CN105238799A - 一种乳腺癌pik3ca突变基因及其应用 - Google Patents
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Abstract
本发明公开了一种乳腺癌PIK3CA突变基因及其应用,该突变基因具有c.1658_1659GT>C突变位点,其核苷酸序列如SEQ?ID?NO:1所示;本发明通过直接测序法,对88例乳腺癌组织、19例乳腺癌癌旁组织以及22例正常人血液样本中PIK3CA基因突变进行检测,首次发现PIK3CA基因S553fs*7突变在乳腺癌组织中高达44.3%,而在乳腺癌癌旁组织和正常人血液样本中均无突变发生;提示此突变可用于辅助乳腺癌的早期诊断;因此,本发明对于临床上防治乳腺癌的发生发展具有重要意义。
Description
技术领域
本发明属于生物技术领域,具体涉及一种乳腺癌中PIK3CA突变基因及其在辅助制备乳腺癌诊断试剂中的应用。
背景技术
乳腺癌,所有女性和家庭的梦魇。全球肿瘤流行病统计数据(GLOBOCAN)显示每年约有130万妇女患乳腺癌,45.8万人死于乳腺癌,无论是发达国家还是发展中国家,乳腺癌都是全世界妇女目前面临的最为常见的恶性肿瘤,也是引起女性死亡的重要病因。自90年代以来,中国的乳腺癌发病率增长速度是全球的2倍多,每年中国乳腺癌新发数量和死亡数量分别占全世界的12.2%和9.6%1。目前,乳腺癌是中国女性发病率最高的癌症,癌症死亡原因位居第六,且年轻化趋势显著,预计到2021年,中国乳腺癌患者将高达250万(Fan,L.etal.2014)。虽然早期检查及诊断技术的提高,手术及综合辅助治疗的进步,使得患者的预后有较明显改善,但是总生存率并不理想(IndependentUKPanelonBreastCancerScreening.2012)。对于特定的分子亚型,由于对药物的原发或继发耐药、甚至缺乏特定的治疗靶点,使得部分中晚期病人缺乏有效的治疗手段(Garcia-Closas,M.etal.2014)。因此,寻找发生有效的肿瘤标志物对于提高乳腺癌的诊断和预测发生、发展及改善预后具有重要的临床指导意义。
肿瘤标志物可以有效地作为临床诊断依据,可以监听高位人群、早期诊断、指导治疗、判断治疗疗效、检测复发及转移,是肿瘤患者的重要检查指标。乳腺癌是一种全身性疾病、其发生和发展是一个涉及多因素、多环节的复杂过程,包括癌基因的激活以及抑癌基因的失活等。因此,基因突变在乳腺癌的发生、发展过程中起着非常重要的作用。PIK3CA基因是近年来被发现除p53突变和Her-2扩增之外最常见和最重要的乳腺癌中的突变基因,突变比率高达40%,其突变可导致多条信号通路在不同水平发生调控异常,从而促进肿瘤细胞的增殖和存活(Bachman,K.E.etal.2004)。
磷脂酰肌醇-3-激酶(PI3K)是一种可使肌醇环第三位羟基磷酸化的磷脂酰肌醇激酶,具有磷脂酰肌醇激酶和丝氨酸-苏氨酸蛋白激酶双重活性(Fruman,D.A.etal.2014),调节细胞增殖、存活、迁移、凋亡和细胞周期等多种生物学功能。PIK3CA基因编码ⅠA类PI3K催化亚单位P110α,其定位于3q26.3,包含21个外显子(Volinia,S.etal.1994)。KANGS等研究发现,PIK3CA基因在多种肿瘤中扮演着癌基因的角色,约30%的人类实体瘤存在PIK3CA基因的突变,其中80%-90%的突变发生在该基因的第9和第20外显子,分别对应该酶的螺旋区和激酶区。该区域的改变引起PIK3CA过表达、P110α活性增加,进而激活下游AKT等信号分子,导致成纤维细胞和乳腺上皮细胞的生长和转化,并抑制细胞凋亡,最终导致肿瘤发生(Vivanco,I.etal.2002)。现有研究显示,应用PCR或基因测序的方法检测组织中PIK3CA基因的突变情况,可提高肿瘤诊断的敏感性;PIK3CA基因突变的肿瘤细胞对PI3K抑制剂更敏感,特异性地针对PIK3CA突变的靶向治疗可有效降低恶性肿瘤的发病率和死亡率。尽管PIK3CA突变对乳腺癌的诊治及预后的影响还需进一步研究,但从目前已有的研究数据可以看出PIK3CA高频突变和突变热点区域的发现对于乳腺癌基因诊断以及靶向治疗的选择具有重要的临床意义。
S553fs*7突变是PIK3CA基因突变之一,然而关于PIK3CA基因S553fs*7突变在乳腺癌中尚未被报道。
发明内容
本发明的目的在于提供一种乳腺癌中PIK3CA突变基因,该突变基因具有c.1658_1659GT>C突变位点,其核苷酸序列如SEQIDNO:1所示,乳腺癌PIK3CA突变基因编码的突变蛋白S553fs*7氨基酸序列如SEQIDNO:2所示。
该突变蛋白具有图5所示的三维空间结构。
本发明另一目的是将上述乳腺癌中PIK3CA突变基因作为一种分子标记,即作为检测乳腺癌的分子标记物。
本发明还可将将乳腺癌PIK3CA突变基因应用在制备乳腺癌临床诊断产品中;
进一步地,在于提供一套检测PIK3CA基因突变的引物组合,一对特异扩增PIK3CA基因9号外显子的正向引物和反向引物,其正向引物的核苷酸序列如SEQIDNO:3所示,反向引物的核苷酸序列如SEQIDNO:4所示。
本发明将乳腺癌PIK3CA突变基因应用在制备用于乳腺癌临床诊断的检测试剂盒中,所述试剂盒包括核苷酸序列如SEQIDNO:3和SEQIDNO:4所示的引物。
所述试剂盒组分包含下列组分中的一种或几种:聚合酶、PCR反应缓冲液、正反向引物,纯水。
上述PCR反应总体系为25μL:其中,PfuMix混合液12.5μL,所述正向引物(5μM)的体积1μL,反向引物(5μM)的体积1μL,所述DNA模板1μL,其余为纯水。PCR反应条件为:①94℃,5min预变性;②94℃,30s变性;③55℃,30s退火;④72℃,35s延伸;循环②至④35次;⑤72℃,5min。
通过对乳腺癌组织、癌旁组织及正常人血液样本中PIK3CA基因突变检测,本发明首次发现了乳腺癌中PIK3CA基因S553fs*7突变,其对于临床乳腺癌早期筛查、指导临床用药以及监测肿瘤预后具有很好的实际应用价值。
附图说明
图1是本发明PIK3CA基因9号外显子突变分布情况示意图;
图2是本发明不同样本中PIK3CA基因第9外显子S553fs*7突变测序图;
图3是本发明不同样本中PIK3CA基因第9外显子S553fs*7突变比率;
图4是本发明PIK3CA基因结构域示意图;
图5是本发明PIK3CA基因S553fs*7突变前后空间构象对比图。
具体实施方式
下面结合附图和实施例对本发明作进一步详细的说明。以下实施例仅用于说明本发明而不用于限制本发明的范围,该领域的技术人员可以根据上述本发明内容对本发明做出一些非本质的改进和调整。下述实施例中,若非特异表明,所用试剂均为分析纯,所有试剂均可从商业渠道获得,百分比均为质量百分比。文中未注明具体条件的实验方法,通常按照《分子克隆实验指南》中所述常规条件,或试剂制造厂商所建议的条件实施。除非另行定义,文中所使用的所有专业和科学用语与本领域熟练人员所熟悉的意义相同。
实施例1:乳腺癌及癌旁组织中DNA提取
1、实验材料
TES缓冲液(将一个50mL离心管洗干净,高温高压灭菌后,加入0.5mL1M的Tris-HCL1mL0.5M的EDTA和2.5mL的10%SDS,加超纯水补足到50mL,调PH=8.0后室温保存),20mg/mL蛋白酶K,3M醋酸钠(pH=5.2),TEBuffer,苯酚,氯仿,异戊醇,无水乙醇,TE溶液。
2、实验方法
2.1组织块解冻,剪取约0.5g组织,放入1.5mL离心管中,剪碎;
2.2加入0.45mLTES缓冲液混匀,再加入50μLSDS(10%),5.0μL蛋白酶K(20mg/mL),充分混匀后,于56℃消化4-6h,每2h摇1次,至溶液清澈;
2.3放置到室温,加入等体积饱和酚(500μL),颠倒混匀,12000rpm,离心10min,分离水相和有机相,小心吸取上层含核酸的水相,到一个新的1.5mL离心管;
2.4加入等体积酚:氯仿:异戊醇=25:24:1的混合液,颠倒混匀,12000rpm,离心10min,取上层转移到新的1.5mL离心管中;
2.5加入等体积氯仿:异戊醇=24:1的溶液,颠倒混匀,12000rpm,离心10min,取上层清液到一个新的1.5mL离心管;
2.6加入1/10体积的3M醋酸钠溶液和2倍体积预冷的无水乙醇沉淀DNA,上下颠倒混匀后放置于-20℃冰箱1h,12000rpm,4℃,离心10min,弃乙醇;
2.7加入75%的乙醇洗涤,8000rpm,离心5min;
2.8小心的倒掉上清,在吸水纸上扣干残液,打开管盖,室温静置10min,待乙醇挥发干净,根据DNA的沉淀量加入适量的TE溶解DNA;4℃过夜溶解,测定浓度后,于-20℃保存待用。
实施例2:血液样本中DNA提取
1、实验材料
白细胞裂解液,红细胞裂解液,蛋白酶K(10mg/mL),苯酚,氯仿,异戊醇,无水乙醇,TE溶液。
2、实验方法
2.1将4mL新鲜血液移至15mL离心管中,加入红细胞裂解液,上下颠倒混匀,4000g,4℃离心,15min;
2.2去上清,反复洗血2-3次;
2.3去上清,加入1mL白细胞裂解液,振荡混匀,均分至两个1.5mL的离心管中,一管于-80℃冻存待用;
2.4另一管加入20μL蛋白酶K(10mg/mL),温和地翻转8次混匀,56℃温育过夜(直至没有肉眼可见的悬浮物),不时翻转混匀;
2.5加入等体积饱和酚(500μL),颠倒混匀,12000rpm,离心10min,分离水相和有机相,小心吸取上层含核酸的水相,到一个新的1.5mL离心管;
2.6加入等体积酚:氯仿:异戊醇=25:24:1的溶液,颠倒混匀,12000rpm,离心10min,取上层转移到新的1.5mL离心管中;
2.7加入等体积氯仿:异戊醇=24:1的溶液,颠倒混匀,12000rpm,离心10min,取上层清液到一个新的1.5mL离心管;
2.8加入1/10体积的3M醋酸钠溶液和2倍体积预冷的无水乙醇沉淀DNA,上下颠倒混匀后放置于-20℃冰箱1h。12000rpm,4℃,离心10min,弃乙醇;
2.9加入75%的乙醇洗涤,8000rpm,离心5min;
3.0小心的倒掉上清,在吸水纸上扣干残液,打开管盖,室温静置10min,待乙醇挥发干净,根据DNA的沉淀量加入适量的TE溶解DNA,4℃过夜溶解,测定浓度后,于-20℃保存待用。
实施例3:PCR扩增PIK3CA基因9号外显子并测序
1、实验材料
2×PfuMix,实施例1、2中所提取的DNA模板,去离子水,扩增PIK3CA基因9号外显子的正反向引物(详见表1),PCR仪,1%的琼脂糖凝胶,电泳仪。
表1
2、实验方法
2.1挑选实施例1、2中质量较好的DNA样本,包括88例乳腺癌组织DNA,19例乳腺癌癌旁组织DNA,22例正常人血液样本DNA;
2.2配置如下表PCR反应体系:
表2
。
2.3反应条件:①94℃,5min预变性;②94℃,30s变性;③55℃,30s退火;④72℃,35s延伸;循环②至④35次;⑤72℃,5min;
2.4PCR反应结束后,铺1%的胶检测,并送至生工生物工程(上海)股份有限公司进行测序;
3、实验结果
测序结果显示,在88例乳腺癌组织标本中共检测到41例PIK3CA基因9号外显子的突变,突变比率高达46.6%,其中E545A/K突变率为43.2%,S553fs*7突变率为44.3%(表3、4、5、6、7)。而在19例乳腺癌癌旁组织和22例正常人血液样本中均未检测到PIK3CA基因9号外显子的突变(见图1、图2、图3)。E545A/K为文献中已报道的热点突变位点,值得注意的是S553fs*7的突变在TCGA数据库中尚未报道。进一步的分析检索,仅有少量文献报道S553fs*7的突变曾在肝癌、子宫内膜癌以及B细胞淋巴癌中发生。而在本研究中S553fs*7双位突变比率在所收集的乳腺癌中高达44.3%,S553fs*7突变位于PIK3CA基因的螺旋区,其突变可以导致PIK3CA基因提前终止翻译,同时影响其螺旋区和激酶区的功能(图4)。
利用Swissmodel数据库(http://swissmodel.expasy.org/repository/)我们对PIK3CA基因发生S553fs*7突变后其结构的改变进行了初步分析(图5)。结果显示,突变后其空间构象发生了明显改变,可能会因此对其下游激酶PI3Ks活性的调节机制以及相关的信号通路改变造成影响。提示PIK3CA基因S553fs*7突变与乳腺癌的发生发展密切相关。
表3
。
表4
。
表5
。
表6
。
表7
。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110>昆明理工大学
<120>一种乳腺癌PIK3CA突变基因及其应用
<160>4
<170>PatentInversion3.3
<210>1
<211>3206
<212>DNA
<213>Homosapiens
<400>1
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gaattttttgatgaaacaagacgactttgtgaccttcggctttttcaaccctttttaaaa300
gtaattgaaccagtaggcaaccgtgaagaaaagatcctcaatcgagaaattggttttgct360
atcggcatgccagtgtgtgaatttgatatggttaaagatccagaagtacaggacttccga420
agaaatattctgaacgtttgtaaagaagctgtggatcttagggacctcaattcacctcat480
agtagagcaatgtatgtctatcctccaaatgtagaatcttcaccagaattgccaaagcac540
atatataataaattagataaagggcaaataatagtggtgatctgggtaatagtttctcca600
aataatgacaagcagaagtatactctgaaaatcaaccatgactgtgtaccagaacaagta660
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aggatgcccaatttgatgttgatggctaaagaaagcctttattctcaactgccaatggac900
tgttttacaatgccatcttattccagacgcatttccacagctacaccatatatgaatgga960
gaaacatctacaaaatccctttgggttataaatagtgcactcagaataaaaattctttgt1020
gcaacctacgtgaatgtaaatattcgagacattgataagatctatgttcgaacaggtatc1080
taccatggaggagaacccttatgtgacaatgtgaacactcaaagagtaccttgttccaat1140
cccaggtggaatgaatggctgaattatgatatatacattcctgatcttcctcgtgctgct1200
cgactttgcctttccatttgctctgttaaaggccgaaagggtgctaaagaggaacactgt1260
ccattggcatggggaaatataaacttgtttgattacacagacactctagtatctggaaaa1320
atggctttgaatctttggccagtacctcatggattagaagatttgctgaaccctattggt1380
gttactggatcaaatccaaataaagaaactccatgcttagagttggagtttgactggttc1440
agcagtgtggtaaagttcccagatatgtcagtgattgaagagcatgccaattggtctgta1500
tcccgagaagcaggatttagctattcccacgcaggactgagtaacagactagctagagac1560
aatgaattaagggaaaatgacaaagaacagctcaaagcaatttctacacgagatcctctc1620
tctgaaatcactgagcaggagaaagattttctatggaccacagacactattgtgtaacta1680
tccccgaaattctacccaaattgcttctgtctgttaaatggaattctagagatgaagtag1740
cccagatgtattgcttggtaaaagattggcctccaatcaaacctgaacaggctatggaac1800
ttctggactgtaattacccagatcctatggttcgaggttttgctgttcggtgcttggaaa1860
aatatttaacagatgacaaactttctcagtatttaattcagctagtacaggtcctaaaat1920
atgaacaatatttggataacttgcttgtgagatttttactgaagaaagcattgactaatc1980
aaaggattgggcactttttcttttggcatttaaaatctgagatgcacaataaaacagtta2040
gccagaggtttggcctgcttttggagtcctattgtcgtgcatgtgggatgtatttgaagc2100
acctgaataggcaagtcgaggcaatggaaaagctcattaacttaactgacattctcaaac2160
aggagaagaaggatgaaacacaaaaggtacagatgaagtttttagttgagcaaatgaggc2220
gaccagatttcatggatgctctacagggctttctgtctcctctaaaccctgctcatcaac2280
taggaaacctcaggcttgaagagtgtcgaattatgtcctctgcaaaaaggccactgtggt2340
tgaattgggagaacccagacatcatgtcagagttactgtttcagaacaatgagatcatct2400
ttaaaaatggggatgatttacggcaagatatgctaacacttcaaattattcgtattatgg2460
aaaatatctggcaaaatcaaggtcttgatcttcgaatgttaccttatggttgtctgtcaa2520
tcggtgactgtgtgggacttattgaggtggtgcgaaattctcacactattatgcaaattc2580
agtgcaaaggcggcttgaaaggtgcactgcagttcaacagccacacactacatcagtggc2640
tcaaagacaagaacaaaggagaaatatatgatgcagccattgacctgtttacacgttcat2700
gtgctggatactgtgtagctaccttcattttgggaattggagatcgtcacaatagtaaca2760
tcatggtgaaagacgatggacaactgtttcatatagattttggacactttttggatcaca2820
agaagaaaaaatttggttataaacgagaacgtgtgccatttgttttgacacaggatttct2880
taatagtgattagtaaaggagcccaagaatgcacaaagacaagagaatttgagaggtttc2940
aggagatgtgttacaaggcttatctagctattcgacagcatgccaatctcttcataaatc3000
ttttctcaatgatgcttggctctggaatgccagaactacaatcttttgatgacattgcat3060
acattcgaaagaccctagccttagataaaactgagcaagaggctttggagtatttcatga3120
aacaaatgaatgatgcacatcatggtggctggacaacaaaaatggattggatcttccaca3180
caattaaacagcatgcattgaactga3206
<210>2
<211>558
<212>PRT
<213>Homosapiens
<400>2
MetProProArgProSerSerGlyGluLeuTrpGlyIleHisLeuMet
151015
ProProArgIleLeuValGluCysLeuLeuProAsnGlyMetIleVal
202530
ThrLeuGluCysLeuArgGluAlaThrLeuIleThrIleLysHisGlu
354045
LeuPheLysGluAlaArgLysTyrProLeuHisGlnLeuLeuGlnAsp
505560
GluSerSerTyrIlePheValSerValThrGlnGluAlaGluArgGlu
65707580
GluPhePheAspGluThrArgArgLeuCysAspLeuArgLeuPheGln
859095
ProPheLeuLysValIleGluProValGlyAsnArgGluGluLysIle
100105110
LeuAsnArgGluIleGlyPheAlaIleGlyMetProValCysGluPhe
115120125
AspMetValLysAspProGluValGlnAspPheArgArgAsnIleLeu
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AsnValCysLysGluAlaValAspLeuArgAspLeuAsnSerProHis
145150155160
SerArgAlaMetTyrValTyrProProAsnValGluSerSerProGlu
165170175
LeuProLysHisIleTyrAsnLysLeuAspLysGlyGlnIleIleVal
180185190
ValIleTrpValIleValSerProAsnAsnAspLysGlnLysTyrThr
195200205
LeuLysIleAsnHisAspCysValProGluGlnValIleAlaGluAla
210215220
IleArgLysLysThrArgSerMetLeuLeuSerSerGluGlnLeuLys
225230235240
LeuCysValLeuGluTyrGlnGlyLysTyrIleLeuLysValCysGly
245250255
CysAspGluTyrPheLeuGluLysTyrProLeuSerGlnTyrLysTyr
260265270
IleArgSerCysIleMetLeuGlyArgMetProAsnLeuMetLeuMet
275280285
AlaLysGluSerLeuTyrSerGlnLeuProMetAspCysPheThrMet
290295300
ProSerTyrSerArgArgIleSerThrAlaThrProTyrMetAsnGly
305310315320
GluThrSerThrLysSerLeuTrpValIleAsnSerAlaLeuArgIle
325330335
LysIleLeuCysAlaThrTyrValAsnValAsnIleArgAspIleAsp
340345350
LysIleTyrValArgThrGlyIleTyrHisGlyGlyGluProLeuCys
355360365
AspAsnValAsnThrGlnArgValProCysSerAsnProArgTrpAsn
370375380
GluTrpLeuAsnTyrAspIleTyrIleProAspLeuProArgAlaAla
385390395400
ArgLeuCysLeuSerIleCysSerValLysGlyArgLysGlyAlaLys
405410415
GluGluHisCysProLeuAlaTrpGlyAsnIleAsnLeuPheAspTyr
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ThrAspThrLeuValSerGlyLysMetAlaLeuAsnLeuTrpProVal
435440445
ProHisGlyLeuGluAspLeuLeuAsnProIleGlyValThrGlySer
450455460
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<211>42
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<213>人工序列
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Claims (5)
1.一种乳腺癌PIK3CA突变基因,其特征在于:该突变基因具有c.1658_1659GT>C突变位点,其核苷酸序列如SEQIDNO:1所示。
2.权利要求1乳腺癌PIK3CA突变基因编码的突变蛋白S553fs*7,其特征在于:氨基酸序列如SEQIDNO:2所示。
3.一种分子标记,其为权利要求1所述的乳腺癌PIK3CA突变基因。
4.权利要求1所述的乳腺癌PIK3CA突变基因在制备乳腺癌临床诊断产品中的应用。
5.权利要求1所述的乳腺癌PIK3CA突变基因在制备用于乳腺癌临床诊断的检测试剂盒中的应用,所述试剂盒包括核苷酸序列如SEQIDNO:3和SEQIDNO:4所示的引物。
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CN107828786A (zh) * | 2017-10-24 | 2018-03-23 | 昆明理工大学 | 靶向敲除PIK3CA基因的sgRNA及应用 |
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CN109913481A (zh) * | 2019-04-24 | 2019-06-21 | 无锡市第五人民医院 | PIK3CA基因g.1792224821G>A突变及其在乳腺癌辅助诊断中的应用 |
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CN107385026A (zh) * | 2017-07-06 | 2017-11-24 | 北京大学深圳医院(北京大学深圳临床医学院) | 一组与乳腺癌相关的突变基因及其辅助诊断试剂盒 |
CN107385026B (zh) * | 2017-07-06 | 2020-08-28 | 北京大学深圳医院(北京大学深圳临床医学院) | 一组与乳腺癌相关的突变基因及其辅助诊断试剂盒 |
CN107828786A (zh) * | 2017-10-24 | 2018-03-23 | 昆明理工大学 | 靶向敲除PIK3CA基因的sgRNA及应用 |
CN109913481A (zh) * | 2019-04-24 | 2019-06-21 | 无锡市第五人民医院 | PIK3CA基因g.1792224821G>A突变及其在乳腺癌辅助诊断中的应用 |
CN112941187A (zh) * | 2021-04-02 | 2021-06-11 | 无锡市第五人民医院 | 一种乳腺癌相关基因pik3ca-q928h突变体及其特异性引物 |
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