WO2020034543A1 - 用于乳腺癌诊断的标记物及其筛选方法 - Google Patents

用于乳腺癌诊断的标记物及其筛选方法 Download PDF

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WO2020034543A1
WO2020034543A1 PCT/CN2018/123905 CN2018123905W WO2020034543A1 WO 2020034543 A1 WO2020034543 A1 WO 2020034543A1 CN 2018123905 W CN2018123905 W CN 2018123905W WO 2020034543 A1 WO2020034543 A1 WO 2020034543A1
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breast cancer
trfs
tirnas
tirna
tdr
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严枫
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江苏省肿瘤医院
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  • the invention belongs to the field of biotechnology, and particularly relates to a marker for breast cancer diagnosis and a screening method thereof.
  • tRF & tiRNA tRNA fragments and tRNA halves
  • TRF & tiRNA related research is a completely new field. Similar to the regulatory functions of miRNA, and can participate in regulating gene transcription and translation, cell proliferation, and cell stress response. Some studies have suggested that tiRNAs are stably expressed in mouse serum, have pre-transcriptional suppression, and may be a new type of signaling molecule.
  • the technical problem to be solved by the present invention is to provide a tRF & tiRNA.
  • the abnormal expression of tRF & tiRNA is related to the occurrence of breast cancer, and can be used as a marker for early diagnosis or prognosis of breast cancer.
  • a marker for breast cancer diagnosis is 5′-tiRNA Val .
  • a screening method for markers for breast cancer diagnosis is as follows:
  • the tRFs & tiRNAs further screened were tested by serum RT-PCR to obtain the tRF & tiRNA with the largest expression difference, which is the target tRF & tiRNA.
  • the sequencing tools are Agilent Bioanalyzer 2100, Illumina NextSeq 500 high-throughput desktop second-generation sequencing system, and high-throughput gene expression database.
  • the number of said tissue specimens from breast cancer patients is six.
  • the number of tRFs & tiRNAs finally selected is six.
  • the RT-PCR detection tool is a tRFs & tiRNA PCR chip.
  • tRFs & tiRNA PCR chips are used to further screen tiRNAs with significant expression differences in breast cancer tissues and serum: 5′-tiRNA Val , 5′-tiRNA Val is a tiRNA that is low in breast cancer and is associated with breast cancer proliferation and metastasis. Closely related, with potential value for early diagnosis and prognostic judgment.
  • FIG. 1 is a structural diagram of a 5′-tiRNA Val of the present invention
  • FIG. 2 is a diagram of differentially expressed tRFs & tiRNAs in tissues of high-throughput sequencing breast cancer patients of the present invention
  • FIG. 3 is a tRFs & tiRNAs map of differentially expressed tissues of breast cancer patients according to the present invention
  • FIG. 4 is a graph showing the expression level of AS-tDR-001430 screened by breast cancer tissues (cancer and paracancerous) of the present invention by RT-PCR;
  • FIG. 5 is a graph showing the expression level of AS-tDR-001130 screened by RT-PCR of breast cancer tissues (cancer and adjacent cancers) according to the present invention.
  • FIG. 6 is a graph showing the expression level of AS-tDR-000779 screened by breast cancer tissues (cancer and adjacent cancer) of the present invention by RT-PCR;
  • FIG. 8 is a sequence diagram of six primers for tRFs & tiRNAs for real-time quantitative PCR of the present invention.
  • FIG. 9 is a graph showing 5′-tiRNA Val levels in breast cancer and adjacent tissues detected by RT-PCR according to the present invention.
  • FIG. 10 is a graph comparing the level of 5′-tiRNA Val in breast cancer serum with normal level detected by RT-PCR according to the present invention.
  • 11 is a comparison chart of 5′-tiRNA Val levels in serum of breast cancer detected by RT-PCR of the present invention at different periods;
  • FIG. 12 is a schematic diagram of a chromosome location where AS-tDR-001430 of the present invention is located.
  • RT-PCR was used to detect the expression levels of the above 6 tRFs & tiRNAs in 16 pairs of tissues (breast cancer and adjacent cancers) (see Table 2 for primer sequences).
  • AS-tDR-000779 (16/16 cases) was found in the high expression group.
  • P 0.000 (as shown in Figure 6)
  • AS-tDR-001130 15/16 cases
  • P 0.000 (such as (Shown in Figure 5)
  • AS-tDR-001430 (16/16 cases) has a maximum down multiple of 4.6 times
  • P 0.000 (shown in Figure 4). Therefore, AS-tDR-000779, AS-tDR-001430, and AS-tDR-001130 were selected for further verification.
  • the serum samples of 30 breast cancer patients and 20 healthy controls were collected, and the expression levels of 3 tRFs & tiRNAs in the serum were detected by RT-PCR.
  • the positive rate of AS-tDR-001430 is high, and the fold is the largest in tissue and serum. Therefore, we focus on this molecule.
  • tRFs & tiRNA PCR chips are used to further screen tiRNAs with significant expression differences in breast cancer tissues and serum: 5′-tiRNA Val , 5′-tiRNA Val is a tiRNA that is low in breast cancer and is associated with breast cancer proliferation and metastasis. Closely related, with potential value for early diagnosis and prognostic judgment.
  • RT-PCR was used to detect the expression levels of the above 6 tRFs & tiRNAs in 16 pairs of tissues (breast cancer and adjacent cancers) (see Table 2 for primer sequences).
  • AS-tDR-000779 (16/16 cases) was found in the high expression group.
  • P 0.000 (as shown in Figure 6)
  • AS-tDR-001130 15/16 cases
  • P 0.000 (such as (Shown in Figure 5)
  • AS-tDR-001430 (16/16 cases) has a maximum down multiple of 4.6 times
  • P 0.000 (shown in Figure 4). Therefore, AS-tDR-000779, AS-tDR-001430, and AS-tDR-001130 were selected for further verification.
  • the serum samples of 30 breast cancer patients and 20 healthy controls were collected, and the expression levels of 3 tRFs & tiRNAs in the serum were detected by RT-PCR.
  • the positive rate of AS-tDR-001430 is high, and the fold is the largest in tissue and serum. Therefore, we focus on this molecule.
  • AS-tDR-001430 The biological properties of AS-tDR-001430 were first analyzed and named. According to the UCSC online software bioinformatics analysis, it was found that AS-tDR-001430 is located on chromosome 6p22.1, the coordinates are 27,248,049-27,248,121, and the length is 73bp.
  • the MINTbase database shows: AS-tDR-001430 in the MINTbase library ID: tRF-32-Q99P9P9NH57SJ, the sequence is 5′-GCTTCTGTAGTGTAGTGGTTATCACGTTCGCC-3 ′, which is the anti-codon loop of mature tRNA-Val-CAC-2-1 by ANG (CTCACAC) fragment generated by specific cleavage, the type is 5'-half, the fragment length is 32nt.
  • AS-tDR-001430 was named 5′-tiRNA Val (as shown in Figure 1).
  • RT-PCR was used to detect the expression levels of the above 6 tRFs & tiRNAs in 16 pairs of tissues (breast cancer and adjacent cancers) (see Table 2 for primer sequences).
  • AS-tDR-000779 (16/16 cases) was found in the high expression group.
  • P 0.000 (as shown in Figure 6)
  • AS-tDR-001130 15/16 cases
  • P 0.000 (such as (Shown in Figure 5)
  • AS-tDR-001430 (16/16 cases) has a maximum down multiple of 4.6 times
  • P 0.000 (shown in Figure 4). Therefore, AS-tDR-000779, AS-tDR-001430, and AS-tDR-001130 were selected for further verification.
  • the serum samples of 30 breast cancer patients and 20 healthy controls were collected, and the expression levels of 3 tRFs & tiRNAs in the serum were detected by RT-PCR.
  • the positive rate of AS-tDR-001430 is high, and the fold is the largest in tissue and serum. Therefore, we focus on this molecule.

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Abstract

本发明提供了一种用于乳腺癌诊断的标记物及其筛选方法，属于生物技术领域，通过筛选乳腺癌患者组织标本，得到一种用于乳腺癌诊断的tRF&tiRNA标记物：5'-tiRNA Val，本发明提供的5'-tiRNA Val是一个低表达于乳腺癌的tiRNA，与乳腺癌的增殖、转移密切相关，具有早期诊断和预后判断的潜在价值。

Description

[根据细则37.2由ISA制定的发明名称]　用于乳腺癌诊断的标记物及其筛选方法 技术领域
本发明属于生物技术领域，特别是涉及一种用于乳腺癌诊断的标记物及其筛选方法。
背景技术
乳腺癌在我国女性恶性肿瘤中发病率位居第一位，死亡率位居第六位，严重危害人们的健康和生活。尽管在药物敏感性和外科技术上已有巨大的突破，但复发和转移的频繁发生，仍是晚期乳腺癌患者死亡的主要原因。目前临床诊断乳腺癌的方法主要依赖于体格检查和影像学检查，但这些方法对于早期乳腺癌的发现敏感性很低，常用来诊断乳腺癌的血清标志物对早期诊断的敏感性和特异性均不理想。
因此，深入理解乳腺癌的发生和发展机制，积极筛选鉴定有效的诊断和预后标志物，对提高乳腺癌检出率和治愈率具有重要意义。
随着恶性肿瘤的研究从功能基因层面(编码蛋白质)逐渐拓展到非编码RNA领域，tRF&tiRNA(tRNA fragments&tRNA halves)引起广泛关注，tRF&tiRNA相关研究是一个全新的领域，在肿瘤方面的研究刚刚起步，具有类似于miRNA的调控功能，并能参与调控基因转录和翻译，细胞增殖以及细胞应激反应。有研究提示tiRNAs在鼠血清中稳定性表达，具有转录前抑制功能，可能是一类新型信号分子。
因此，从tRF&tiRNA角度挖掘乳腺癌新型分子靶标并探讨分子机制十分必要。
发明内容
本发明要解决的技术问题时提供一种tRF&tiRNA，该tRF&tiRNA的异常表达与乳腺癌的发生相关，能够作为乳腺癌早期诊断或预后判断的标记物。
一种用于乳腺癌诊断的标记物，所述的标记物为5′-tiRNA Val。
一种用于乳腺癌诊断的标记物的筛选方法，方法如下：
A.对若干乳腺癌患者组织标本进行测序，芯片检测的原始数据进行均一化处理，初筛出差异倍数2倍以上的tRFs&tiRNAs；筛选出差异倍数＞2，并且P＜0.05的tRFs&tiRNAs；完全配对、不存在错配，删除同一个tRFs&tiRNAs的重复注释项目，剔除具有编码蛋白潜能、表达丰度过低的tRFs&tiRNAs，再进行聚类分析；进一步缩小研究范围，筛选差异倍数＞2.5、P＜0.05的tRFs&tiRNAs、并结合基因本体功能富集分析和京都基因与基因组百科全书通路分析以及与乳腺癌相关文献检索，最终选出若干个tRFs&tiRNAs；
B.将筛选出的tRFs&tiRNAs进行扩大组织样本RT-PCR检测，进一步筛选出表达差异倍数≥2、P＝0.000的tRFs&tiRNAs；
C.将进一步筛选出的tRFs&tiRNAs进行血清RT-PCR检测，得到表达差异倍数最大的tRF&tiRNA，即为目标tRF&tiRNA。
作为优选，所述的测序的工具为安捷伦生物分析仪2100、Illumina NextSeq 500高通量台式二代测序系统及高通量基因表达数据库。
作为优选，所述的乳腺癌患者组织标本的例数为6。
作为优选，步骤A中，最终选出的tRFs&tiRNAs的个数为6。
作为优选，所述的RT-PCR检测工具为tRFs&tiRNA PCR芯片。
本发明采用tRFs&tiRNA PCR芯片进一步筛选出乳腺癌组织和血清中具有显著表达差异的tiRNA：5′-tiRNA Val，5′-tiRNA Val是一个低表达于乳腺癌的tiRNA，与乳腺癌的增殖、转移密切相关，具有早期诊断和预后判断的潜在价值。
附图说明
图1为本发明的5′-tiRNA Val的结构图；
图2为本发明高通量测序乳腺癌患者组织中差异表达的tRFs&tiRNAs图；
图3为本发明聚类分析乳腺癌患者组织中差异表达的tRFs&tiRNAs图；
图4为本发明乳腺癌组织(癌和癌旁)RT-PCR筛选出的AS-tDR-001430的表达水平图；
图5为本发明乳腺癌组织(癌和癌旁)RT-PCR筛选出的AS-tDR-001130的表达水平图；
图6为本发明乳腺癌组织(癌和癌旁)RT-PCR筛选出的AS-tDR-000779的表达水平图；
图7为本发明乳腺癌组织(癌和癌旁)RT-PCR筛选出的AS-tDR-001430、AS-tDR-001130和AS-tDR-000779的表达水平对比图；
图8为本发明6条待验证tRFs&tiRNAs实时定量PCR引物序列图；
图9为本发明RT-PCR检测乳腺癌和癌旁组织中5′-tiRNA Val水平图；
图10为本发明RT-PCR检测乳腺癌血清中5′-tiRNA Val水平与正常水平对比图；
图11为本发明RT-PCR检测乳腺癌血清中5′-tiRNA Val水平在不同时期的对比图；
图12为本发明AS-tDR-001430所在的染色体位置示意图。
具体实施方式
以下实施例仅用于说明本发明，但不限制本发明的保护范围。
实施例一
1.目标tRF&tiRNA筛选
1)从生物样本库中获取6例乳腺癌患者组织标本(癌及癌旁)，应用安捷伦生物分析仪2100(Agilent BioAnalyzer 2100)文库准备、Illumina NextSeq 500高通量台式二代测序系统测序及高通量基因表达数据库(Gene Expression Omnibus)大数据分析筛选tRFs&tiRNAs，结果显示：组织中差异表达2倍以上的tRFs&tiRNAs共1135个(上调496个、下调639个，如图2所示)；
2)根据差异倍数FC(Fold change)＞2，P＜0.05(FC越大，说明两个样本之间的差异越大，P值(P value)当原假设为真时所得到的样本观察结果或更极端结果出现的概率，是用来判定假设检验结果的一个参数,是最常用的统计指标之一，P越小，说明差异基因的可靠性越高)，完全配对，不存在错配，删除同一个tRF&tiRNA的重复注释，剔除具有编码蛋白潜能、表达丰度过低的tRFs&tiRNAs，通过聚类分析，得出符合条件指标的tRFs&tiRNAs：上调17个、下调14个(如图3所示)；
3)以FC＞2.5、P＜0.05、并结合基因本体功能富集分析(Gene Ontology功能富集分析)和京都基因与基因组百科全书通路分析(Kyoto Encyclopedia of Genes and Genomes通路分析)以及与乳腺癌相关文献检索，最终选出6个差异表达的tRFs&tiRNAs：上调4个(AS-tDR-001356，FC＝3.66，P＝0.000；
AS-tDR-000882，FC＝2.90，P＝0.008；
AS-tDR-000014，FC＝4.89，P＝0.000；
AS-tDR-000779，FC＝6.58，P＝0.023)，
下调2个(AS-tDR-001430，FC＝-2.65，P＝0.019；
AS-tDR-001130，FC＝-4.90，P＝0.028)。
2.测序筛选的6条tRFs&tiRNAs的组织RT-PCR检测(如图8所示)
采用RT-PCR检测16对组织(乳腺癌癌及癌旁)中的上述6个tRFs&tiRNAs的 表达水平(引物序列见表2)，结果发现：高表达组中AS-tDR-000779(16/16例)上调2倍，P＝0.000(如图6所示)，其他3个tRFs&tiRNAs表达差异没有统计学意义；低表达组AS-tDR-001130(15/16例)下调2.1倍，P＝0.000(如图5所示)；AS-tDR-001430(16/16例)下调倍数最大为4.6倍，P＝0.000(如图4所示)。所以选出AS-tDR-000779、AS-tDR-001430、AS-tDR-001130进一步验证。
3.组织RT-PCR筛选的3条tRFs&tiRNAs的血清RT-PCR检测
收集30例乳腺癌、20例健康对照者血清标本，采用RT-PCR分别检测血清中3条tRFs&tiRNAs的表达水平，结果显示乳腺癌患者血清AS-tDR-000779表达无差异；AS-tDR-001130下调2.32倍，P＝0.002；AS-tDR-001430下调倍数较大为2.50倍，P＝0.022(如图9所示)。AS-tDR-001430的阳性率高，且在组织和血清中差异倍数皆最大。因此，我们重点关注该分子。
本发明采用tRFs&tiRNA PCR芯片进一步筛选出乳腺癌组织和血清中具有显著表达差异的tiRNA：5′-tiRNA Val，5′-tiRNA Val是一个低表达于乳腺癌的tiRNA，与乳腺癌的增殖、转移密切相关，具有早期诊断和预后判断的潜在价值。
实施例二
1.目标tRF&tiRNA筛选
1)从生物样本库中获取6例乳腺癌患者组织标本(癌及癌旁)，应用安捷伦生物分析仪2100(Agilent BioAnalyzer 2100)文库准备、Illumina NextSeq 500高通量台式二代测序系统测序及高通量基因表达数据库(Gene Expression Omnibus)大数据分析筛选tRFs&tiRNAs，结果显示：组织中差异表达2倍以上的tRFs&tiRNAs共1135个(上调496个、下调639个，如图2所示)；
2)根据差异倍数FC(Fold change)＞2，P＜0.05(FC越大，说明两个样本之间的差异越大，P值(P value)当原假设为真时所得到的样本观察结果或 更极端结果出现的概率，是用来判定假设检验结果的一个参数,是最常用的统计指标之一，P越小，说明差异基因的可靠性越高)，完全配对，不存在错配，删除同一个tRF&tiRNA的重复注释，剔除具有编码蛋白潜能、表达丰度过低的tRFs&tiRNAs，通过聚类分析，得出符合条件指标的tRFs&tiRNAs：上调17个、下调14个(如图3所示)；
3)以FC＞2.5、P＜0.05、并结合基因本体功能富集分析(Gene Ontology功能富集分析)和京都基因与基因组百科全书通路分析(Kyoto Encyclopedia of Genes and Genomes通路分析)以及与乳腺癌相关文献检索，最终选出6个差异表达的tRFs&tiRNAs：上调4个(AS-tDR-001356，FC＝3.66，P＝0.000；
AS-tDR-000882，FC＝2.90，P＝0.008；
AS-tDR-000014，FC＝4.89，P＝0.000；
AS-tDR-000779，FC＝6.58，P＝0.023)，
下调2个(AS-tDR-001430，FC＝-2.65，P＝0.019；
AS-tDR-001130，FC＝-4.90，P＝0.028)。
2.测序筛选的6条tRFs&tiRNAs的组织RT-PCR检测(如图8所示)
采用RT-PCR检测16对组织(乳腺癌癌及癌旁)中的上述6个tRFs&tiRNAs的表达水平(引物序列见表2)，结果发现：高表达组中AS-tDR-000779(16/16例)上调2倍，P＝0.000(如图6所示)，其他3个tRFs&tiRNAs表达差异没有统计学意义；低表达组AS-tDR-001130(15/16例)下调2.1倍，P＝0.000(如图5所示)；AS-tDR-001430(16/16例)下调倍数最大为4.6倍，P＝0.000(如图4所示)。所以选出AS-tDR-000779、AS-tDR-001430、AS-tDR-001130进一步验证。
3.组织RT-PCR筛选的3条tRFs&tiRNAs的血清RT-PCR检测
收集30例乳腺癌、20例健康对照者血清标本，采用RT-PCR分别检测血清 中3条tRFs&tiRNAs的表达水平，结果显示乳腺癌患者血清AS-tDR-000779表达无差异；AS-tDR-001130下调2.32倍，P＝0.002；AS-tDR-001430下调倍数较大为2.50倍，P＝0.022(如图9所示)。AS-tDR-001430的阳性率高，且在组织和血清中差异倍数皆最大。因此，我们重点关注该分子。
4.目标tRF&tiRNA的生物学属性和命名
首先分析AS-tDR-001430生物学属性并命名。通过UCSC在线软件生物信息学分析发现：AS-tDR-001430位于染色体6p22.1，坐标为27,248,049-27,248,121，长度为73bp。MINTbase数据库显示：AS-tDR-001430在MINTbase库中ID：tRF-32-Q99P9P9NH57SJ，序列为5′-GCTTCTGTAGTGTAGTGGTTATCACGTTCGCC-3′，是由ANG在成熟tRNA-Val-CAC-2-1的反密码子环(CTCACAC)处特异性切割产生的片段，类型为5′-half，片段长度为32nt。根据上述生物信息再参照文献，把AS-tDR-001430命名为5′-tiRNA Val(如图1所示)。
实施例三
1.目标tRF&tiRNA筛选
1)从生物样本库中获取6例乳腺癌患者组织标本(癌及癌旁)，应用安捷伦生物分析仪2100(Agilent BioAnalyzer 2100)文库准备、Illumina NextSeq 500高通量台式二代测序系统测序及高通量基因表达数据库(Gene Expression Omnibus)大数据分析筛选tRFs&tiRNAs，结果显示：组织中差异表达2倍以上的tRFs&tiRNAs共1135个(上调496个、下调639个，如图2所示)；
2)根据差异倍数FC(Fold change)＞2，P＜0.05(FC越大，说明两个样本之间的差异越大，P值(P value)当原假设为真时所得到的样本观察结果或更极端结果出现的概率，是用来判定假设检验结果的一个参数,是最常用的统计 指标之一，P越小，说明差异基因的可靠性越高)，完全配对，不存在错配，删除同一个tRF&tiRNA的重复注释，剔除具有编码蛋白潜能、表达丰度过低的tRFs&tiRNAs，通过聚类分析，得出符合条件指标的tRFs&tiRNAs：上调17个、下调14个(如图3所示)；
3)以FC＞2.5、P＜0.05、并结合基因本体功能富集分析(Gene Ontology功能富集分析)和京都基因与基因组百科全书通路分析(Kyoto Encyclopedia of Genes and Genomes通路分析)以及与乳腺癌相关文献检索，最终选出6个差异表达的tRFs&tiRNAs：上调4个(AS-tDR-001356，FC＝3.66，P＝0.000；
AS-tDR-000882，FC＝2.90，P＝0.008；
AS-tDR-000014，FC＝4.89，P＝0.000；
AS-tDR-000779，FC＝6.58，P＝0.023)，
下调2个(AS-tDR-001430，FC＝-2.65，P＝0.019；
AS-tDR-001130，FC＝-4.90，P＝0.028)。
2.测序筛选的6条tRFs&tiRNAs的组织RT-PCR检测(如图8所示)
采用RT-PCR检测16对组织(乳腺癌癌及癌旁)中的上述6个tRFs&tiRNAs的表达水平(引物序列见表2)，结果发现：高表达组中AS-tDR-000779(16/16例)上调2倍，P＝0.000(如图6所示)，其他3个tRFs&tiRNAs表达差异没有统计学意义；低表达组AS-tDR-001130(15/16例)下调2.1倍，P＝0.000(如图5所示)；AS-tDR-001430(16/16例)下调倍数最大为4.6倍，P＝0.000(如图4所示)。所以选出AS-tDR-000779、AS-tDR-001430、AS-tDR-001130进一步验证。
3.组织RT-PCR筛选的3条tRFs&tiRNAs的血清RT-PCR检测
收集30例乳腺癌、20例健康对照者血清标本，采用RT-PCR分别检测血清中3条tRFs&tiRNAs的表达水平，结果显示乳腺癌患者血清AS-tDR-000779表 达无差异；AS-tDR-001130下调2.32倍，P＝0.002；AS-tDR-001430下调倍数较大为2.50倍，P＝0.022(如图9所示)。AS-tDR-001430的阳性率高，且在组织和血清中差异倍数皆最大。因此，我们重点关注该分子。
4.初步分析5′-tiRNA Val与临床病理参数的关系
16对组织，根据癌组织中5′-tiRNA Val表达水平的中位数将患者分为高表达和低表达两组，与癌旁组织相比，乳腺癌组织中5′-tiRNA Val的表达水平显著下降。癌组织中5′-tiRNA Val表达上调者淋巴结转移率(1/9)明显低于表达下调者淋巴结转移率(5/7)(χ2＝5.730,P＝0.035)。分析前述30例乳腺癌患者血清5′-tiRNA Val与临床病理参数的关系，发现其表达水平与乳腺癌患者肿瘤的TNM分期以及淋巴结转移显著负相关。提示5′-tiRNA Val的作用机制可能贯穿了包括发生、增殖、转移在内的乳腺癌恶性进程(cancer progression)。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (6)

  1. 一种用于乳腺癌诊断的标记物，其特征在于：所述的标记物为5′-tiRNA Val。
  2. 一种用于乳腺癌诊断的标记物的筛选方法，其特征在于：
    方法如下：
    A.对若干乳腺癌患者组织标本进行测序，芯片检测的原始数据进行均一化处理，初筛出差异倍数2倍以上的tRFs&tiRNAs；筛选出差异倍数＞2，并且P＜0.05的tRFs&tiRNAs；完全配对、不存在错配，删除同一个tRFs&tiRNAs的重复注释项目，剔除具有编码蛋白潜能、表达丰度过低的tRFs&tiRNAs，再进行聚类分析；进一步缩小研究范围，筛选差异倍数＞2.5、P＜0.05的tRFs&tiRNAs、并结合基因本体功能富集分析和京都基因与基因组百科全书通路分析以及与乳腺癌相关文献检索，最终选出若干个tRFs&tiRNAs；
    B.将筛选出的tRFs&tiRNAs进行扩大组织样本RT-PCR检测，进一步筛选出表达差异倍数≥2、P＝0.000的tRFs&tiRNAs；
    C.将进一步筛选出的tRFs&tiRNAs进行血清RT-PCR检测，得到表达差异倍数最大的tRF&tiRNA，即为目标tRF&tiRNA。
  3. 根据权利要求2所述的一种用于乳腺癌诊断的标记物的筛选方法，其特征在于：所述的测序的工具为安捷伦生物分析仪2100、Illumina NextSeq 500高通量台式二代测序系统及高通量基因表达数据库。
  4. 根据权利要求2所述的一种用于乳腺癌诊断的标记物的筛选方法，其特征在于：所述的乳腺癌患者组织标本的例数为6。
  5. 根据权利要求2所述的一种用于乳腺癌诊断的标记物的筛选方法，其特征在于：步骤A中，最终选出的tRFs&tiRNAs的个数为6。
  6. 根据权利要求2所述的一种用于乳腺癌诊断的标记物的筛选方法，其特征在于：所述的RT-PCR检测工具为tRFs&tiRNA PCR芯片。
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