CN104531878B - 一种联合lncRNA和AR3检测试剂盒及应用 - Google Patents
一种联合lncRNA和AR3检测试剂盒及应用 Download PDFInfo
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Abstract
本发明涉及一种预测和评价雄激素受体(AR)的激素去势治疗后去势抵抗或复发的方法、试剂盒及应用,所述的方法联合检测PCGEM1或PCAT1及雄激素受体异构体AR3(AR‑V7)。其优点表现在:一方面,直观地将目标检测基因与其病理变化结合起来;另一方面,能够高效、准确的检测该特异性目标检测基因,并放大该信号,较常规的染色检测更为灵敏和精确,并有利于保存显色后的组织切片。最重要的是联合检测PCGEM1及AR异构体AR3可以有效的预测和评价AR相关肿瘤的去势抵抗或复发转移情况。
Description
技术领域
本发明涉及分子生物学技术领域,具体地说,是一种联合lncRNA如PCGEM1和AR3(AR-V7)检测试剂盒及用于诊断AR相关肿瘤的去势抵抗及复发。
背景技术
癌症占人类死亡病因的近1/4(www.cancer.org)。在过去的几十年里肿瘤复发和转移仍是改善整体存活率的主要障碍,这可能主要归因于人们对于癌症生物学仍然缺乏全面的了解。例如,功能基因组学研究的最新进展表明,参与人类基因转录的绝大多数是非编码RNA。然而,对于lncRNAs在调控癌症基因表达及机制等方面人类仍然所知甚少[1]。
近期我们通过对肿瘤表观遗传机制的研究发现lncRNAs可以通过多种机制调控肿瘤的进展。新近有研究表明lncRNA PCGEM1可以与AR结合作用[2]。PCGEM1在前列腺癌上调[3],可促进细胞增殖[4],而我们则通过临床标本研究发现,PCGEM1的基因表达与前列腺癌恶性程度及转移复发呈高度相关性,PCGEM1不但可以通过与AR直接作用,更重要的是PCGEM1与AR异构体(如AR3)的表达呈高度正相关,我们进一步证明了PCGEM1通过选择性转录修饰调控了AR的异构体如AR3的表达。由此发现lncRNAs如PCGEM1可能通过调控AR基因转录的表观遗传机制参与AR相关肿瘤的增殖、恶性侵袭复发及转移驱动。因为在一定程度上是由于AR的异构体如AR-V7(AR3)介导了该类肿瘤的激素去势抵抗,而并非野生型全长AR。而研究已表明AR-V7在AR在很大程度上诱导形成了相关肿瘤如前列腺癌的激素去势抵抗[5]。
所以,联合检测PCGEM1或PCAT1及雄激素受体异构体AR3,是建立在lncRNAs(PCGEM)通过本专利证明的表观遗传调控机制的基础上。即lncRNAs(PCGEM1)通过调控AR-V7(AR3)pre-mRNA的转录后修饰及共定位,直接决定了表达雄激素受体的肿瘤如前列腺癌、肺癌及乳腺癌等的激素去势治疗后去势抵抗或复发转移。
针对上述AR相关肿瘤的特异性调节lncRNAs如PCGEM1和该类肿瘤的激素去势抵抗的高度相关AR异构体(如AR3)的表达呈高度,通过荧光原位杂交及免疫组织化学技术联合检测临床组织标本的上述指标,可以高度有效的评估和预测诊断AR相关肿瘤的激素去势抵抗和复发转移,并在激素去势抵抗和复发转移的早期做出诊断。该项申请的技术及试剂盒是基于当前最新的研究结果而建立。这是一种目前其他方法都无法替代的检测方法,迄今为止尚未见相关报道。
(参考文献:1.Xie C,Yuan J,Li H,Li M,Zhao G,Bu D,et al.NONCODEv4:exploring the world of long non-coding RNA genes.Nucleic acids research.2013;doi:10.1093/nar/gkt1222.2.Yang L,Lin C,Jin C,Yang JC,Tanasa B,Li W,etal.lncRNA-dependent mechanisms of androgen-receptor-regulated gene activationprograms.Nature.2013;29:598-602.3.Srikantan V,Zou Z,Petrovics G,Xu L,AugustusM,Davis L,et al.PCGEM1,a prostate-specific gene,is overexpressed in prostatecancer.Proceedings of the National Academy of Sciences of the United Statesof America.2000;97:12216-21.4.Petrovics G,Zhang W,Makarem M,Street JP,Connelly R,Sun L,et al.Elevated expression of PCGEM1,a prostate-specific genewith cell growth-promoting function,is associated with high-risk prostatecancer patients.Oncogene.2004;23:605-11.5.Antonarakis ES,et al.AR-V7andresistance to enzalutamide and abiraterone in prostate cancer.The New Englandjournal of medicine.2014;371,1028-1038.)
发明内容
本发明的目的是针对现有技术中的不足,提供一种预测和评价雄激素受体的激素去势治疗后去势抵抗或复发的方法。
本发明的再一的目的是,提供一种预测和评价雄激素受体的激素去势治疗后去势抵抗或复发的试剂盒。
本发明的另一的目的是,提供一种预测和评价雄激素受体的激素去势治疗后去势抵抗或复发的试剂盒的应用。
为实现上述目的,本发明采取的技术方案是:一种预测和评价雄激素受体的激素去势治疗后去势抵抗或复发的方法,所述的方法联合检测长链非编码RNA如lncRNA PCGEM1或PCAT1及雄激素受体异构体AR3(AR-V7)。
所述的PCGEM1通过原位杂交检测,扩大信号后,经过酶反应或荧光显色,原位杂交检测的寡核苷酸探针用地高辛标记(显色为红色荧光),所述的探针序列PCGEM1-1:T+T+T+TCCAAAGGG+T+CCGCTGTCCCTG+G+A+G、PCGEM1-2:A+T+T+CCCCTCAGA+A+ATCTCAGGGCTT+G+T+C,其中+选自β-D-氧-LNA核苷酸类似物中的任一种。
所述的雄激素受体异构体AR3通过荧光免疫组织化学检测,然后经过酶反应或荧光显色并扩大信号,荧光二抗为绿色荧光蛋白EGFP(显色为绿色荧光)。
为实现上述第二个目的,本发明采取的技术方案是:一种预测和评价雄激素受体的激素去势治疗后去势抵抗或复发的试剂盒,所述的试剂盒通过荧光原位杂交探针(FISH)检测PCGEM1并通过荧光免疫组织化学检测雄激素受体异构体AR3,所述的试剂盒包括PCGEM1的核酸原位杂交检测探针,所述核酸原位杂交检测探针的序列为PCGEM1-1:T+T+T+TCCAAAGGG+T+CCGCTGTCCCTG+G+A+G、PCGEM1-2:A+T+T+CCCCTCAGA+A+ATCTCAGGGCTT+G+T+C,其中+选自β-D-氧-LNA核苷酸类似物中的任一种,所述的检测AR3的抗体及荧光二抗为绿色荧光蛋白EGFP(显色为绿色荧光)。
所述的核酸原位杂交检测探针和可药用的稀释剂、载体或辅剂组合使用。
所述的载体包含水性载体,所述水性载体包含用于将pH维持在7.35-7.45范围内的缓冲剂。
为实现上述第三个目的,本发明采取的技术方案是:所述的试剂盒制备诊断癌症的试剂中的应用。
所述的癌症为实体肿瘤形式。
所述的癌症选自具有PCGEM1表达的肿瘤及相关性疾病,所述的疾病包括前列腺癌、肺癌、乳腺癌、睾丸癌、子宫颈癌和结肠癌等。
所述的癌症选自具有AR及AR3表达的肿瘤及相关性疾病,所述的疾病包括前列腺癌、肺癌、乳腺癌、睾丸癌、子宫颈癌和结肠癌等。
本发明优点在于:
1、本发明目的是在于提供一种高效、高度特异性、灵敏性及高可信度,同时又非常直观并具有临床实用性和应用价值的用于雄激素受体相关肿瘤激素去势抵抗及复发的诊断试剂盒。
2、本发明具有显著的特异性,且能够将其检测的荧光信号与其病变相关的组织病理变化结合起来。进一步明确该病变或组织部位的良恶性程度,了解并评估其肿瘤激素去势抵抗及复发的潜在性及可能程度。
3、联合上述方法具有直观、准确、高效性。
4、本发明具有特异性强,具有切实的临床使用价值。联合lncRNA PCGEM1原位杂交检测探针及AR3荧光免疫化学检测双重检测增加了临床应用的可行性和可信度,能够直接应用于临床,这是目前其他分子生物学检测方法不能比拟的特点。
5、由于所设计的核酸探针与目标核酸的杂交是特异的,且信号经过逐渐放大,所以较常规的染色方法检测更为灵敏,具有更高的区分度和准确性。
6、一方面,直观地将目标检测基因与其病理变化结合起来;另一方面,能够高效、准确的检测该特异性目标检测基因,并放大该信号,较常规的染色检测更为灵敏和精确,并有利于保存显色后的组织切片。
7、本发明中,显色后的切片可以长期保存。
附图说明
附图1是原位核酸杂交检测表明PCGEM1在肿瘤尤其是高度恶性及转移性前列腺癌中表达显著上调。
附图2是实时定量PCR及蛋白印迹检测表明在雄激素去势条件下PCGEM1及AR3表达显著增高。
附图3是蛋白印迹检测表明下调PCGEM1表达引起AR3表达同向显著降低。
附图4是荧光原位杂交表明PCGEM1在肿瘤,尤其是在转移性前列腺癌中表达显著上调,且具有核内定位表现。
附图5是通过细胞培养,联合荧光原位杂交及荧光免疫组织化学检测表明,和正常条件下比较,在激素去势条件下,PCGEM1及AR3被诱导显著上调,且二者之间存在细胞核内共定位现象。
附图6是通过组织切片,联合荧光原位杂交及荧光免疫组织化学检测表明,与低度恶性肿瘤比较,在高度恶性肿瘤中PCGEM1及AR3被诱导显著上调,且二者之间存在细胞核内共定位现象。
具体实施方式
下面结合附图对本发明提供的具体实施方式作详细说明。
本发明目的是在于提供一种高效、高度特异性、灵敏性及高可信度,同时又直观并具有临床实用性和应用价值的用于雄激素受体相关肿瘤激素去势抵抗及复发的诊断试剂盒。
本发明根据长链非编码RNA(lncRNAs),如PCGEM1核酸序列中两段特有的RNA序列设计合成了一条特异性标记的LNA寡核苷酸探针,并建立了组织切片的荧光原位杂交方法,该探针对照组RNA序列无特异性信号,而lncRNA PCGEM1表达的组织样本中具有显著特异的深红色荧光信号,具有显著的特异性,且能够将其检测的荧光信号与其病变相关的组织病理变化结合起来。进一步明确该病变或组织部位的良恶性程度,了解并评估其肿瘤激素去势抵抗及复发的潜在性及可能程度。
组织切片的荧光免疫组织化学检测人体组织切片中的雄激素受体异构体AR3,然后经过酶反应或荧光显色并扩大信号,荧光二抗为绿色荧光蛋白EGFP(显色为绿色荧光)。雄激素受体相关肿瘤激素去势抵抗及复发的联合诊断试剂盒:通过联合检测上述荧光原位杂交探针(FISH)检测PCGEM1及荧光免疫组织化学检测雄激素受体异构体AR3。包括PCGEM1的核酸原位杂交检测探针。联合检测的AR3抗体及荧光二抗为绿色荧光蛋白EGFP(显色为绿色荧光)。
联合上述方法具有直观、准确、高效及灵敏和特异性强,具有切实的临床使用价值。故lncRNA PCGEM1原位杂交检测探针及AR3荧光免疫化学的联合检测用于雄激素受体相关肿瘤激素去势抵抗及复发的联合诊断试剂盒。
本发明所述的两条长链非编码RNA(lncRNAs)探针,其序列如下:
PCGEM1-1:T+T+T+TCCAAAGGG+T+CCGCTGTCCCTG+G+A+G;PCGEM1-2:A+T+T+CCCCTCAGA+A+ATCTCAGGGCTT+G+T+C。其中,+选自β-D-氧-LNA核苷酸类似物中的任一种。
其中,两种具体的探针为:
PCGEM1-1:TTTTTTTCCAAAGGGGTTCCGCTGTCCCTGGGGAAG;PCGEM1-2:AATTTTCCCCTCAGAAAAATCTCAGGGCTTTGGTTC。
PCGEM1-1:TGTCTATCCAAAGGGTTACCGCTGTCCCTGCGCATG;PCGEM1-2:AGTATCCCCCTCAGAGAAATCTCAGGGCTTCGATCC。
其中,G为G-β-D-氧-LNA核苷酸类似物,C为C-β-D-氧-LNA核苷酸类似物,A为A-β-D-氧-LNA核苷酸类似物,T为T-β-D-氧-LNA核苷酸类似物。
阴性对照
PCGEM1-blocker-1:CTCCAGGGACAGCGGACCCTTTGGAAAA(SEQ ID NO.1);PCGEM1-blocker-2:GACAAGCCCTGAGATTTCTGAGGGGAAT(SEQ ID NO.2)。
本发明提供了一种通过联合lncRNAs原位杂交检测探针及AR3荧光免疫化学检测用于雄激素受体相关肿瘤激素去势抵抗及复发的诊断技术及试剂盒。
用地高辛标记的特异性探针与组织切片中的lncRNA PCGEM1核酸原位杂交结合;
进行原位杂交检测;
a.所述的地高辛标记探针结合生物素化抗地高辛,再结合链霉亲和素-生物素-过氧化物酶复合物,最后再结合生物素化过氧化物酶;
b.经酶反应显色,荧光二抗显色。
荧光免疫组织化学检测
所述的核酸特异性探针是根据组织中特异表达的lncRNAs如PCGEM1核酸序列中的两段特有的RNA序列设计合成的两条寡核苷酸探针,合成后于每一探针的3·末端标记地高辛;所述的两条寡核苷酸探针的序列PCGEM1-1:T+T+T+TCCAAAGGG+T+CCGCTGTCCCTG+G+A+G;PCGEM1-2:A+T+T+CCCCTCAGA+A+ATCTCAGGGCTT+G+T+C。
lncRNAs如PCGEM1原位杂交检测探针及AR3荧光免疫组织化学检测
组织样本的荧光原位杂交及荧光免疫组织化学检测需要固定、切片、灭活内源性过氧化物酶以及消化蛋白以便暴露核酸等处理,进行常规处理。地高辛表达的核酸探针可以采用免疫学抗原抗体反应、二甲基联苯胺(DAB)酶反应显色。本发明为达到荧光原位显色及共定位的目的,联合荧光免疫组织化学检测,采用二步法,并分别应用不同颜色的荧光二抗进行显色,以达到组织样本中荧光双重标记显色的目的,本发明在该方面进行了有效的改进。
本发明提供了一种通过联合lncRNAs原位杂交检测探针及AR3荧光免疫化学检测用于雄激素受体相关肿瘤激素去势抵抗及复发的诊断技术及试剂盒,该试剂盒包括了上述特异性探针和原位杂交试剂,以及荧光免疫组织化学检测试剂。
实施例1 合成lncRNA PCGEM1-LNA寡核苷酸和对照探针
LNA寡核苷酸对靶核酸表达的影响,可在多种细胞类型中的任何一种中检测,条件是目标核酸以可测量的水平存在。靶物可内源性表达,或通过编码所述核酸的瞬时或稳定转染表达。以上序列均由美国Exiqon合成。
a.合成特异性探针:
PCGEM1-1:T+T+T+TCCAAAGGG+T+CCGCTGTCCCTG+G+A+G;PCGEM1-2:A+T+T+CCCCTCAGA+A+ATCTCAGGGCTT+G+T+C
b.阴性对照:
PCGEM1-blocker-1:CTCCAGGGACAGCGGACCCTTTGGAAAA(SEQ ID NO.1);PCGEM1-blocker-2:GACAAGCCCTGAGATTTCTGAGGGGAAT(SEQ ID NO.2)。
实施例2 组织切片准备及检测(图1)
a.临床肿瘤组织样本切片。
b.组织样本来源于临床实验课题。
c.组织切片样本经过常规HE染色,确定结构良好,来源确切。分别选取肿瘤样本检测组以及选取阴性对照组。
d.样品固定及切片制备。
e.荧光原位杂交检测。
f.荧光免疫组织化学检测。
实施例3 实时定量PCR检测PCGEM1或AR3的表达量(图2,图3)
靶基因的表达水平可使用例如定量PCR、核糖核酸酶保护测试按常规确定。为示例目的提供以下细胞类型,但是其它细胞类型也可按常规使用,条件是靶物在所选细胞类型中表达。
细胞培养在如以下所述的合适培养基中,并保持在37℃、95-98%湿度和5%CO2中。当在低氧或缺氧下培养时,O2水平分别保持在1-2%或0-0.5%。细胞每周按常规传代2-3次。
(1)收集细胞。
(2)提取细胞中的总RNA。采用Ambion公司的RNA提取试剂盒(AM1560),按照试剂盒说明书方法进行操作。
(3)取100ng的总RNA,分别用U6和PCGEM1或AR3引物反转总RNA里面对应的PCGEM1或AR3。
(4)取第一步反转得到的产物,根据Ambion公司的试剂盒说明书进行后续的real-time实验。
实时定量PCR引物序列如下,
PCGEM1:5.1-TTTTTGCCCTATGCCGTAAC(SEQ ID NO.3),
3.1-GGAGACTCCCAACCTGATGA(SEQ ID NO.4)。
AR3:5.1-GCAATTGCAAGCATCTCAAA(SEQ ID NO.5),
3.1-GGAGGGAGTCAGCAATCAAG(SEQ ID NO.6)。
(5)RT-PCR试剂盒完成逆转录(Takara)。
(6)荧光定量PCR试剂盒检测(Takara)。
(7)数据及结果分析。
实施例4 AR3蛋白印迹分析(图3)
AR3蛋白的表达水平可使用例如蛋白印迹分析。为示例目的提供以下细胞类型,但是其它细胞类型也可按常规使用,条件是靶物在所选细胞类型中表达。
细胞培养在如以下所述的合适培养基中,并保持在37℃、95-98%湿度和5%CO2中。当在低氧或缺氧下培养时,O2水平分别保持在1-2%或0-0.5%。细胞每周按常规传代2-3次。
22RV1:人前列腺癌细胞系22RV1,培养在含有谷氨酰胺的RPMI-1640(Gibco)+10%FBS+庆大霉素中。
LNCaP:人前列腺癌细胞系LNCaP,培养在含有Glutamax+10%FBS的RPMI中。
实施例5 联合荧光原位杂交及荧光免疫组织化学染色(图4,图5,图6)
一、荧光原位杂交
1、脱蜡:
1)二甲苯脱蜡3次,每次5min;
2)100%酒精两次,每次2min;
3)移出酒精,斜置切片,标记末段向下,空气干燥。
2、蛋白酶处理:
1)每个染色缸40ml蛋白酶K消化溶液,配制方法如下:2×SSC 40ml倒入Facal管,在水浴槽中预热。将消化酶液加入管内,摇动直到酶溶解。
2)37℃水浴槽中预热染色缸和蛋白酶K溶液。37℃孵育20min。
3)×SSC在室温下漂洗切片3次,每次1min。
4)梯度酒精脱水(-20℃预冷)。
3、变性:
1)每一个立式染色缸配制40ml变性溶液;
2)78℃水浴槽中平衡预热混合液染色缸;
3)78℃孵育8min;
4)即移入-20℃预冷70%酒精的染色缸内2min,再依次移入80%、90%和100%的-20℃预冷酒精内,每缸2min;
5)空气干燥。
4、杂交:
1)准备探针;
2)取一个较大的湿盒,交叉放置切片;
3)滴10μl探针在切片的组织上,加盖玻片;
4)盖上湿盒盖,37℃孵育12h~16h。
杂交后的水洗:
5)镊子小心去除盖玻片;
6)43℃预热杂交后水洗溶液40ml水洗切片15min;
7)2×SSC(37℃)洗两次,每次10min;
8)切片放入染色缸的1×PBS内待检测,勿使切片干燥。
检测:
9)从1×PBS中取出切片,除去过多的水分,避免标本干燥。把切片放入湿盒内,同时处理4张切片。
10)每张切片使用30μl~60μl罗丹明抗-地高辛抗体或FITC卵白素,室温下孵育20min。
11)去掉塑料盖膜,把切片放入含1×PBS的染色缸。1×PBS室温下洗3次,每次2min。
扩增:
12)从1×PBS中取出切片,斜置切片使液体排出;
13)每张切片滴30μl~60μl抗-卵白素抗体,加塑料盖膜,室温孵育20min;
14)去掉塑料盖膜,把切片放入含1×PBS的染色缸。1×PBS室温下洗3次,每次2min;
15)从1×PBS中取出切片,斜置切片使液体排出;
16)每张切片滴30μl~60μl抗-卵白素抗体,加塑料盖膜,室温孵育20min;
17)1×PBS室温下洗3次,每次2min。
5、细胞核染色:
1)每张切片加10μl~20μl DAPI,覆盖盖玻片并在室温下孵育2~5ml;
2)尽可能快的在荧光显微镜下观察或封闭盒内保存在-20℃冰箱。切片在染色之后1h内可以在显微镜下观察。
二、荧光原位免疫组织化学
(1)标本处理。
(2)滴加适当稀释的特异性抗体于标本上,置湿盒中,37℃30~60分钟或4℃过夜。
(3)滴加适当稀释的间接荧光抗体,置湿盒中,37℃30~60分钟。
(5)PBS洗2次,双蒸水洗1次。
(6)甘油缓冲液封片,荧光显微镜下观察。
(7)对照染色:可用空白对照和阴性对照等。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明方法的前提下,还可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
Claims (1)
1.一种预测和评价雄激素受体的激素去势治疗后去势抵抗或复发的试剂盒在制备诊断高度恶性及转移性前列腺癌的试剂中的应用,其特征在于,所述的试剂盒通过荧光原位杂交探针检测PCGEM1并通过荧光免疫组织化学检测雄激素受体异构体AR3,所述的试剂盒包括PCGEM1的核酸原位杂交检测探针,所述核酸原位杂交检测探针用地高辛标记,所述核酸原位杂交检测探针的序列为PCGEM1-1:TTTTTTTCCAAAGGGGTTCCGCTGTCCCTGGGGAAG、PCGEM1-2:AATTTTCCCCTCAGAAAAATCTCAGGGCTTTGGTTC,其中G为G-β-D-氧-LNA核苷酸类似物,C为C-β-D-氧-LNA核苷酸类似物,A为A-β-D-氧-LNA核苷酸类似物,T为T-β-D-氧-LNA核苷酸类似物,所述的检测AR3的抗体及荧光二抗为绿色荧光蛋白EGFP。
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