对亚硫酸盐转化DNA直接进行Sanger测序相比较，定量准确率更高，扩增子更长和通量更高，成本相对较低。
背景技术
亚硫酸盐扩增子测序技术是把现有的亚硫酸盐转化和片段特异PCR扩增与文库构建及二代测序技术集合起来一种方法，可同时对多个样本和多个基因的单个碱基进行甲基化分析。
发明内容
本发明采用亚硫酸盐扩增子测序对AJUBA启动子上的两个CpG进行甲基化差异位点分析，其中一个CpG岛转录起始位点（TSS）上游-652bp — -153bp处，序列为GGGTCCAGAAGGTGGTGCCTTTTTATTGCAGTTTATCTGTTTGTCTCTCATTCTCTCTGGACAAGCTTGTCCTTTCCTTTGTCTGCGCACCCTTCGGTCTCTGCATCGTCCACACTCCTCTTTGCTCCTCAGCTCTCCCTTTCTTTCGCTACCTCTTCCCTCTTCCACCTCTCTCTGTGGGCTTCGTCGCCTTTCAGGGCTATCTTTACGTACACGTCTCTGAACCTCTTTATGCATTTGTCTTCTTTCTCTCTGTCTTCCTCTTTATGGCTCTCTCCTTGTCTCACCATTTCTGTACCAGTCTCTCTCCATCTCTCCCACAGTCCCCACGCTGGGAACAAAGTCACGGTGCTAGCGCAGAACATGCCTCCTGTCGTCCGGTCCCGGGACGCGACACATTCCACCATGCCCCTCCGCGCATTCCAACTGCAGACAAAGAGTCCCCGTCCCCTACCCCCACACTCTTTCCTATTCCAATTCTTCAGTGTTGTAGGGGCAGC，扩增该序列的正向引物mAF1为5’-GGGTTTAGAAGGTGGTGTTT-3’，反向引物mAR1为5’-ACTACCCCTACAACACTAAAAA-3’；另一个CpG岛位于TSS下游+9bp — +293bp处，序列为CCAGGCAGACAAAAGTTTGGGAGAGAAGTTCGGTCTGTGGCTAGCGTGAGAGCTAGGGGGGCGGGGGCCGGGGAGAGTGGGGCAGTCGGGCGAGTCGACGCGTGAACAGATAGACCTGCGGACTGGACAGCCGCGGCCAGAGACCCTGCTAGCCCCGCTCAGCCCCAGATGCGCGGGGGGACGCAGCCCCTCCCGCTGGGGGATGCTGTGGGATTCCTGGCGCAGGGCATCCAGGCCGCCCGCTAAGCCCCTGTGCCTCCCCTGTGCCCCTGGGGAACCAGAGTC，扩增该序列的正向引物mAF2为5’-TTAGGTAGATAAAAGTTTGGGAGAGAAG-3’，反向引物mAR2为5’-AACTCTAATTCCCCAAAAACACA-3’，通过亚硫酸盐扩增子测序的方法得到这两段序列在非小细胞肺癌和正常组织之间的差异甲基化位点。具体实施方式为：
一、非小细胞肺癌和正常石蜡组织DNA提取：采用凯杰FFPE组织提取试剂盒进行基因组DNA提取（QIAamp DNA FFPE Tissue Kit,Cat No:56404）。
二、DNA甲基化转化反应：采用Zymo Research DNA 甲基化转化试剂盒进行此步骤（EZ DNA Methylation-GoldTM Kit, Cat No. D5005）。
三、PCR反应：1.反应体系配制
PCR Mix 配制 体积（μl）
Takara Ex Taq HS (RR006A) 0.25
10* Ex Taq Buffer 2.5
dNTP Mixture (2.5mM each) 2
DNA甲基化转化溶液(Template) 1.5
Forward Primer (10 μM) 0.5
Reverse Primer (10 μM) 0.5
Nuclease-Free Water Up to25
2.PCR反应条件
Figure 973004DEST_PATH_IMAGE002
四、建库：采用诺唯赞的DNA建库试剂盒（VAHTS Turbo DNA Library Prep Kitfor Illumina®，Vazyme）进行此步骤。
五、上机测序
1、将测序仪开启处于使用状态。
2、DNA文库取5 ul (4 um)放入1.5ml离心管内添加5 ulNaOH(0.2N)变性5 min。
3、将变性后的文库用测序稀释液稀释至1000 ul。
4、将稀释后的文库加入到测序试剂盒指定区域内。
5、将测序试剂盒及测序液放入仪器内启动测序程序进行测序反应。
六、数据分析及结果
1. AJUBA基因启动子CpG岛在基因组上的相对位置及第一个和第二个CpG岛上各个CG位点的相对位置（图1）。
2. 我们发现AJUBA启动子第一个CpG岛上的CG95 (GL 2345199 bp)和CG415 (GL2345231 bp)以及第二个CpG岛上CG69 (GL 23452626 bp)和CG156 (GL 23452713 bp)位点的甲基化水平在非小细胞肺癌组织和正常组织之间差异显著（图2）。
3. 我们通过对27位非小细胞肺癌病人的肿瘤和正常组织进行Sanger测序分析发现AJUBA启动子上存在28个单核苷酸突变，其中21.43%（6/28）的位点位于CG位置（表1）。进一步根据突变类型把这些病人分成突变组和非突变组，生存分析发现突变组患者具有相对较差的预后（图3）。
附图说明：
图1A. AJUBA启动子CpG岛在基因组上的相对位置，1stCpG岛位于TSS上游，2ndCpG岛位于TSS下游; B. 1stCpG岛和2ndCpG岛（C）上各个CG位点的相对位置
图2 A. AJUBA启动子1stCpG岛和2ndCpG岛（C）在非小细胞肺癌不同时期和正常组织（N）中甲基化水平；B. 1stCpG和2ndCpG岛（D）各CG位点在肿瘤（T）和正常组织中甲基化水平
图3 Kaplan-Meier生存分析。
序列表
<110> 闫池/河南省肿瘤医院
<120> 一种确定AJUBA基因启动子CpG岛差异甲基化位点的方法
<130> 20200831
<141> 2020-08-31
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 500
<212> DNA
<213> 人(Homo sapiens)
<220>
<221> promoter
<222> (652)..(153)
<223> 第一个CpG岛位于转录起始位点（TSS)的上游。<400> 1
gggtccagaa ggtggtgcct ttttattgca gtttatctgt ttgtctctca ttctctctgg 60
acaagcttgt cctttccttt gtctgcgcac ccttcggtct ctgcatcgtc cacactcctc 120
tttgctcctc agctctccct ttctttcgct acctcttccc tcttccacct ctctctgtgg 180
gcttcgtcgc ctttcagggc tatctttacg tacacgtctc tgaacctctt tatgcatttg 240
tcttctttct ctctgtcttc ctctttatgg ctctctcctt gtctcaccat ttctgtacca 300
gtctctctcc atctctccca cagtccccac gctgggaaca aagtcacggt gctagcgcag 360
aacatgcctc ctgtcgtccg gtcccgggac gcgacacatt ccaccatgcc cctccgcgca 420
ttccaactgc agacaaagag tccccgtccc ctacccccac actctttcct attccaattc 480
ttcagtgttg taggggcagc 500
<210> 2
<211> 285
<212> DNA
<213>人(Homo sapiens)
<220>
<221> promoter
<222> (9)..(293)
<223>第二个CpG岛位于转录起始位点（TSS)的下游。
<400> 2
ccaggcagac aaaagtttgg gagagaagtt cggtctgtgg ctagcgtgag agctaggggg 60
gcgggggccg gggagagtgg ggcagtcggg cgagtcgacg cgtgaacaga tagacctgcg 120
gactggacag ccgcggccag agaccctgct agccccgctc agccccagat gcgcgggggg 180
acgcagcccc tcccgctggg ggatgctgtg ggattcctgg cgcagggcat ccaggccgcc 240
cgctaagccc ctgtgcctcc cctgtgcccc tggggaacca gagtc 285
<210> 3
<211> 1300
<212> DNA
<213>人(Homo sapiens)
<220>
<221> variation
<400> 3
gcgtgaacag atagacctgc ggactggaca gccgcggcca gagaccctgc tagccccgct 60
cagccccaga tgcgcggggg gacgcagccc ctcccgctgg gggatgctgt gggattcctg 120
gcgcagggca tccaggccgc ccgctaagcc cctgtgcctc ccctgtgccc ctggggaacc 180
agagtccggc tgcagggaaa gagaaccggc cgccgagacg ccgcagggtg ccaggcgggg 240
agggggcgag aggccccagg cccgagggca tggagcggtt aggagagaaa gccagtcgcc 300
tgctggagaa gttcggccgc agaaagggtg aatctagccg gtctgggtct gacgggaccc 360
ccgggccggg caaggggcgc ctaagtgggt tggggggacc taggaagtca gggccccgag 420
gagctactgg gggacctggg gatgagccgt tggagccggc ccgggagcaa ggttccctgg 480
acgctgagcg aaatcagcgc ggctcctttg aggcgccgcg ctacgaaggc tcttttcccg 540
cggggccgcc gcccacccgg gccttgcctc tacctcagtc gttgcccccc gattttcggc 600
tggagcccac ggccccggcc ctcagccccc gctctagctt cgccagtagc tcggccagcg 660
acgcgagcaa gccgtccagc ccccggggca gcctgctgct ggacggggcg ggggctggcg 720
gagctggagg tagccggccc tgcagcaatc gcaccagcgg catcagcatg ggctacgacc 780
agcgccacgg gagccccttg ccagcggggc cgtgcctgtt tggcccaccc ctggccggag 840
caccggcagg ctattctccc ggaggggtcc cgtccgccta cccggagctc cacgccgccc 900
tggaccgatt gtacgctcag cggcccgcgg ggttcggctg ccaggaaagc cgccactcgt 960
atcccccggc cctgggcagc cctggagctc tagccggggc cggagtggga gcggcggggc 1020
ccttggagag acggggggcg caacccggac gacactctgt gaccggctac ggggactgcg 1080
ccgtgggcgc ccggtaccag gacgagctaa cagctttgct tcgcctgacg gtgggcaccg 1140
gtgggcgaga agccggagcc cgcggagaac cctcggggat tgagccgtcg ggtctggagg 1200
agccaccagg tcctttcgtt ccggaggccg cccgggcccg gatgcgggag ccagaggcca 1260
gggaggacta cttcggtgag tgagagaagt ggttagggac 1300

Claims (2)

1.一种确定AJUBA基因启动子CpG岛差异甲基化位点的方法，其特征在于：AJUAB基因启动子区域存在两个CpG岛，一个CpG岛位于ATG上游-652bp — -153bp处，序列为GGGTCCAGAAGGTGGTGCCTTTTTATTGCAGTTTATCTGTTTGTCTCTCATTCTCTCTGGACAAGCTTGTCCTTTCCTTTGTCTGCGCACCCTTCGGTCTCTGCATCGTCCACACTCCTCTTTGCTCCTCAGCTCTCCCTTTCTTTCGCTACCTCTTCCCTCTTCCACCTCTCTCTGTGGGCTTCGTCGCCTTTCAGGGCTATCTTTACGTACACGTCTCTGAACCTCTTTATGCATTTGTCTtctttctctctgtcttcctctttatggctctctccttgtctcaccatttctgtaccagtctctctccatctctcccACAGTCCCCACGCTGGGAACAAAGTCACGGTGCTAGCGCAGAACATGCCTCCTGTCGTCCGGTCCCGGGACGCGACACATTCCACCATGCCCCTCCGCGCATTCCAACTGCAGACAAAGAGTCCCCGTCCCCTACCCCCACACTCTTTCCTATTCCAATTCTTCAGTGTTGTAGGGGCAGC，扩增该序列的正向引物mAF1为5’-GGGTTTAGAAGGTGGTGTTT-3’，反向引物mAR1为5’-ACTACCCCTACAACACTAAAAA-3’；另一个CpG岛位于ATG下游+9bp — +293bp处，序列为CCAGGCAGACAAAAGTTTGGGAGAGAAGTTCGGTCTGTGGCTAGCGTGAGAGCTAGGGGGGCGGGGGCCGGGGAGAGTGGGGCAGTCGGGCGAGTCGACGCGTGAACAGATAGACCTGCGGACTGGACAGCCGCGGCCAGAGACCCTGCTAGCCCCGCTCAGCCCCAGATGCGCGGGGGGACGCAGCCCCTCCCGCTGGGGGATGCTGTGGGATTCCTGGCGCAGGGCATCCAGGCCGCCCGCTAAGCCCCTGTGCCTCCCCTGTGCCCCTGGGGAACCAGAGTC，扩增该序列的正向引物mAF2为5’-TTAGGTAGATAAAAGTTTGGGAGAGAAG-3’，反向引物mAR2为5’-AACTCTAATTCCCCAAAAACACA-3’，通过亚硫酸盐扩增子测序的方法得到这两段序列在非小细胞肺癌和正常组织之间的差异甲基化位点。
2.一种确定AJUBA基因启动子单核苷酸变异的方法，特征如下：该调控区域的序列为GCGTGAACAGATAGACCTGCGGACTGGACAGCCGCGGCCAGAGACCCTGCTAGCCCCGCTCAGCCCCAGATGCGCGGGGGGACGCAGCCCCTCCCGCTGGGGGATGCTGTGGGATTCCTGGCGCAGGGCATCCAGGCCGCCCGCTAAGCCCCTGTGCCTCCCCTGTGCCCCTGGGGAACCAGAGTCCGGCTGCAGGGAAAGAGAACCGGCCGCCGAGACGCCGCAGGGTGCCAGGCGGGGAGGGGGCGAGAGGCCCCAGGCCCGAGGGCATGGAGCGGTTAGGAGAGAAAGCCAGTCGCCTGCTGGAGAAGTTCGGCCGCAGAAAGGGTGAATCTAGCCGGTCTGGGTCTGACGGGACCCCCGGGCCGGGCAAGGGGCGCCTAAGTGGGTTGGGGGGACCTAGGAAGTCAGGGCCCCGAGGAGCTACTGGGGGACCTGGGGATGAGCCGTTGGAGCCGGCCCGGGAGCAAGGTTCCCTGGACGCTGAGCGAAATCAGCGCGGCTCCTTTGAGGCGCCGCGCTACGAAGGCTCTTTTCCCGCGGGGCCGCCGCCCACCCGGGCCTTGCCTCTACCTCAGTCGTTGCCCCCCGATTTTCGGCTGGAGCCCACGGCCCCGGCCCTCAGCCCCCGCTCTAGCTTCGCCAGTAGCTCGGCCAGCGACGCGAGCAAGCCGTCCAGCCCCCGGGGCAGCCTGCTGCTGGACGGGGCGGGGGCTGGCGGAGCTGGAGGTAGCCGGCCCTGCAGCAATCGCACCAGCGGCATCAGCATGGGCTACGACCAGCGCCACGGGAGCCCCTTGCCAGCGGGGCCGTGCCTGTTTGGCCCACCCCTGGCCGGAGCACCGGCAGGCTATTCTCCCGGAGGGGTCCCGTCCGCCTACCCGGAGCTCCACGCCGCCCTGGACCGATTGTACGCTCAGCGGCCCGCGGGGTTCGGCTGCCAGGAAAGCCGCCACTCGTATCCCCCGGCCCTGGGCAGCCCTGGAGCTCTAGCCGGGGCCGGAGTGGGAGCGGCGGGGCCCTTGGAGAGACGGGGGGCGCAACCCGGACGACACTCTGTGACCGGCTACGGGGACTGCGCCGTGGGCGCCCGGTACCAGGACGAGCTAACAGCTTTGCTTCGCCTGACGGTGGGCACCGGTGGGCGAGAAGCCGGAGCCCGCGGAGAACCCTCGGGGATTGAGCCGTCGGGTCTGGAGGAGCCACCAGGTCCTTTCGTTCCGGAGGCCGCCCGGGCCCGGATGCGGGAGCCAGAGGCCAGGGAGGACTACTTCGGTGAGTGAGAGAAGTGGTTAGGGAC，扩增该序列的正向引物AF为5’-GCGTGAACAGATAGACCTGC-3’，反向引物AR为5’-GTCCCTAACCACTTCTCTCACT-3’， 通过Sanger测序法对非小细胞肺癌组织和正常组织进行DNA测序，并比较他们之间的序列多态性，本研究结果说明该段序列单核苷酸突变与非小细胞肺癌病人的术后不良预后相关。
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110287967A1 (en) * 2009-01-28 2011-11-24 Ait Austrian Institute Of Technology Gmbh Lung Cancer Methylation Markers
CN108148910A (zh) * 2017-12-18 2018-06-12 广东省人民医院(广东省医学科学院) 一种肺癌相关的285基因靶向捕获测序试剂盒及其应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110287967A1 (en) * 2009-01-28 2011-11-24 Ait Austrian Institute Of Technology Gmbh Lung Cancer Methylation Markers
CN108148910A (zh) * 2017-12-18 2018-06-12 广东省人民医院(广东省医学科学院) 一种肺癌相关的285基因靶向捕获测序试剂盒及其应用

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