CN110218792A - 一种用于乳腺癌诊断及预后的标志物以及其获得方法 - Google Patents
一种用于乳腺癌诊断及预后的标志物以及其获得方法 Download PDFInfo
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Abstract
本发明提供一种用于乳腺癌诊断及预后的标志物以及其获得方法,该标志物为5′‑tiRNAVal;该方法包括:(1)预测靶基因:通过基因本体功能富集分析、京都基因与基因组百科全书通路分析以及将各数据库进行交集比较找到交集基因;(2)靶基因筛选及验证:(21)通过分析判断得到前述交集基因存在互补结合5′‑tiRNAVal种子区域的MER;(22)通过检测转染5′‑tiRNAVal模拟物后两株细胞中基因的相对表达量,找到FZD3在两株细胞中显著降低;(23)通过荧光素酶报告基因检测,得到5′‑tiRNAVal是与FZD3的3′‑UTR结合。本发明的标志物以及负向调控通路的作用机制迄今为止国内外未见报道,具有源头创新性,可为乳腺癌诊断和预后的新分子标志物提供理论实验依据。
Description
技术领域
本发明涉及生物技术领域,尤其是涉及一种用于乳腺癌诊断及预后的标志物以及其获得方法。
背景技术
乳腺癌是全球女性最常见的恶性肿瘤之一,发病率仅次于卵巢癌和宫颈癌。乳腺癌也是女性死于癌症的第二大原因,仅次于肺癌。目前分子生物学的进展对乳腺癌的早期诊断和治疗提供了很大的帮助,但对于晚期广泛侵袭和转移的乳腺癌的治疗仍然效果不佳,是乳腺癌患者死亡的主要原因。临床诊断乳腺癌的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。
发明内容
本发明的目的在于:针对现有技术存在的问题,提供一种用于乳腺癌诊断及预后的标志物以及其获得方法,解决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的问题。
本发明的发明目的通过以下技术方案来实现:
一种用于乳腺癌诊断及预后的标志物,该标志物为5′-tiRNAVal(MINTbase数据库唯一ID:tRF-32-Q99P9P9NH57SJ)。
一种用于乳腺癌诊断及预后的标志物的获得方法,该方法包括:
(1)预测靶基因:通过基因本体功能富集分析、京都基因与基因组百科全书通路分析以及将GO(基因本体功能富集分析)、KEGG Pathway(京都基因与基因组百科全书通路分析)数据库进行交集比较找到交集基因;
(2)靶基因筛选及验证:
(21)通过分析判断得到前述交集基因存在互补结合5′-tiRNAVal种子区域的MER(单体单元);
(22)通过检测转染5′-tiRNAVal模拟物后MDA-MB-231(乳腺癌细胞MDA-MB-231)和MCF-7(乳腺癌细胞MCF-7)两株细胞中基因的相对表达量,找到FZD3(卷曲蛋白家族受体3)在两株细胞中显著降低;
(23)通过荧光素酶报告基因检测,得到5′-tiRNAVal是与FZD3(卷曲蛋白家族受体3)的3′-UTR(3’非翻译区)结合。
步骤(1)具体包括:通过基因本体功能富集分析显示5′-tiRNAVal作用的靶标基因功能显著富集的GO主要分布在生物过程类别,细胞成分和分子功能分类;通过京都基因与基因组百科全书通路分析显示,17种生物学途径在乳腺癌组织中显著改变,涉及Wnt、mTOR(哺乳动物雷帕霉素靶蛋白)、VEGF(血管内皮生长因子)、P53(肿瘤蛋白p53)信号通路;GO(基因本体功能富集分析)和KEGG Pathway(京都基因与基因组百科全书通路分析)中有显著差异的共同交集的是Axon guidance(轴突导向)功能通路,选取相交的10条基因,再次与KEGG Pathway(京都基因与基因组百科全书通路分析)数据库中癌症通路Pathways incancer(癌症途径)相交,交集基因4条:FZD3(卷曲蛋白家族受体3)、PIK3CB(磷脂酰肌醇-4,5-二磷酸3-激酶催化亚单位β)、PRKCA(蛋白激酶Cα)、PTCH1(蛋白质修补同系物1)。
步骤(21)具体为通过microRNA.org(非编码单链RNA分子.org)、TargetScan(目标扫描)分析判断得到前述交集基因存在互补结合5′-tiRNAVal种子区域的MER(单体单元)。
5′-tiRNAVal通过结合FZD3负向调控Wnt/β-catenin信号通路进而影响乳腺癌的进展。
5′-tiRNAVal使MDA-MB-231和MCF-7细胞株中FZD3、β-catenin、c-Myc以及cyclinD1的蛋白水平降低,APC蛋白水平升高。
与现有技术相比,本发明采用GO以及KEGG生物学分析,结合miRanda数据库以及TargetScan分析软件筛选出5′-tiRNAVal可能靶基因,同时结合qRT-PCR(实时荧光定量PCR)、Westernblot(蛋白免疫印迹)、荧光素酶报告基因验证靶基因并查阅相关文献推测5′-tiRNAVal参与Wnt/β-catenin信号通路。进一步验证5′-tiRNAVal通过结合FZD3对其下游基因的调控。5′-tiRNAVal具有肿瘤标志物潜能以及5′-tiRNAVal/FZD3/Wnt/β-catenin负向调控通路的作用机制迄今为止国内外未见报道,具有源头创新性,可为乳腺癌诊断和预后提供新的理论实验依据。
附图说明
图1为GO分析生物过程类别;
图2为GO分析细胞成分;
图3为GO分析分子功能分类;
图4为KEGG分析;
图5为GO和KEGG共同交集筛选可能靶基因;
图6为5′-tiRNAVal种子区域的MER;
图7为乳腺癌细胞中靶基因的表达量;
图8为荧光素酶报告;
图9为5′-tiRNAVal结合FZD3负向调控wnt/β-catenin信号通路的机制图;
图10为wnt/β-catenin通路相关基因蛋白水平。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明。
实施例
tRF&tiRNA(tRNA fragments & tRNA halves)是近年来发现的一类非编码RNA,具有类似于microRNA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。本发明通过测序分析乳腺癌组织和血清中差异表达的tRFs&tiRNAs,发现5′-tiRNAVal的表达存在显著性差异,生物信息分析发现FZD3是5′-tiRNAVal潜在靶基因,参与Wnt/β-Catenin信号通路调控。5′-tiRNAVal肿瘤标志物潜能以及5′-tiRNAVal/FZD3/Wnt/β-catenin负向调控通路的作用机制迄今为止国内外未见报道,具有源头创新性,可为乳腺癌诊断和预后的新分子标志物提供理论实验依据。
本发明具体过程如图1~图10所示:
1.预测靶基因
1)通过基因本体功能富集分析(Gene Ontology)显示5′-tiRNAVal作用的靶标基因功能显著富集的GO主要分布在生物过程类别(Biological Process categories),细胞成分(Cellular Component)和分子功能分类(Molecular Function Classification)。富含BP类别的靶基因主要涉及以下术语:生物调节,代谢过程,生物过程调节,细胞过程调节和代谢过程等(图1)。在CC中,主要差异涉及以下术语:细胞,细胞部分,细胞内,细胞内部分细胞器部分,膜结合细胞器和细胞质等(图2)。在MF中,主要差异涉及以下术语:结合,蛋白质结合,离子结合和有机环状化合物结合等(图3)。
2)通过京都基因与基因组百科全书通路分析(KEGG Pathway)显示,17种生物学途径在乳腺癌组织中显著改变,涉及Wnt、mTOR、VEGF、P53等信号通路(图4)。
3)GO和Pathway中有显著差异的共同交集的是Axon guidance功能通路(GOID:0007411,P=8.90494E-06,包含基因29条;PathwayID:hsa04360,P=0.02502919,包含基因16条),选取相交的10条基因,再次与KEGG Pathway数据库中癌症通路Pathways in cancer(PathwayID:hsa05200;P=0.03227842,包含基因共30条)相交,交集基因4条:FZD3、PIK3CB、PRKCA、PTCH1(图5)。
2.靶基因筛选及验证
1)采用microRNA.org(http://www.microrna.org/microrna/);TargetScan(http://www.targetscan.org/)进一步分析,发现上述FZD3、PIK3CB、PRKCA、PTCH1,4条可能的靶基因序列中都存在互补结合5′-tiRNAVal种子区域的MER,提示5′-tiRNAVal可能通过与之结合而发挥作用(图6)。图6中各标注含义如下表:
编号 | 靶基因名 | Rrange | Seed Match |
NM_017412 | FZD3 | (529,558) | 8mer |
NM_006219 | PIK3CB | (480,514) | 7mer-A1 |
NM_002737 | PRKCA | (6333,6364) | 7mer-m8 |
NM_000264 | PTCH1 | (2642,2679) | 7mer-m8 |
2)检测转染5′-tiRNAVal模拟物后MDA-MB-231和MCF-7两株细胞中基因的相对表达量,发现只有FZD3在两株细胞中显著降低。结果提示:5′-tiRNAVal主要是通过结合FZD3的调控在乳腺癌发生发展中起关键作用(图7)。
3)荧光素酶报告基因检测结果显示,pmirGLO-FZD3-wt(野生型)3′UTR质粒和5′-tiRNAVal模拟物共转染,可明显降低荧光素酶的活性,差异具有统计学意义,突变质粒pmirGLO-FZD3-mut(突变型)3′UTR与5′-tiRNAVal模拟物共转染,荧光素酶活性没有明显变化。结果提示:5′-tiRNAVal与FZD3的3′-UTR结合(图8)。本步骤中用到的各引物的序列如下:
表1各引物序列
3.5′-tiRNAVal结合FZD3负向调控Wnt/β-catenin信号通路可能机制
1)文献报道FZD3(Frizzled family receptor 3)属于跨膜卷曲蛋白家族一员,FZD3是乳腺癌发生的潜在标志物,参与Wnt/β-Catenin信号通路调控。Wnt/β-Catenin信号通路是一条在生物进化中极为保守的通路,与肿瘤关系密切,在乳腺癌的发生和发展中发挥重要作用,当Wnt/β-catenin通路被激活时,β-catenin降解减少而在细胞质中积聚,过量的β-catenin转移进入核内,诱导下游的促癌明星分子c-myc、CyclinD1等的转录,引起细胞异常增殖,促进肿瘤的发生。因此,我们推测5′-tiRNAVal可能通过结合FZD3负向调控Wnt/β-catenin信号通路进而影响乳腺癌的进展(图9)。
2)5′-tiRNAVal使MDA-MB-231和MCF-7细胞株中FZD3、β-catenin、c-Myc以及cyclinD1的蛋白水平降低,APC蛋白水平升高(图10)。
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,应当指出的是,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及预后的标志物,其特征在于,该标志物为5′-tiRNAVal。
2.一种用于乳腺癌诊断及预后的标志物的获得方法,其特征在于,该方法包括:
(1)预测靶基因:通过基因本体功能富集分析、京都基因与基因组百科全书通路分析以及将GO、KEGG Pathway数据库进行交集比较找到交集基因;
(2)靶基因筛选及验证:
(21)通过分析判断得到前述交集基因存在互补结合5′-tiRNAVal种子区域的MER;
(22)通过检测转染5′-tiRNAVal模拟物后MDA-MB-231和MCF-7两株细胞中基因的相对表达量,找到FZD3在两株细胞中显著降低;
(23)通过荧光素酶报告基因检测,得到5′-tiRNAVal是与FZD3的3′-UTR结合。
3.根据权利要求2所述的用于乳腺癌诊断及预后的标志物的获得方法,其特征在于,步骤(1)具体包括:通过基因本体功能富集分析显示5′-tiRNAVal作用的靶标基因功能显著富集的GO主要分布在生物过程类别,细胞成分和分子功能分类;通过京都基因与基因组百科全书通路分析显示,17种生物学途径在乳腺癌组织中显著改变,涉及Wnt、mTOR、VEGF、P53信号通路;GO和KEGG Pathway中有显著差异的共同交集的是Axon guidance功能通路,选取相交的10条基因,再次与KEGG Pathway数据库中癌症通路Pathways in cancer相交,交集基因4条:FZD3、PIK3CB、PRKCA、PTCH1。
4.根据权利要求2所述的用于乳腺癌诊断及预后的标志物的获得方法,其特征在于,步骤(21)具体为通过microRNA.org、TargetScan分析判断得到前述交集基因存在互补结合5′-tiRNAVal种子区域的MER。
5.根据权利要求2所述的用于乳腺癌诊断及预后的标志物的获得方法,其特征在于,5′-tiRNAVal通过结合FZD3负向调控Wnt/β-catenin信号通路进而影响乳腺癌的进展。
6.根据权利要求5所述的用于乳腺癌诊断及预后的标志物的获得方法,其特征在于,5′-tiRNAVal使MDA-MB-231和MCF-7细胞株中FZD3、β-catenin、c-Myc以及cyclinD1的蛋白水平降低,APC蛋白水平升高。
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