CN113897437B - 检测样本中标志物表达水平的试剂在制备用于诊断乳腺癌的试剂盒中的应用 - Google Patents
检测样本中标志物表达水平的试剂在制备用于诊断乳腺癌的试剂盒中的应用 Download PDFInfo
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Abstract
本发明公开了检测样本中标志物表达水平的试剂在制备用于诊断乳腺癌的试剂盒中的应用,涉及生物医药技术领域。具体而言,本发明提供的标志物为侵袭性乳腺癌转录因子标志物,具体为在乳腺癌组织中低表达的IRX1、ATOH8、IRX6、KLF15、ALX4、ZBTB16以及在癌组织中高表达的E2F8和MYBL中的至少5种,利用这些标志物的在侵袭性乳腺癌癌组织及癌旁组织的差异表达,可用于侵袭性乳腺癌的有效诊断、预后评估或筛药。
Description
技术领域
本发明涉及生物医药技术领域,具体而言,涉及检测样本中标志物表达水平的试剂在制备用于诊断乳腺癌的试剂盒中的应用。
背景技术
乳腺癌(Breast cancer)是全球女性死亡的第二大癌症,其治疗的主要挑战之一是乳腺癌细胞具有异质性,从而决定治疗策略的选择。根据受体类型的不同,乳腺癌可被分为4种亚型:***/孕激素受体阳性(ER/PR)、人类表皮生长因子受体阳性(HER2)和三阴性乳腺癌(Triple negative breast cancer, TNBC);而根据病理及分子分型的不同可分为:管腔样A型 [Luminal A,(ER/PR阳性,HER2阴性)]、管腔样B型 [Luminal B,(ER/PR阳性,HER2阳性)]、HER2阳性类型及基底样型(Basal);而Basal类型中大部分由三阴性乳腺癌(Triple negative breast cancer, TNBC)是组成。三阴性乳腺癌内部仍具有异质性,占所有乳腺癌亚型的12%-17%。三阴性乳腺癌细胞表面不表达***受体(ER)、孕激素受体(PR)及表皮生长因子受体(HER2),目前可选手术及化学治疗。
然而,由于三阴性乳腺癌转移及复发性较其他乳腺癌高,长期应用化疗药物会导致患者耐受,因此,探索有效的三阴性乳腺癌发生发展的分子机制,开发新型靶向治疗药物,对三阴性乳腺癌患者的治疗提供有潜力地临床治疗前景。
鉴于此,特提出本发明。
发明内容
本发明的目的在于提供检测样本中标志物表达水平的试剂在制备用于诊断乳腺癌的试剂盒中的应用。
本发明是这样实现的:
第一方面,本发明实施例提供了检测样本中标志物表达水平的试剂在制备用于诊断乳腺癌的试剂盒中的应用,所述标志物包括IRX1、ATOH8、IRX6、KLF15、ALX4、ZBTB16、E2F8和MYBL2中的至少5种。
第二方面,本发明实施例提供了检测样本中标志物表达水平的试剂在制备用于乳腺癌预后评估的试剂盒中的应用,所述标志物包括IRX1、ATOH8、IRX6、KLF15、ALX4、ZBTB16、E2F8和MYBL2中的至少5种。
第三方面,本发明实施例提供了检测样本中标志物表达水平的试剂在制备用于防治乳腺癌的药物中的应用,所述标志物包括IRX1、ATOH8、IRX6、KLF15、ALX4、ZBTB16、E2F8和MYBL2中的至少5种。
第四方面,本发明实施例提供了一种试剂盒,应用于乳腺癌的诊断、预后评估或药物筛选,其由以下组分组成:分别用于检测IRX1、ATOH8、IRX6、KLF15、ALX4、ZBTB16、E2F8和MYBL2中的至少5种标志物表达水平的试剂。
本发明具有以下有益效果:
本发明公开了一种侵袭性乳腺癌
本发明提供一种侵袭性乳腺癌转录因子标志物,具体为在乳腺癌组织中低表达的IRX1、ATOH8、IRX6、KLF15、ALX4、ZBTB16以及在癌组织中高表达的E2F8和MYBL中的至少5种,利用这些标志物的在侵袭性乳腺癌癌组织及癌旁组织的差异表达,可用于侵袭性乳腺癌的有效诊断、预后评估或筛药。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1为四种亚型乳腺癌癌组织癌组织与癌旁差异表达TFs;其中,Luninal A Ca代表Luminal A型癌组织,Luninal A P代表癌旁组织;Luminal A Ca代表Luminal B型癌组织,Luninal B P代表癌旁组织;HER2 Ca代表HER2阳性亚型癌组织,HER2 P代表癌旁组织;Basal Ca代表TNBC亚型癌组织,Basal P代表癌旁组织;
图2~4均为Luminal A型乳腺癌差异表达的转录因子与侵袭性乳腺癌患者无复发生存期相关性分析;HR(Hazard ratio)为风险率;
图5为Luminal B型乳腺癌差异表达的转录因子与侵袭性乳腺癌患者无复发生存期相关性分析;HR(Hazard ratio)为风险率;
图6为HER2型乳腺癌差异表达的转录因子与侵袭性乳腺癌患者无复发生存期相关性分析;HR(Hazard ratio)为风险率;
图7为Basal型乳腺癌差异表达的转录因子与侵袭性乳腺癌患者无复发生存期相关性分析;HR(Hazard ratio)为风险率;
图8为qRT-PCR方法验证侵袭性乳腺癌癌组织与癌旁组织中差异TFs的相对表达量;
图9~21依次为标志物组0~12的ROC曲线图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
首先,本发明提供了检测样本中标志物表达水平的试剂在制备用于诊断乳腺癌的试剂盒中的应用,所述标志物包括IRX1、ATOH8、IRX6、KLF15、ALX4、ZBTB16、E2F8和MYBL2中的至少5种。
上述标志物均为转录因子(TFs),是细胞信号转导过程中参与基因转录表达调控。检测标志物的表达水平可以是RNA层面的表达水平检测和/或蛋白层面的表达水平检测,优选为RNA的表达水平。
在乳腺癌组织和癌旁组织差异表达的转录因子有很多,但是其中很多差异的转录因子不是或不仅是由乳腺癌引发的,还与其他信号通路相关,因此,并非是能够特异性作为诊断乳腺癌的标志物,如果不进行筛选和分析,则基于标志物的表达水平的检测结果,也无法有效准确地实现乳腺癌的诊断或预测。本发明实施例提供的标志物组合是经一系列创造性劳动,分析筛选获得的、与乳腺癌(尤其是侵袭性乳腺癌)的发生或严重程度相关的标志物组合,通过对该标志物组合的表达水平的检测与分析,能够实现对乳腺癌的诊断或辅助诊断。
上述标志物的核酸序列可基于现有的数据库获得。具体地,IRX1编码区的核酸序列如下:5’- ATGTCCTTCCCGCAGCTGGGCTACCCGCAGTACCTGAGCGCCGCGGGGCCGGGCGCCTACGGCGGCGAGCGCCCGGGGGTGCTGGCCGCGGCCGCTGCGGCGGCTGCCGCCGCCTCGTCGGGCCGACCGGGGGCCGCGGAGCTGGGCGGCGGGGCAGGCGCGGCTGCAGTCACCTCGGTGCTGGGCATGTACGCGGCGGCGGGGCCGTACGCGGGCGCGCCCAACTACAGCGCCTTCCTGCCCTACGCCGCGGATCTCAGCCTCTTCTCGCAGATGGGCTCGCAGTATGAACTGAAGGACAACCCTGGGGTGCACCCCGCCACCTTCGCAGCCCACACGGCGCCGGCTTATTACCCCTACGGCCAGTTCCAATACGGGGACCCCGGGCGGCCCAAGAACGCCACCCGCGAGAGCACCAGCACGCTCAAGGCCTGGCTCAACGAGCACCGCAAGAATCCCTACCCCACCAAGGGCGAGAAGATCATGCTGGCCATCATCACCAAGATGACCCTCACGCAGGTCTCCACCTGGTTCGCCAACGCGCGCCGGCGCCTCAAGAAGGAGAACAAGGTGACATGGGGAGCGCGCAGCAAGGACCAGGAAGATGGAGCGCTCTTCGGCAGCGACACCGAGGGCGACCCGGAGAAGGCCGAGGACGACGAGGAGATCGACCTGGAAAGCATCGACATTGACAAGATCGACGAGCACGATGGCGACCAGAGCAACGAGGATGACGAGGACAAGGCCGAGGCTCCGCACGCGCCCGCAGCCCCTTCTGCTCTTGCCCGGGACCAAGGCTCGCCGCTGGCAGCAGCCGACGTTCTCAAGCCCCAGGACTCGCCCTTGGGCCTGGCAAAGGAGGCCCCAGAGCCGGGCAGCACGCGCCTGCTGAGCCCCGGCGCTGCAGCGGGCGGCCTGCAGGGTGCGCCGCACGGCAAGCCCAAGATCTGGTCGCTGGCGGAGACAGCCACGAGCCCCGACGGTGCGCCCAAGGCTTCGCCACCACCACCCGCGGGCCACCCCGGCGCGCACGGGCCCTCCGCCGGGGCGCCGCTGCAACACCCCGCCTTCCTGCCTAGCCACGGACTGTACACCTGCCACATCGGCAAGTTCTCCAACTGGACCAACAGCGCATTCCTCGCACAGGGCTCCCTGCTCAACATGCGCTCCTTCCTGGGCGTTGGCGCTCCCCACGCCGCGCCCCATGGCCCTCACCTTCCTGCACCTCCACCACCGCAGCCGCCGGTCGCTATTGCCCCGGGGGCACTCAATGGAGACAAGGCCTCGGTCCGCAGCAGCCCCACGCTCCCAGAGAGAGACCTCGTCCCCAGGCCAGATTCGCCGGCACAGCAGTTAAAGTCGCCCTTCCAGCCGGTACGCGACAACTCTCTGGCCCCGCAGGAGGGAACGCCGCGGATCCTAGCAGCCCTCCCGTCCGCCTGA -3’。
ATOH8编码区的核酸序列如下:5’- ATGAAGCACATCCCGGTCCTCGAGGACGGGCCGTGGAAGACCGTGTGCGTGAAGGAGCTGAACGGCCTTAAGAAGCTCAAGCGGAAAGGCAAGGAGCCGGCGCGGCGCGCGAACGGCTATAAAACTTTCCGACTGGACTTGGAAGCGCCCGAGCCCCGCGCCGTAGCCACCAACGGGCTGCGGGACAGGACCCATCGGCTGCAGCCGGTCCCGGTACCGGTGCCGGTGCCAGTCCCAGTGGCGCCGGCCGTTCCCCCAAGAGGGGGCACGGACACAGCCGGGGAGCGCGGGGGCTCTCGGGCGCCCGAGGTCTCCGACGCGCGGAAACGCTGCTTCGCCCTAGGCGCAGTGGGGCCAGGACTCCCCACGCCGCCGCCGCCGCCGCCTCCTGCGCCCCAGAGCCAGGCACCTGGGGGCCCAGAGGCACAGCCTTTCCGGGAGCCGGGTCTGCGTCCTCGCATCTTGCTGTGCGCACCGCCCGCGCGCCCCGCGCCGTCAGCACCCCCAGCACCGCCAGCGCCCCCGGAGTCCACTGTGCGCCCTGCGCCCCCGACGCGCCCCGGGGAAAGTTCCTACTCGTCAATTTCACACGTAATTTACAATAACCACCAGGATTCCTCCGCGTCGCCTAGGAAACGACCGGGCGAAGCGACTGCCGCCTCCTCCGAGATCAAAGCCCTGCAGCAGACCCGGAGGCTCCTGGCGAACGCCAGGGAGCGGACGCGGGTGCACACCATCAGCGCAGCCTTCGAGGCGCTCAGGAAGCAGGTGCCGTGCTACTCATATGGGCAGAAGCTGTCCAAACTGGCCATCCTGAGGATCGCCTGTAACTACATCCTGTCCCTGGCGCGGCTGGCTGACCTTGACTACAGTGCCGACCACAGCAACCTCAGCTTCTCCGAGTGTGTGCAGCGCTGCACCCGCACCCTGCAGGCCGAGGGACGTGCCAAGAAGCGCAAGGAGTGA -3’。
IRX6编码区的核酸序列如下:5’- ATGTCCTTCCCACACTTTGGACACCCGTACCGCGGCGCTTCCCAGTTTCTGGCGTCGGCAAGTTCCAGCACCACATGCTGCGAATCTACCCAACGCTCTGTCTCAGATGTGGCATCAGGCTCCACCCCAGCGCCCGCTCTCTGCTGCGCACCCTACGATAGTCGACTGCTGGGCAGTGCGCGACCGGAGCTGGGCGCCGCCTTGGGCATCTATGGAGCACCCTATGCGGCCGCTGCAGCTGCCCAGAGCTACCCTGGCTACCTGCCCTATAGCCCAGAGCCCCCCTCACTGTATGGGGCACTGAATCCACAGTATGAATTTAAGGAGGCTGCAGGGAGTTTTACATCCAGCCTGGCACAACCAGGAGCCTATTATCCCTATGAGCGGACTCTGGGGCAGTACCAATATGAACGGTATGGCGCAGTGGAATTGAGTGGCGCCGGTCGCCGAAAGAACGCGACCCGGGAGACCACCAGTACACTCAAGGCCTGGCTCAACGAGCACCGCAAAAACCCCTACCCCACTAAGGGTGAGAAGATCATGCTGGCCATCATCACCAAGATGACCCTCACCCAGGTGTCCACCTGGTTCGCCAACGCACGCCGGCGCCTCAAGAAAGAGAACAAAATGACATGGGCGCCCAAGAACAAAGGTGGGGAGGAGAGGAAGGCAGAGGGAGGAGAGGAGGACTCACTAGGCTGCCTAACTGCTGACACCAAAGAAGTTACTGCTAGCCAGGAGGCCCGGGGGCTCCGGCTGAGTGACCTGGAAGACCTGGAGGAAGAGGAGGAGGAGGAGGAGGAAGCTGAAGACGAGGAGGTAGTGGCCACAGCTGGGGACAGGCTGACGGAGTTCCGAAAGGGCGCGCAGTCACTGCCTGGGCCGTGCGCTGCAGCTCGAGAGGGCCGATTGGAGCGCAGGGAGTGCGGCCTGGCTGCGCCCCGCTTCTCCTTCAATGACCCTTCCGGATCGGAAGAAGCTGACTTCCTCTCGGCGGAGACAGGCAGCCCTAGGTTGACCATGCACTACCCATGCTTGGAGAAACCGCGCATCTGGTCTCTGGCGCACACCGCGACAGCCAGCGCTGTTGAAGGTGCACCCCCAGCCCGGCCTAGGCCACGAAGTCCTGAGTGCCGTATGATTCCTGGACAGCCTCCTGCCTCTGCCCGGCGACTCTCAGTCCCCAGAGACTCCGCGTGCGACGAGTCTTCCTGCATACCCAAAGCCTTTGGAAACCCCAAGTTTGCCCTGCAGGGACTACCGCTGAACTGTGCGCCGTGCCCGCGGAGGAGCGAGCCTGTAGTGCAGTGCCAGTACCCGTCTGGAGCAGAAGCAGGTTAG -3’。
KLF15编码区的核酸序列如下:5’- ATGGTGGACCACTTACTTCCAGTGGACGAGAACTTCTCGTCGCCAAAATGCCCAGTTGGGTATCTGGGTGATAGGCTGGTTGGCCGGCGGGCATATCACATGCTGCCCTCACCCGTCTCTGAAGATGACAGCGATGCCTCCAGCCCCTGCTCCTGTTCCAGTCCCGACTCTCAAGCCCTCTGCTCCTGCTATGGTGGAGGCCTGGGCACCGAGAGCCAGGACAGCATCTTGGACTTCCTATTGTCCCAGGCCACGCTGGGCAGTGGCGGGGGCAGCGGCAGTAGCATTGGGGCCAGCAGTGGCCCCGTGGCCTGGGGGCCCTGGCGAAGGGCAGCGGCCCCTGTGAAGGGGGAGCATTTCTGCTTGCCCGAGTTTCCTTTGGGTGATCCTGATGACGTCCCACGGCCCTTCCAGCCTACCCTGGAGGAGATTGAAGAGTTTCTGGAGGAGAACATGGAGCCTGGAGTCAAGGAGGTCCCTGAGGGCAACAGCAAGGACTTGGATGCCTGCAGCCAGCTCTCAGCTGGGCCACACAAGAGCCACCTCCATCCTGGGTCCAGCGGGAGAGAGCGCTGTTCCCCTCCACCAGGTGGTGCCAGTGCAGGAGGTGCCCAGGGCCCAGGTGGGGGCCCCACGCCTGATGGCCCCATCCCAGTGTTGCTGCAGATCCAGCCCGTGCCTGTGAAGCAGGAATCGGGCACAGGGCCTGCCTCCCCTGGGCAAGCCCCAGAGAATGTCAAGGTTGCCCAGCTCCTGGTCAACATCCAGGGGCAGACCTTCGCACTCGTGCCCCAGGTGGTACCCTCCTCCAACTTGAACCTGCCCTCCAAGTTTGTGCGCATTGCCCCTGTGCCCATTGCCGCCAAGCCTGTTGGATCGGGACCCCTGGGGCCTGGCCCTGCCGGTCTCCTCATGGGCCAGAAGTTCCCCAAGAACCCAGCCGCAGAACTCATCAAAATGCACAAATGTACTTTCCCTGGCTGCAGCAAGATGTACACCAAAAGCAGCCACCTCAAGGCCCACCTGCGCCGGCACACGGGTGAGAAGCCCTTCGCCTGCACCTGGCCAGGCTGCGGCTGGAGGTTCTCGCGCTCTGACGAGCTGTCGCGGCACAGGCGCTCGCACTCAGGTGTGAAGCCGTACCAGTGTCCTGTGTGCGAGAAGAAGTTCGCGCGGAGCGACCACCTCTCCAAGCACATCAAGGTGCACCGCTTCCCGCGGAGCAGCCGCTCCGTGCGCTCCGTGAACTGA -3’。
ALX4编码区的核酸序列如下:5’- ATGAATGCTGAGACTTGCGTCTCTTACTGCGAGTCGCCGGCCGCTGCCATGGACGCCTACTACAGCCCGGTGTCGCAGAGTCGGGAGGGCTCGTCGCCTTTTAGGGCATTTCCCGGAGGCGACAAGTTCGGCACAACTTTCCTGTCGGCCGCCGCCAAAGCACAGGGATTCGGGGACGCCAAGAGCCGGGCCCGTTACGGCGCTGGGCAGCAGGACCTGGCGACACCCCTGGAGAGTGGAGCTGGGGCGCGGGGCTCCTTTAACAAGTTCCAGCCCCAGCCGTCGACCCCGCAGCCCCAGCCGCCGCCGCAGCCGCAGCCGCAGCAGCAGCAGCCGCAGCCCCAGCCGCCCGCGCAACCGCATCTTTACTTGCAGCGAGGCGCCTGCAAGACGCCCCCGGACGGCAGCCTCAAACTCCAGGAAGGCAGCAGCGGCCACAGCGCGGCCTTGCAGGTTCCCTGCTACGCTAAAGAGAGCTCCCTGGGTGAGCCAGAGTTACCCCCTGACTCTGACACTGTGGGGATGGACAGCAGCTACCTGAGTGTCAAGGAGGCTGGGGTGAAGGGGCCCCAGGACCGGGCCAGCTCAGACCTCCCCAGCCCATTGGAGAAGGCCGACTCAGAGAGCAACAAGGGCAAGAAGCGGCGGAACCGGACCACCTTCACCAGCTACCAGCTGGAGGAGCTGGAGAAGGTCTTCCAGAAGACCCACTACCCAGACGTGTATGCGCGGGAACAGCTGGCCATGAGGACAGACCTCACTGAGGCCCGCGTGCAGGTCTGGTTCCAGAACCGAAGGGCCAAGTGGAGGAAGCGGGAGCGTTTTGGGCAGATGCAGCAGGTTCGAACCCACTTCTCCACTGCATATGAGCTGCCCCTCCTCACCCGAGCTGAGAACTACGCCCAGATTCAGAACCCGTCCTGGCTCGGCAACAACGGGGCTGCCTCACCAGTGCCAGCCTGCGTGGTCCCCTGCGACCCGGTGCCTGCCTGCATGTCCCCTCATGCCCACCCCCCTGGCTCTGGGGCCAGCAGCGTCACCGACTTCCTGAGTGTGTCTGGGGCTGGCAGTCACGTGGGCCAGACGCACATGGGCAGCCTGTTTGGAGCAGCCAGCCTCAGCCCAGGCCTCAATGGCTACGAGCTCAACGGCGAGCCGGACCGCAAGACCTCGAGCATCGCGGCCCTCCGCATGAAGGCCAAGGAGCACAGTGCGGCCATTTCCTGGGCCACATGA -3’。
ZBTB16编码区的核酸序列如下:5’- ATGGATCTGACAAAAATGGGCATGATCCAGCTGCAGAACCCTAGCCACCCCACGGGGCTACTGTGCAAGGCCAACCAGATGCGGCTGGCCGGGACTTTGTGCGATGTGGTCATCATGGTGGACAGCCAGGAGTTCCACGCCCACCGGACGGTGCTGGCCTGCACCAGCAAGATGTTTGAGATCCTCTTCCACCGCAATAGTCAACACTATACTTTGGACTTCCTCTCGCCAAAGACCTTCCAGCAGATTCTGGAGTATGCATATACAGCCACGCTGCAAGCCAAGGCGGAGGACCTGGATGACCTGCTGTATGCGGCCGAGATCCTGGAGATCGAGTACCTGGAGGAACAGTGCCTGAAGATGCTGGAGACCATCCAGGCCTCAGACGACAATGACACGGAGGCCACCATGGCCGATGGCGGGGCCGAGGAAGAAGAGGACCGCAAGGCTCGGTACCTCAAGAACATCTTCATCTCGAAGCATTCCAGCGAGGAGAGTGGGTATGCCAGTGTGGCTGGACAGAGCCTCCCTGGGCCCATGGTGGACCAGAGCCCTTCAGTCTCCACTTCATTTGGTCTTTCAGCCATGAGTCCCACCAAGGCTGCAGTGGACAGTTTGATGACCATAGGACAGTCTCTCCTGCAGGGAACTCTTCAGCCACCTGCAGGGCCCGAGGAGCCAACTCTGGCTGGGGGTGGGCGGCACCCTGGGGTGGCTGAGGTGAAGACGGAGATGATGCAGGTGGATGAGGTGCCCAGCCAGGACAGCCCTGGGGCAGCCGAGTCCAGCATCTCAGGAGGGATGGGGGACAAGGTTGAGGAAAGAGGCAAAGAGGGGCCTGGGACCCCGACTCGAAGCAGCGTCATCACCAGTGCTAGGGAGCTACACTATGGGCGAGAGGAGAGTGCCGAGCAGGTGCCACCCCCAGCTGAGGCTGGCCAGGCCCCCACTGGCCGACCTGAGCACCCAGCACCCCCGCCTGAGAAGCATCTGGGCATCTACTCCGTGTTGCCCAACCACAAGGCTGACGCTGTATTGAGCATGCCGTCTTCCGTGACCTCTGGCCTCCACGTGCAGCCTGCCCTGGCTGTCTCCATGGACTTCAGCACCTATGGGGGGCTGCTGCCCCAGGGCTTCATCCAGAGGGAGCTGTTCAGCAAGCTGGGGGAGCTGGCTGTGGGCATGAAGTCAGAGAGCCGGACCATCGGAGAGCAGTGCAGCGTGTGTGGGGTCGAGCTTCCTGATAACGAGGCTGTGGAGCAGCACAGGAAGCTGCACAGTGGGATGAAGACGTACGGGTGCGAGCTCTGCGGGAAGCGGTTCCTGGATAGTTTGCGGCTGAGAATGCACTTACTGGCTCATTCAGCGGGTGCCAAAGCCTTTGTCTGTGATCAGTGCGGTGCACAGTTTTCGAAGGAGGATGCCCTGGAGACACACAGGCAGACCCATACTGGCACTGACATGGCCGTCTTCTGTCTGCTGTGTGGGAAGCGCTTCCAGGCGCAGAGCGCACTGCAGCAGCACATGGAGGTCCACGCGGGCGTGCGCAGCTACATCTGCAGTGAGTGCAACCGCACCTTCCCCAGCCACACGGCTCTCAAACGCCACCTGCGCTCACATACAGGCGACCACCCCTACGAGTGTGAGTTCTGTGGCAGCTGCTTCCGGGATGAGAGCACACTCAAGAGCCACAAACGCATCCACACGGGTGAGAAACCCTACGAGTGCAATGGCTGTGGCAAGAAGTTCAGCCTCAAGCATCAGCTGGAGACGCACTATAGGGTGCACACAGGTGAGAAGCCCTTTGAGTGTAAGCTCTGCCACCAGCGCTCCCGGGACTACTCGGCCATGATCAAGCACCTGAGAACGCACAACGGCGCCTCGCCCTACCAGTGCACCATCTGCACAGAGTACTGCCCCAGCCTCTCCTCCATGCAGAAGCACATGAAGGGCCACAAGCCCGAGGAGATCCCGCCCGACTGGAGGATAGAGAAGACGTACCTCTACCTGTGCTATGTGTGA -3’。所述标志物为IRX1、ATOH8、IRX6、ALX4、KLF15、ZBTB16和E2F8中的至少6种,针对这些标志物组合进行检测,能够更有效准确地提高诊断乳腺癌的有效性和准确率。优选地,所述标志物为IRX1、ATOH8、IRX6、ALX4、ZBTB16和E2F8,基于该组合进行浸润性乳腺癌诊断的AUC值更高,能达到0.9653。
可选地,所述试剂包括引物和/或探针。本发明不对引物和探针的条数和序列进行具体限定,在靶序列已知的情况下,引物和探针的序列和反应条件均可基于现有的技术知识获取。优选地,所述引物包括引物对1~8中的至少一对,引物对1~8的序列依次如SEQ IDNo.1~16所示,具体信息可参照后续表3。引物对包括上游引物和下游引物,因此,引物对1的上游引物和下游引物对应的序列如SEQ ID No.1~2所示,引物对2的上游引物和下游引物对应的序列如SEQ ID No.3~4所示,依此类推。
优选地,所述乳腺癌为侵袭性导管乳腺癌。浸润性乳腺癌是指癌细胞已穿破乳腺导管或小叶腺泡的基底膜并侵入间质的一种恶性肿瘤。乳腺癌的类型包括:***/孕激素受体阳性(ER/PR)、人类表皮生长因子受体阳性(HER2)和三阴性乳腺癌(TNBC)。或根据病理及分子分型的不同分为的:管腔样A型、管腔样B型、HER2阳性类型及基底样型(Basal)。
可选地,所述样本为生物组织样本或含有生物组织样本的环境样本。优选的,所述生物样本选自乳腺癌癌组织和乳腺癌癌旁组织中的至少一种。
本发明实施例还提供了检测样本中标志物表达水平的试剂在制备用于乳腺癌预后评估的试剂盒中的应用,所述标志物包括IRX1、ATOH8、IRX6、KLF15、ALX4、ZBTB16、E2F8和MYBL2中的至少5种。
可以理解的是,本实施例以及后续实施例中所述的样本、标志物以及试剂均可同前述对应的实施例所述,不再赘述。
本发明实施例还提供了检测样本中标志物表达水平的试剂在制备用于防治乳腺癌的药物中的应用,所述标志物包括IRX1、ATOH8、IRX6、KLF15、ALX4、ZBTB16、E2F8和MYBL2中的至少5种。
本文中的“防治”是指预防或治疗,治疗可以指治愈或使病症得到一定程度的好转。
优选地,所述“制备用于防治乳腺癌的药物”包括对药物的筛选。
此外,本发明实施例还提供了一种试剂盒,应用于乳腺癌的诊断、预后评估或药物筛选,其由以下组分组成:分别用于检测IRX1、ATOH8、IRX6、KLF15、ALX4、ZBTB16、E2F8和MYBL2中的至少5种标志物表达水平的试剂。
可选地,所述试剂包括引物和/或探针。优选地,所述引物包括引物对1~8中的至少一对,引物对1~8的序列依次如SEQ ID No.1~16所示。
可选地,所述试剂盒还包括:RNA提取试剂、RNA反转录试剂以及PCR反应试剂中的至少一种。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例1
一、 实施对象的选择
选取12例深圳市人民医院甲乳外科已确诊的侵袭性导管乳腺癌的患者,其中包括3例Luminal A型,3例Luminal B型,3例HER2阳性,3例TNBC型。手术切除肿瘤组织,癌旁组织为距肿瘤组织约3-5cm处切除。患者年龄在34岁至70岁。
二、 组织RNA提取及反转录建库
1. RNA提取
(1)在将组织在液氮中速冻,使用研磨器将组织磨碎,每管组织内加入1ml TRIzol后用振荡器中涡旋,在水平摇床上慢摇10min,充***解组织;(2)将TRIzol-细胞裂解液加入到1.5mL的无酶管中,每个样品中加入200μl氯仿,剧烈震荡15sec后,室温静置2-3 min,10000 rcf, 4°C离心15 min;(3)将上层水相转移至新的1.5mL的无酶管中,加入500μl异丙醇,混合均匀后室温放置40min,10000 rcf, 4°C离心15min;(4)小心移除上清液体,管底可见RNA白色沉淀,使用预冷的无酶水配制的75%乙醇清洗一次,10000 g, 4°C离心1min。(5)室温干燥RNA白色沉淀,加入30-60μL 无酶水水溶解沉淀。-80°C保存备用。
2. 反转录建库
(1)首先进行RNA样本质检:琼脂糖凝胶电泳分析样本RNA完整性及是否存在DNA污染;NanoDrop:检测RNA纯度(OD260/280及OD260/230比值);Agilent 2100 bioanalyzer:精确检测RNA完整性。
(2)通过Oligo(dT磁珠)富集带有poly A尾的RNA(包括mRNA和lncRNA),从总RNA中去除核糖体RNA,在NEB Fragmentation Buffer中用二价阳离子打断RNA,片段化的RNA为模板,引物选择随机寡核苷酸,在M-MuLV逆转录酶体系中合成cDNA第一条链,随后用RNaseH降解RNA链,并在DNA polymerase I 体系下,以dNTPs为原料合成cDNA第二条链。纯化后的双链cDNA经过末端修复、加A尾并连接测序接头,用AMPure XP beads筛选250-300bp左右的cDNA,进行PCR扩增并再次使用AMPure XP beads纯化PCR产物,最终获得文库。建库用试剂盒为NEBNextUltra RNA Library Prep Kit for Illumina。
三、 质检及RNA-seq上机测序
1. 文库构建完成后,先使用Qubit2.0 Fluorometer进行初步定量,稀释文库至1.5ng/ul,随后使用Agilent 2100 bioanalyzer对文库的insert size进行检测,insertsize符合预期后,qRT-PCR对文库有效浓度进行准确定量。
2. 把不同文库按照有效浓度及目标下机数据量的需求pooling后进行Illumina测序。测序的基本原理是边合成边测序。在测序的flow cell中加入四种荧光标记的dNTP、DNA聚合酶以及接头引物进行扩增,在每一个测序簇延伸互补链时,每加入一个被荧光标记的dNTP就能释放出相对应的荧光,测序仪通过捕获荧光信号,并通过计算机软件将光信号转化为测序峰,从而获得待测片段的序列信息。
四、 RNA-seq数据生物信息学分析
1. 数据质控:测序片段被高通量测序仪测得的图像数据经CASAVA碱基识别转化为序列数据(reads),去除带接头(adapter)的reads;去除含N(N表示无法确定碱基信息)的reads;去除低质量reads(Qphred<=20的碱基数占整个read长度的50%以上的reads)。
2. 数据分析:基因表达水平分析:计算各样本所有基因的表达值(Expectednumber of Fragments Per Kilobase of transcript sequence per Millions basepairs sequenced,FPKM)后,FPKM是指每百万fragments中来自某一基因每千碱基长度的fragments成对的reads数目,然后通过盒形图展示不同样本基因表达水平的分布情况;基因差异表达分析,首先对原始的readcount进行标准化(normalization),主要是对测序深度的校正。然后统计学模型进行假设检验概率(Pvalue)的计算,最后进行多重假设检验校正,得到FDR值(错误发现率)。对不同组别间的基因做差异分析,|log2(FoldChange)|>1&padj<0.05的候选基因进一步分析;此外,采用GO富集分析和KEGG信号通路富集性分析进行细胞功能及差异基因通路分析。
五、 实时荧光定量PCR反应(qRT-PCR)
1. RNA反转录实验:
在RNase-Free microtube 管中配制下列混合液,10μl体系:首先使用DNase降解样本中DNA。
表1 10μl体系
PCR反应条件:37°C, 30min。
2. 添加25mM EDTA 1μl至混合液,65°C热激反应10min。向混合液中加入50μM 0.5μL Random primer(随机引物)和4uM 1ul dNTP。
PCR反应条件:72°C,10min,然后置于冰上。
3. RNA反转录合成 cDNA,反应体系见表2。
表2 反应体系
PCR反应条件:37°C,60min;70°C,15min。
得到的20μl cDNA 用DEPCH2O 稀释20倍后,-20°C冰箱贮存。
4. qRT-PCR
由Invitrogen公司设计合成表3所示引物。
表3 引物
在EP管中配制表4所示的PCR反应液。
表4 PCR反应液
在 PCR 仪上按表5所示反应条件进行qRT- PCR反应。
表5 反应条件
5. 实验结果分析:
反应结束后确认qRT-PCR的扩增曲线和熔解曲线,基因的表达量=2-ΔΔCt,即ΔΔct=(实验组目的基因的Ct的平均值-实验组管家基因GAPDH的Ct的平均值)-(对照组目的基因的Ct的平均值一对照管家基因的Ct的平均值)。
(1)四种乳腺癌亚型中分别筛选表达差异转录因子。
对上述12例乳腺癌样本的RNA-seq数据进行分析,将4种乳腺癌亚型(Luminal A、Lunimal B、HER2和Basal)分别与各自亚型的癌旁(P)进行比较,结果发现,与癌旁比较,Luminal A癌组织中差异表达上调的TFs是30个,下调的TFs是65个;Luminal B癌组织中差异表达上调的TFs是62个,下调的TFs是268个;HER2癌组织中差异表达上调的TFs是74个,下调的TFs是131个;Basal癌组织中差异表达上调的TFs是118个,下调的TFs是182个,如图1所示。
(2)4种亚型乳腺癌中特异性表达差异转录因子。
提高差异表达基因可信度,将log2(fold change)倍数调至5倍且P<0.05为具有显著性差异的条件。将4种亚型Luminal A、Luminal B、HER2和Basal分别与各自癌旁进行比较,筛选差异表达的TFs,包括上调与下调的TFs。Luminal A型乳腺癌患者癌与癌旁比较(表6),表达上调的转录因子有4个,分别为HOXB13、ZNF385B、MYBL2、PITX1;表达下调的转录因子有9个,分别为ETV3L、ZIC4、MLXIPL、SOX10、SOX8、ATOH8、IRX1、ELF5、HLF。
表6 Luminal A型乳腺癌患者癌与癌旁比较表达差异的TFs
Luminal B型乳腺癌患者癌与癌旁比较(表7),表达上调的转录因子有7个,分别为ZNF716、HOXB13、GATA4、HOXC12、INSM1、KLF7和MYT1;表达下调的转录因子有18个,分别为;ZNF536、ETV3L、POU3F3、MLXIPL、SOX10、IRX6、OSR1、MEOX1、KLF15、ATOH8、IRX1、HLF、RFX6、DBX2、ALX1、HIF3A、FIGLA和SHOX。
表7 Luminal B型乳腺癌患者癌与癌旁比较表达差异的TFs
HER2型乳腺癌患者癌与癌旁比较(表8),表达上调的转录因子有20个,分别为LIN28A、ZNF716、HOXC11、TERB1、POU4F3、RFX4、ONECUT1、DMRTC2、HOXB13、SOX11、HOXC12、MNX1、HOXC13、NKX2-2、SIM2、PITX1、PAX7、ONECUT2、ISX和LHX8;表达下调的转录因子有12个,分别为ZNF536、ZNF804B、ALX4、ETV3L、SOX10、OSR1、POU5F1B、IRX1、TCF23、LMX1A、ZBTB16和ALX1。
表8 HER2型乳腺癌患者癌与癌旁比较表达差异的TFs
Basal型乳腺癌患者癌与癌旁比较(表9),表达上调的转录因子有28个,分别为TLX3、LIN28B、PRDM12、DMRTA2、TLX1、PRDM9、PAX6、WT1、VAX1、POU3F2、HOXB13、SIX3、DMRT1、SOX11、GATA4、ZIC1、NKX2-5、SPIB、MYBL2、SIM2、E2F8、SALL3、ESX1、PAX5、ZNF695、LHX2、SCRT2和CTCFL;表达下调的转录因子有5个;分别为ZNF804B、FOXI2、FOXN4、RFX6和LMX1A。
表9 Basal型乳腺癌患者癌与癌旁比较表达差异的TFs
(3)上述转录因子在4种亚型乳腺癌患者中的生存期
通过Gene card 数据库(https://www.genecards.org/)筛选已知lncRNA名称的lncRNA;并通过Kaplan-Meier数据库(https://kmplot.com/analysis)分别筛选出差异表达的转录因子与4种亚型乳腺癌患者预后的表达相关性。
见图2~4,Luminal A中上调的TFs:MYBL2表达越高,侵袭性乳腺癌患者的无复发生存期(Relapse-free survival,RFS)越短(MYBL2:HR=1.75,P<1E-16);Luminal A中下调的TFs:HLF、IRX1、MLXIPL和ATOH8表达越低,侵袭性乳腺癌患者的RFS越短(HLF:HR=0.9,P=0.042;IRX1:HR=0.78,P=0.042;MLXIPL:HR=0.82,P=0.00011;ATOH8:HR=0.68,P=8.1e-07)。
见图5,Luminal B中下调的TFs:ATOH8、HIF3A、IRX1、IRX6、HLF、KLF15、MEOX1和MLXIPL;这些TFs表达越低,侵袭性乳腺癌患者的RFS越短(ATOH8:HR=0.68,P=8.1e-07;HIF3A:HR=0.86,P=0.0046;IRX1:HR=0.78,P=0.0011;IRX6:HR=0.7,P=3.1e-06;HLF:HR=0.9,P=0.042;KLF15:HR=0.67,P=2.7e-07;MEOX1:HR=0.7,P=2.6e-12;MLXIPL:HR=0.82,P=0.00011)。
见图6,在乳腺癌HER2亚型中,表达上调的TFs:HOXC13,表达越高,侵袭性乳腺癌患者的RFS越短(HOXC13:HR=1.12,P=0.026);下调的TFs:ALX4和ZBTB16;这些TFs表达越低,侵袭性乳腺癌患者的RFS越短(ALX4:HR=0.89,P=0.018;ZBTB16:HR=0.65,P<1E-16)。
见图7,在乳腺癌Basal亚型中,表达上调的TFs:E2F8和MYBL2;这些TFs表达越高,侵袭性乳腺癌患者的RFS越短(E2F8:HR=1.74,P<1E-16;MYBL2:HR=1.75,P<1E-16)。
(4)实时荧光反转录定量PCR(qRT-PCR)验证上述差异的转录因子在侵袭性乳腺癌患者中mRNA表达水平。
通过qRT-PCR方法在2对侵袭性乳腺癌癌组织和癌旁组织中进行检测,如图8所示,与癌旁比较,癌组织中表达上调的TFs为E2F8和MYBL2;在癌组织中表达下调的TFs为IRX1、ATOH8、IRX6、KLF15、ALX4和ZBTB16,*P<0.05,**P<0.01,***P<0.001为显著性差异。
实施例2
验证标志物组合对于诊断乳腺癌的准确性。
采用实施例1公开的标志物及其检测方法,设置13组试验组,每组试验组的区别在于标志物组合的不同,见表10。
表10 标志物组合
分别基于组0~组12的标志物组合在12例浸润性乳腺癌患者的癌组织和癌旁组织中进行受试者工作特征曲线(receiver operating characteristic curve, ROC)描绘,多个TFs标志物ROC曲线绘制的步骤包括:共12例浸润性乳腺癌组织,每个样本分为2组:癌组织和癌旁组织,将标志物组合的相对表达量在癌组织中的均值记为癌(Ca),标志物组合的相对表达量在癌旁组织中的均值记为癌(P),共2列,分别使用GraphPad Prism 7.00软件中Analyze绘制ROC曲线。
ROC曲线如图9~21所示。
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> 深圳市人民医院
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Claims (7)
1.针对检测样本中标志物ATOH8、IRX6、KLF15、ALX4、ZBTB16及E2F8表达水平的6对检测引物在制备用于诊断乳腺癌的试剂盒中的应用。
2.根据权利要求1所述的应用,其特征在于,所述引物包括引物对2~7,引物对2~7的序列依次如SEQ ID No.3~14所示。
3.根据权利要求1所述的应用,其特征在于,所述乳腺癌为侵袭性导管乳腺癌。
4.根据权利要求1~3任一项所述的应用,其特征在于,所述样本为生物组织样本或含有生物组织样本的环境样本。
5.一种用于乳腺癌的诊断、预后评估或药物筛选的试剂盒,其特征在于,其包括用于检测标志物表达水平的6对检测引物;所述标志物为:ATOH8、IRX6、KLF15、ALX4、ZBTB16以及E2F8。
6.根据权利要求5所述的试剂盒,其特征在于,所述引物包括引物对2~7,引物对2~7的序列依次如SEQ ID No.3~14所示。
7.根据权利要求5或6所述的试剂盒,其特征在于,所述试剂盒还包括:RNA提取试剂、RNA反转录试剂以及PCR反应试剂中的至少一种。
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