CN108315415A - CYP1B1rs162549在制备预测前列腺癌根治术后生化复发风险的试剂中的应用 - Google Patents
CYP1B1rs162549在制备预测前列腺癌根治术后生化复发风险的试剂中的应用 Download PDFInfo
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Abstract
本发明公开了CYP1B1 rs162549在制备预测前列腺癌根治术后生化复发风险的试剂中的应用。检测CYP1B1基因多态性变异位点rs162549的引物或探针在制备预测前列腺癌根治术后生化复发风险的试剂中的应用。一种用于预测前列腺癌根治术后生化复发风险的试剂盒，包含用于检测CYP1B1基因多态性变异位点rs162549基因型的引物和探针。CYP1B1 SNP rs162549与前列腺癌根治术后生化复发风险有关。该位点TT基因型携带者复发风险显著高于其他基因型。对于行前列腺癌根治术的患者进行该位点的基因分型有助于判断复发风险，针对高危患者积极采取辅助治疗以改善预后。
Description
技术领域
本发明属于分子生物学领域，涉及CYP1B1rs162549在制备预测前列腺癌根治术后生化复发风险的试剂中的应用。
背景技术
目前前列腺癌根治术是临床局限性前列腺癌的标准治疗，但10年内术后复发率高达30％。前列腺特异抗原(prostate specific antigen，PSA)和病理指标对于术后复发的预测效果特异度和敏感度较低，临床亟需能够预测术后复发的生物标记物，理想的生物标志物将有助于对患者复发风险分层进行精准辅助治疗从而改善预后。
此前的研究显示雌激素通过氧化代谢在前列腺癌的进展过程中起重要作用。CYP1B1是一种在雌激素代谢羟基化过程中的关键酶。雌激素(如雌激素酮E1、雌二醇E2)可经氧化代谢生成儿茶酚雌激素和雌激素醌，CYP1B1催化儿茶酚雌激素生成致癌性4-O HE。这些氧化代谢产物具有DNA损伤效应，导致前列腺癌发生及持续进展。CYP1B1蛋白表达量在前列腺癌组织中明显高于良性前列腺增生组织。
考虑到CYP1B1在雌激素代谢过程中的重要作用，CYP1B1基因的单核苷酸多态性(Single Nucleotide Polymorphisms，SNP)有可能通过改变基因表达水平导致前列腺癌进展。目前尚无CYP1B1基因多态性与前列腺癌根治术后复发的关联性研究。
发明内容
本发明的目的是针对现有技术的上述不足，提供CYP1B1基因多态性变异位点rs162549作为检测靶点在制备预测前列腺癌根治术后生化复发风险的试剂中的应用。
本发明的另一目的是提供一种用于预测前列腺癌根治术后生化复发风险的试剂盒。
本发明的目的可通过以下技术方案实现：
CYP1B1基因多态性变异位点rs162549作为检测靶点在制备预测前列腺癌根治术后生化复发风险的试剂中的应用；rs162549位点TT基因型携带者术后复发风险显著高于其他基因型。
检测CYP1B1基因多态性变异位点rs162549的引物或探针在制备预测前列腺癌根治术后生化复发风险的试剂中的应用。
一种用于预测前列腺癌根治术后生化复发风险的试剂盒，包含用于检测CYP1B1基因多态性变异位点rs162549基因型的引物和探针。
所述的引物和探针序列为：所述的引物选自：rs162549_For：SEQ ID NO.21，rs162549_Rev:SEQ ID NO.22；所述的探针选自：rs162549_P1:SEQ ID NO.23，rs162549_P2:SEQ ID NO.24。
有益效果：
本发明在426例行前列腺癌根治术的患者中采用Taqman探针法检测CYP1B1基因8个标签SNP的分布情况。采用RT-PCR方法检测127例前列腺癌旁组织中CYP1B1mRNA的表达。采用Cox比例风险模型和Kaplan–Meier法进行关联性和生存分析。结果显示CYP1B1rs162549是前列腺癌根治术后生化复发的独立预测因素(HR:0.65,95％CI:0.33–0.93,P＝0.025)，并且与CYP1B1mRNA的表达相关，该位点TT基因型携带者复发风险显著高于其他基因型。CYP1B1SNP rs162549与前列腺癌根治术后生化复发风险有关。对于行前列腺癌根治术的患者进行该位点的基因分型有助于判断复发风险，针对高危患者积极采取辅助治疗以改善预后。
附图说明
图1、CYP1B1rs162549各基因型的无复发生存曲线
图2、CYP1B1rs162549各基因型的mRNA表达量的比较
具体实施方式
实施例1
1.研究对象
纳入2006年至2009年间在复旦大学附属肿瘤医院接受前列腺癌根治术的426例局限性前列腺癌患者，中位随访时间37.7个月，术后每3月随访PSA变化。将血清PSA水平连续两次≥0.2ng/ml定义为生化复发。临床病理资料和随访信息通过电子病历系统检索获得。排除术后接受辅助内分泌或放疗的患者。
2.候选SNP选择及基因分型检测
利用人类基因组数据库NCBI，查找CYP1B1基因，确定研究区域。通过HapMap数据库查找本研究区域的SNP位点基因分型数据信息，获取汉族人群中CYP1B1基因及其延伸区域内所有SNP位点基因分型数据。将获取的基因分型数据导入Haploview 4.2软件，筛选出最小等位基因频率(minor allele frequency，MAF)大于0.05的SNPs，导出连锁不平衡图谱及数据。通过对连锁不平衡图谱的数据比对，挑选出每个单倍域内r2≥0.8且LOD>3的SNPs，选取平均r2值最大的一个SNP作为该单倍域标签SNP。共确定8个标签SNP位点(rs10916、rs162562、rs2551188、rs9341266、rs9341248、rs162549、rs1056827、rs1056836)。
3.基因分型检测方法
外周血样本来自复旦大学附属肿瘤医院组织库，采用德国Qiagen公司试剂盒，按照操作步骤提取全基因组DNA。琼脂糖凝胶电泳法检测DNA质量，德国Eppendorf公司的BioPhotometer核酸蛋白测定仪检测DNA的浓度和纯度，定量标化至50ng/μl，于4℃下分装备用。采用TaqMan探针法进行基因分型,设计引物和探针购于美国ABI公司，序列见表1。反应体系5μl，包括1μl DNA模板、0.125μl引物探针、2.5μl TaqMan Universal Master Mix以及1.375μl去离子水。在每个384孔反应板中设置阴性空白对照。扩增条件为95℃预变性10min；95℃变性15S，60℃退火30S，60℃延伸30S共40个循环。扩增之后用ABI PRISM7900HT荧光定量PCR仪检测荧光分布情况，并应用SDS 2.4软件进行基因分型。随机抽取10％样本重复试验进行验证，基因型符合率100％。
表1.8个标签SNP位点的引物、探针信息
3.CYP1B1mRNA检测
127例前列腺癌旁组织样本来自复旦大学附属肿瘤医院组织库。按照TRIzol试剂(Invitrogen公司，美国)的说明提取总RNA。PCR反应体系总体积20μL，其中模板cDNA 1μg，10×PCR缓冲液(15mmol/L MgCl2)2.4μL，Taq-DNA聚合酶1U，10mmol/L dNTPs 0.4μL，5mmol/L上游引物和下游引物各lμL，加ddH2O至20μL。依次预变性，变性，退火，延伸。扩增产物为GAPDH，452bp；CYP1B1，297bp。引物CYP1B1上游GCTGCAGTGGCTGCTCCT；下游CCCACGACCTGATCCAATTCT。
4.统计学方法
计量资料以均数±标准差表示，数值变量用非配对的t检验，分类变量用x2检验。组间基因型频率及等位基因频率比较采用x2检验。采用Cox比例风险回归模型，对可能的混杂因素(年龄、临床病理信息)进行调整，计算风险比(hazard ratio，HR)和95％可信区间(95％confidence interval，95％CI)来评估各种基因型与前列腺癌术后复发风险的相关性。Kaplan-Meier法进行生存分析。P<0.05为差异有统计学意义。所有统计分析使用SAS9.1软件完成。
5.结果
患者基线特征见表2，随访期间共有100例患者出现生化复发(23.5％)。术前PSA、病理分期、淋巴结侵犯、Gleason评分与生化复发相关(P<0.01)。
表2.研究对象的临床病理特征
426例患者基因型分布见表3，对上述临床病理特征进行调整后分析显示，CYP1B1rs162549是前列腺癌根治术后复发的独立预后因素(HR:0.65,95％CI:0.33–0.93,P＝0.025)，TT基因型携带者术后复发风险显著升高(图1，P＝0.036)。
表3.CYP1B1标签SNPs与前列腺癌根治术后生化复发的关系
进一步分析CYP1B1各SNP位点与mRNA表达的关系(图2)，与AT或AA基因型相比，rs162549TT基因型的CYP1B1mRNA表达量显著升高，差异具有统计学意义(P＝0.025)。
序列表
<110> 复旦大学附属肿瘤医院
<120> CYP1B1rs162549在制备预测前列腺癌根治术后生化复发风险的试剂中的应用
<160> 34
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tttgccttct ggtatacttc cttac 25
<210> 2
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
caagcaaaag aggtacaaca tcac 24
<210> 3
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gagtatagtg gggttccatg agttat 26
<210> 4
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gagtatagtg gggttccatg atttat 26
<210> 5
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gaaggtagca ttctttggag tt 22
<210> 6
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tgtctatttc tgcatatggg ag 22
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<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
actgaatgaa gaagtatttt ggtaac 26
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
actgaatgaa gcagtatttt ggtaac 26
<210> 9
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
tgtaacccag cgccaaacc 19
<210> 10
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
cgggtggcgt ggtaggtac 19
<210> 11
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
taacggttcc tgcaatctgg ggaca 25
<210> 12
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
taacggttcc tgcaatttgg ggaca 25
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
agggagtttt tgatagttgt gtgta 25
<210> 14
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
tctttcttct aaatgtccat gctt 24
<210> 15
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
ggttaaacat tttcccactc attctga 27
<210> 16
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
ggttaaacat tttcccactt attctga 27
<210> 17
<211> 16
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ctggtggcgc tgctgg 16
<210> 18
<211> 16
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
ctcggggtcg tcgtgg 16
<210> 19
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
gccaacgtca tgagtgccg 19
<210> 20
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
gccaacgtca tgaatgccg 19
<210> 21
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
gtcgtcatca gatgcttgcg 20
<210> 22
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
cgttttttgt tttcttttcc tttc 24
<210> 23
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
aggtgcttgg agtttacctg gcttatttaa 30
<210> 24
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
aggtgcttgg agtttacctg gcttttttaa 30
<210> 25
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
ccccatagtg gtgctgaatg 20
<210> 26
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
tccgagtagt ggccgaaag 19
<210> 27
<211> 17
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
ccgaccggcc ggccttc 17
<210> 28
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
cgccgaccgg cctgccttc 19
<210> 29
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
accacattcc caaggacact g 21
<210> 30
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
ctggtcaggt ccttgttgat g 21
<210> 31
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
tggtctgtga atcatgaccc actgaagtg 29
<210> 32
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
tggtctgtga atcatgaccc agtgaagtg 29
<210> 33
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
gctgcagtgg ctgctcct 18
<210> 34
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
cccacgacct gatccaattc t 21
Claims (4)
1.CYP1B1基因多态性变异位点rs162549作为检测靶点在制备预测前列腺癌根治术后生化复发风险的试剂中的应用；rs162549位点TT基因型携带者术后复发风险显著高于其他基因型。
2.检测CYP1B1基因多态性变异位点rs162549的引物或探针在制备预测前列腺癌根治术后生化复发风险的试剂中的应用。
3.一种用于预测前列腺癌根治术后生化复发的试剂盒，其特征在于包含用于检测CYP1B1基因多态性变异位点rs162549基因型的探针或引物。
4.根据权利要求3所述的试剂盒，其特征在于所述的引物选自：rs162549_For：SEQ IDNO.21，rs162549_Rev:SEQ ID NO.22；所述的探针选自：rs162549_P1:SEQ ID NO.23，rs162549_P2:SEQ ID NO.24。
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| NCBI.NLM.NIH.GOV/SNP/: "rs162549", 《DBSNP》 * |
| 郭云良等主编: "《中西医结合医学人才培养【M】》", 31 May 2013 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182485A (zh) * | 2018-10-22 | 2019-01-11 | 北京华夏时代生物工程有限公司 | Cyp基因单核苷酸多态性的荧光原位杂交测序检测方法 |
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