CN114657244A - 一组诊断宫颈癌变的指标及应用 - Google Patents
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Abstract
本发明公开了一组用于宫颈癌检测的基因及应用,属生物和医学检验领域。所涉及的基因包括:LGALS7B,ANXA8,CDKN2A,CALML3,PKP1,SERPINB5,SERPINB4,KIAA0101,DSG3,DSC3,GPR87,CLCA2,PITX1,IRF6,FAM83A,AURKB,CKMT1A,MMP13,ALG1L,RHOV,SDC1,TRIM29,LYPD3,FGFBP1,PVRL4,KRT5,KRT6A,KRT7,KRT16,KRT6B,KRT15,S100A2,S100A14,S100A9,S100A8,SFN,SERPINB3,LAD1,FXYD3,TACSTD2,PI3,CXCL17,MAL2,CSTA,PDZK1IP1,LAMB3,RAB25,C19orf33,ELF3,GJB3,SPINT1,PKP3,SERINC2,CENPW,CDH3等55个基因。涉及检测上述基因(单个或任意组合)产物表达量的改变来筛查、鉴别宫颈是否癌变的一种分子生物学方法。联合应用上述基因鉴别宫颈癌,在特异性大于99%时,灵敏度可达95%以上;为分子生物学技术检测宫颈癌变提供了一种可靠的方法。
Description
技术领域
本发明属生物和医学检验领域,通过检测单个或任意组合的相关基因产物(蛋白质、RNA)在宫颈病变组织、体液等中表达量的改变来鉴别宫颈是否发生癌变的一种分子生物学方法及应用。所涉及的基因包括:LGALS7B,ANXA8,CDKN2A,CALML3,PKP1,SERPINB5,SERPINB4,KIAA0101,DSG3,DSC3,GPR87,CLCA2,PITX1,IRF6,FAM83A,AURKB,CKMT1A,MMP13,ALG1L,RHOV,SDC1,TRIM29,LYPD3,FGFBP1,PVRL4,KRT5,KRT6A,KRT7,KRT16,KRT6B,KRT15,S100A2,S100A14,S100A9,S100A8,SFN,SERPINB3,LAD1,FXYD3,TACSTD2,PI3,CXCL17,MAL2,CSTA,PDZK1IP1,LAMB3,RAB25,C19orf33,ELF3,GJB3,SPINT1,PKP3,SERINC2,CENPW,CDH3等55个基因。
背景技术
宫颈癌(Cervical squamous cell carcinoma and endocervicaladenocarcinoma,CESC)作为最常见的妇科恶性肿瘤,早发现、早治疗是降低该病死亡率的重要手段。目前,宫颈癌的早期筛查、诊断,主要采取“宫颈三步诊断法”。第一步,宫颈脱落细胞学检查,即宫颈刮片筛查,通过在显微镜下检查宫颈刮片中的脱落细胞中是否存在异形细胞或可疑癌细胞。该方法简单、快速,但主观性强,检查效率较低。出现可疑细胞后,需进行第二步的检查-阴道镜检查来准确地判断它来自宫颈的什么部位,在阴道镜对图像放大功能的指导下,钳取可疑病变组织作标本。然后第三步就是将钳取的组织送病理活检,进行确诊。这个三步法耗时长、主观性强,而且病理形态改变往往滞后于癌前病变,容易错过早发现的时机。因此,寻找早期、快速鉴别宫颈癌的分子生物学方法迫在眉睫。在本发明中,我们基于宫颈癌变相关基因产物(蛋白、RNA)表达量的改变来诊断宫颈癌,为宫颈癌的大规模筛查提供一种简单、快速、高通量的方法。本发明中,我们通过对宫颈癌基因表达谱的大规模筛查,找到一组与宫颈癌变相关的基因:LGALS7B,ANXA8,CDKN2A,CALML3,PKP1,SERPINB5,SERPINB4,KIAA0101,DSG3,DSC3,GPR87,CLCA2,PITX1,IRF6,FAM83A,AURKB,CKMT1A,MMP13,ALG1L,RHOV,SDC1,TRIM29,LYPD3,FGFBP1,PVRL4,KRT5,KRT6A,KRT7,KRT16,KRT6B,KRT15,S100A2,S100A14,S100A9,S100A8,SFN,SERPINB3,LAD1,FXYD3,TACSTD2,PI3,CXCL17,MAL2,CSTA,PDZK1IP1,LAMB3,RAB25,C19orf33,ELF3,GJB3,SPINT1,PKP3,SERINC2,CENPW,CDH3等55个基因。与正常宫颈组织相比,上述基因在癌变宫颈组织中的表达量极度增高,上述部分基因与宫颈癌的类型、预后等具有紧密联系。上述基因的联合应用有利于宫颈癌的筛查和鉴别。
基于现有技术的研究基础与现状,本申请的发明人拟提供一种用于宫颈癌筛查和鉴别的指标和方法。本发明涉及通过上述基因组(单个或二种及以上任意组合)在宫颈病变组织中的表达量改变,来检测宫颈组织是否发生癌变、癌组织类型、预后等。尤其是通过多种基因联合形成阵列,再与提取、检测宫颈病变组织中蛋白质(如免疫比浊、胶体金、免疫化学发光等)或RNA(如RT-PCR、荧光定量PCR、RNA array、RNA Sequencing等)相结合,可以为宫颈癌的预测、分类、分子分型及预后评估等提供一种快速、高通量、有效、客观的方法。
发明内容
本发明的目的在于为“快速、高效的鉴别宫颈癌、分子分型及预后评估等”提供指标及方法。通过检测与宫颈癌变相关基因的表达谱,来辅助宫颈癌变的鉴别、分子分型及预后评估等的一种分子学方法。
本发明基于大规模基因表达谱的分析,筛选出与正常宫颈组织相比,在宫颈癌组织中表达量显著性增高的一组基因,包括:LGALS7B,ANXA8,CDKN2A,CALML3,PKP1,SERPINB5,SERPINB4,KIAA0101,DSG3,DSC3,GPR87,CLCA2,PITX1,IRF6,FAM83A,AURKB,CKMT1A,MMP13,ALG1L,RHOV,SDC1,TRIM29,LYPD3,FGFBP1,PVRL4,KRT5,KRT6A,KRT7,KRT16,KRT6B,KRT15,S100A2,S100A14,S100A9,S100A8,SFN,SERPINB3,LAD1,FXYD3,TACSTD2,PI3,CXCL17,MAL2,CSTA,PDZK1IP1,LAMB3,RAB25,C19orf33,ELF3,GJB3,SPINT1,PKP3,SERINC2,CENPW,CDH3等55个基因。所述的宫颈癌筛查、鉴别、分子分型及预后评估与上述基因(单个或任意组合)在宫颈组织癌变前后出现表达量改变相关,可用于预测宫颈组织是否发生癌变、分子分型、耐药性、药物筛选、病人预后评估等。
S1单独利用上述基因中“LGALS7B,ANXA8,PKP1,SERPINB5,SERPINB4,KIAA0101,DSC3,GPR87,CKMT1A,RHOV,LYPD3,FGFBP1,PVRL4,KRT6A,KRT6B,KRT15,LAD1,C19orf33,ELF3,GJB3,PKP3,SERINC2,CENPW,CDH3”等表达量改变来判断宫颈是否癌变、癌症分类、分子分型、肿瘤恶性程度、病人预后等;
S2联合上述基因(2个及2个以上的任意组合)表达量改变来检测宫颈癌变、分子分型、肿瘤恶性程度、病人预后等;
S3上述单个基因或任意组合后再与其它指标相结合后,来预测宫颈癌变、癌症分类、分子分型、肿瘤恶性程度、病人预后等;
本发明涉及到检测上述基因表达产物包括:蛋白质和RNA。
本发明涉及到检测的上述基因产物,既包括肿瘤细胞中的,也包括各种原因进入体液中的mRNA、蛋白质以及外泌体中的,包括是完整和片段。
本发明所述检测对象包括:子宫组织、肿瘤组织、体液(包括血液、尿液、穿刺液等)、宫颈脱落细胞、穿刺、宫颈刮片等,作为预测宫颈癌变、肿瘤恶性程度、靶向药物筛选、转移能力、分子分型、生存期等预后评估指标;
本发明所涉及的体液包括血液、尿液、盆腔积液、肿瘤穿刺液、淋巴液等。
本发明所涉及的血液包括全血、血清、血浆、分离有核细胞、血液中的循环肿瘤细胞、外泌体等。
本发明所涉及的上述基因蛋白检测包括直接提取宫颈病变组织蛋白经化学或免疫学方法进行检测和免疫组化分析。
本发明所涉及的上述基因RNA检测包括经直接提取宫颈病变组织RNA后结合RT-PCR技术、荧光定量PCR技术、RNA Sequencing、RNA array等进行检测。
本发明涉及基因产物的检测包括蛋白水平和RNA水平,检测方法涉及:免疫组化、免疫荧光、Western blot、ELISA、流式细胞术检测、RT-PCR、胶体金、免疫学检测、化学法检测、荧光定量PCR、RNA Sequencing、RNA array等检测技术(但不仅限于这些技术)等。
本发明所涉及所述的RT-PCR检测上述基因的RNA产物,逆转录后,涉及到PCR包括:普通PCR、荧光定量PCR、巢式PCR、多重PCR、数字PCR等。
本发明的有益效果是:
微量、快速、高通量是技术发展的大势所趋;病理形态学虽然是诊断宫颈癌的金标准,但近年来病理医生紧缺而且病理诊断耗时长、主观性强;而且病理上的改变往往滞后于分子生物学上的改变。因此,分子生物学指标有望成为肿瘤诊断的重要手段。在本发明中,我们提供了一种基于肿瘤基因组表达谱改变,来对宫颈癌变、分子分型、药物筛选、预后评估等判定的分子学方法。应用上述基因的组合表达谱鉴别宫颈癌与正常宫颈组织时,当特异性大于99%时,灵敏度可达95%以上。另一方面,根据不同需求通过多种肿瘤标志物联合形成阵列,再与提取、检测宫颈病变组织中蛋白质(如免疫比浊、胶体金、免疫化学发光等)或RNA(如RT-PCR、荧光定量PCR、RNA array、RNA Sequencing等)等相结合,可以为宫颈癌的预测、分子分型及预后评估提供一种快速、高通量、有效、客观的方法。
本发明涉及的肿瘤标志物也可用于宫颈癌的疗效评估、药物筛选、耐药监测等。
附图说明
图1.mRNA Sequencing结果的热图显示:本发明涉及的基因LGALS7B,ANXA8,CDKN2A,CALML3,PKP1,SERPINB5,SERPINB4,KIAA0101,DSG3,DSC3,GPR87,CLCA2,PITX1,IRF6,FAM83A,AURKB,CKMT1A,MMP13,ALG1L,RHOV,SDC1,TRIM29,LYPD3,FGFBP1,PVRL4等的mRNA在正常宫颈组织和宫颈癌(CESC)组织中总体表达情况。
图2.mRNA Sequencing结果的热图显示:本发明涉及的基因KRT5,KRT6A,KRT7,KRT16,KRT6B,KRT15,S100A2,S100A14,S100A9,S100A8,SFN,SERPINB3,LAD1,FXYD3,TACSTD2,PI3,CXCL17,MAL2,CSTA,PDZK1IP1,LAMB3,RAB25,C19orf33,ELF3等的mRNA在正常宫颈组织和宫颈癌(CESC)组织中总体表达情况。
图3.mRNA Sequencing结果的热图显示:本发明涉及的基因GJB3,SPINT1,PKP3,SERINC2,CENPW,CDH3等的mRNA在正常宫颈组织和宫颈癌(CESC)组织中总体表达情况。
图4.生存曲线结果显示:SDC1,SPINT1,SERINC2,CDKN2A,SERPINB4,AURKB,CKMT1A等基因的mRNA表达量与病人总体生存时间之间的关系。SDC1,SPINT1,SERINC2等基因的表达量增加提示总体上肿瘤恶性程度较高、病人预后较差;而CDKN2A,SERPINB4,AURKB,CKMT1A等基因表达量较高者肿瘤恶性程度较低、病人预后较好。
图5.mRNA Sequencing结果显示:与正常宫颈组织相比,宫颈癌各亚型在ANXA8,LGALS7B,CALML3,PKP1,CDKN2A,KIAA0101,CKMT1A,KRT7,ELF3等基因中的表达情况。
具体实施方式
实施例1
在本发明中,我们以宫颈癌(Cervical squamous cell carcinoma andendocervical adenocarcinoma,CESC)为实施对象,以RNA Sequencing技术分析宫颈癌与正常宫颈组织中mRNA表达量的差异;以及这些差异与宫颈癌的鉴别、分类及预后等之间的关系为例。我们基于The Cancer Genome Atlas(TCGA)数据库研究中宫颈癌与宫颈正常组织之间基因表达谱差异,筛选出mRNA表达量具有显著性差异的目标基因,包括:LGALS7B,ANXA8,CDKN2A,CALML3,PKP1,SERPINB5,SERPINB4,KIAA0101,DSG3,DSC3,GPR87,CLCA2,PITX1,IRF6,FAM83A,AURKB,CKMT1A,MMP13,ALG1L,RHOV,SDC1,TRIM29,LYPD3,FGFBP1,PVRL4,KRT5,KRT6A,KRT7,KRT16,KRT6B,KRT15,S100A2,S100A14,S100A9,S100A8,SFN,SERPINB3,LAD1,FXYD3,TACSTD2,PI3,CXCL17,MAL2,CSTA,PDZK1IP1,LAMB3,RAB25,C19orf33,ELF3,GJB3,SPINT1,PKP3,SERINC2,CENPW,CDH3等基因。
通过分析TCGA数据库中mRNA Sequencing结果,我们发现:与其对应的正常组织相比,在宫颈癌组织中LGALS7B,ANXA8,CDKN2A,CALML3,PKP1,SERPINB5,SERPINB4,KIAA0101,DSG3,DSC3,GPR87,CLCA2,PITX1,IRF6,FAM83A,AURKB,CKMT1A,MMP13,ALG1L,RHOV,SDC1,TRIM29,LYPD3,FGFBP1,PVRL4,KRT5,KRT6A,KRT7,KRT16,KRT6B,KRT15,S100A2,S100A14,S100A9,S100A8,SFN,SERPINB3,LAD1,FXYD3,TACSTD2,PI3,CXCL17,MAL2,CSTA,PDZK1IP1,LAMB3,RAB25,C19orf33,ELF3,GJB3,SPINT1,PKP3,SERINC2,CENPW,CDH3等基因的总体表达量显著性增高(图1-3)。可根据需要对上述基因进行不同组合形成阵列,以提高检出效率。
通过上述基因的组合,可以提高结宫颈癌鉴别的灵敏度和特异性,显著性提高结宫颈癌的诊断效率。例如,用LGALS7B,ANXA8,CDKN2A,CALML3,PKP1,KRT5,KRT6A,KRT7,KRT16,KRT6B,KRT15,S100A2,S100A14,S100A9基因组合的mRNA表达量差异谱,来区分正常宫颈组织和宫颈癌(CESC)时,特异性大于99%时,灵敏度可达95%以上(图1,2)。
通过对TCGA数据库中mRNA Sequencing结果与病人总体生存曲线间关系的研究,我们发现:SDC1,SPINT1,SERINC2等基因的表达量增加提示总体上肿瘤恶性程度较高、病人预后较差;而CDKN2A,SERPINB4,AURKB,CKMT1A等基因表达量较高者肿瘤恶性程度较低、病人预后较好(图4)。
在本发明中,我们通过对TCGA数据库中mRNA Sequencing结果与宫颈癌组织类型的关系分析,发现:上述基因在不同类型的宫颈癌具有不同的表达谱(图5)。例如:ANXA8,LGALS7B,CALML3,PKP1等基因mRNA表达量增高,只在Cervical squamous cell carcinoma组织型中增高(图5),可用以区分该类型的宫颈癌。
在本发明中,我们通过对TCGA数据库中mRNA Sequencing结果显示:即使同一病理类型的宫颈癌中,也存在着上述基因表达谱的差异。可以依据不同上述基因表达谱的差异来对宫颈癌进行分子分型;例如:ANXA8+/LGALS7B+/CALML3+/PKP1+型;ANXA8-/LGALS7B-/CALML3-/PKP1-型等,将宫颈癌分为不同分子亚型(图1,2)。
小结
我们的结果表明:LGALS7B,ANXA8,CDKN2A,CALML3,PKP1,SERPINB5,SERPINB4,KIAA0101,DSG3,DSC3,GPR87,CLCA2,PITX1,IRF6,FAM83A,AURKB,CKMT1A,MMP13,ALG1L,RHOV,SDC1,TRIM29,LYPD3,FGFBP1,PVRL4,KRT5,KRT6A,KRT7,KRT16,KRT6B,KRT15,S100A2,S100A14,S100A9,S100A8,SFN,SERPINB3,LAD1,FXYD3,TACSTD2,PI3,CXCL17,MAL2,CSTA,PDZK1IP1,LAMB3,RAB25,C19orf33,ELF3,GJB3,SPINT1,PKP3,SERINC2,CENPW,CDH3等55个基因与宫颈癌之间具有紧密联系,上述基因组mRNA表达量的增高可用于宫颈癌的鉴别、分类、分子分型、预后评估等。而且联合应用LGALS7B,ANXA8,CDKN2A,CALML3,PKP1,KRT5,KRT6A,KRT7,KRT16,KRT6B,KRT15,S100A2,S100A14,S100A9基因组合的mRNA表达量差异谱,来区分正常宫颈组织和宫颈癌时,特异性大于99%时,灵敏度达95%以上。提示:本发明提供的基因组为宫颈癌的快速鉴定、分型及预后评估等提供了一个可靠的方法。而且可以根据不同的需求选用不同的上述基因进行组合。
Claims (10)
1.一组宫颈癌(Cervical squamous cell carcinoma and endocervicaladenocarcinoma,CESC)诊断、分子分型及预后评估等的分子生物学指标及应用,所涉及的基因包括:LGALS7B,ANXA8,CDKN2A,CALML3,PKP1,SERPINB5,SERPINB4,KIAA0101,DSG3,DSC3,GPR87,CLCA2,PITX1,IRF6,FAM83A,AURKB,CKMT1A,MMP13,ALG1L,RHOV,SDC1,TRIM29,LYPD3,FGFBP1,PVRL4,KRT5,KRT6A,KRT7,KRT16,KRT6B,KRT15,S100A2,S100A14,S100A9,S100A8,SFN,SERPINB3,LAD1,FXYD3,TACSTD2,PI3,CXCL17,MAL2,CSTA,PDZK1IP1,LAMB3,RAB25,C19orf33,ELF3,GJB3,SPINT1,PKP3,SERINC2,CENPW,CDH3等55个基因,通过检测宫颈病变组织、癌组织、体液(血液、尿液等)、宫颈刮片、脱落细胞等生物样品中上述基因产物(蛋白、RNA)表达量,来诊断宫颈是否发生癌变、癌症分类、分子分型、耐药性、药物筛选、肿瘤恶性程度、病人预后评估等;
S1单独利用上述基因中“LGALS7B,ANXA8,PKP1,SERPINB5,SERPINB4,KIAA0101,DSC3,GPR87,CKMT1A,RHOV,LYPD3,FGFBP1,PVRL4,KRT6A,KRT6B,KRT15,LAD1,C19orf33,ELF3,GJB3,PKP3,SERINC2,CENPW,CDH3”等表达量改变来确定宫颈癌变、癌症分类、分子分型、肿瘤恶性程度、病人预后等;
S2联合上述基因(2个及2个以上的任意组合)表达量改变来检测宫颈癌变、分子分型、肿瘤恶性程度、病人预后等;
S3上述单个基因或任意组合后再与其它指标相结合后,来检测宫颈癌变、癌症分类、分子分型、肿瘤恶性程度、病人预后等。
2.权利要求1所述的用途,其特征在于,所述检测对象包括:子宫组织、肿瘤组织、体液(包括血液、尿液、穿刺液等)、宫颈脱落细胞、穿刺、宫颈刮片等,作为预测宫颈癌变、肿瘤恶性程度、靶向药物筛选、转移能力、分子分型、生存期等预后评估指标。
3.权利要求1所述的用途,其特征在于,所述检测基因表达产物包括:蛋白质和RNA,既包括肿瘤细胞中的,也包括各种原因进入体液中的游离的RNA、蛋白及外泌体中的RNA、蛋白,包括完整和片段(RNA、蛋白)。
4.权利要求1,2所述的用途,其特征在于,所述本发明所涉及的血液包括全血、血清、血浆、分离有核细胞、血液中的循环肿瘤细胞、血液中的外泌体等。
5.权利要求1所述的用途,其特征在于所述的蛋白浓度检测包括直接提取宫颈病变组织蛋白经化学或免疫学方法进行检测和免疫组化分析,所述的RNA浓度检测包括直接提取宫颈病变组织RNA进行检测。
6.权利要求1,3,5所述的用途,其特征在于,所述检测的基因包括:基因表达的检测(包括蛋白水平和RNA水平,检测方法涉及:免疫组化、免疫荧光、Western blot、ELISA、流式细胞术检测、RT-PCR、免疫学检测、胶体金、化学法检测、荧光定量PCR、RNA Sequencing、RNAarray等检测技术,但不仅限于这些技术)等。
7.权利要求1所述的用途,其特征在于,可以直接检测上述基因的绝对量;也可以和正常组织(包括癌旁组织)的比值,来预测宫颈癌变、肿瘤类型、分子分型、恶性程度、转移能力、耐药性、生存期等恶性指标的评估等。
8.权利要求1、6所述的用途,其特征在于所述的RT-PCR检测上述基因的RNA产物,逆转录后,涉及到PCR包括:普通PCR、荧光定量PCR、巢式PCR、多重PCR、数字PCR、芯片微阵列等。
9.权利要求1、5、6、8所述的用途,其特征在于所述的上述基因的表达量包括:和自身内参的比较、和自身内参的比较后再与癌旁组织、和自身内参的比较后再与正常组织相比较等。
10.权利要求1所述的用途,其特征在于通过上述基因的组合应用形成肿瘤标志物矩阵(芯片)模式,以提高宫颈癌预测、分型和预后判断的效率。
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