CN112176066A - 一种宫颈病变早期筛查和诊断的分子标志物及其应用 - Google Patents

一种宫颈病变早期筛查和诊断的分子标志物及其应用 Download PDF

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CN112176066A
CN112176066A CN202011194681.1A CN202011194681A CN112176066A CN 112176066 A CN112176066 A CN 112176066A CN 202011194681 A CN202011194681 A CN 202011194681A CN 112176066 A CN112176066 A CN 112176066A
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杨武林
崔倩文
刘雨
赵加荣
王宏志
戴海明
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Abstract

本发明属于分子生物医学技术领域，具体涉及高迁移率蛋白HMGB3及其基因作为宫颈病变早期阶段的筛查和诊断的分子标志物和该分子标志物的应用。本发明通过在GEO数据库中筛选出宫颈病变各个进展阶段中的差异基因，再根据对差异基因在病变组织中的表达情况作统计，筛选出优先级较高的基因HMGB3作后续验证。验证实验证明，在宫颈上皮CIN1级即宫颈初级病变中，HMGB3的表达阳性率远高于现有诊断方法的阳性率，说明HMGB3作为标志物的灵敏度相对于现有诊断方法有明显优势，可用于宫颈病变的早期检测，筛查与诊断。

Description

一种宫颈病变早期筛查和诊断的分子标志物及其应用
技术领域
本发明属于分子生物医学技术领域，具体涉及高迁移率蛋白HMGB3作为宫颈病变早期阶段的筛查和诊断的分子标志物和该分子标志物的应用。
技术背景
宫颈癌是仅次于乳腺癌的第二大女性高发肿瘤，据统计，全球每年约有20万妇女因宫颈癌去世。且近年来，宫颈癌的发病年龄更趋于年轻化(Haimiti A,Hailiman Y,Gulina A,et al.Reduced expression of members of the mhc-i antigen processingmachinery in ethnic Uighur women with cervical cancer in the Xinjiang regionof China.Curr Oncol 2014；21:67–74)，宫颈癌的早期不易察觉，往往被发现时已经出现了病变甚至已经发展成癌，因此其早期筛查尤为重要。
宫颈癌和宫颈病变产生的主要原因是人乳头瘤病毒(HPV)的感染，主要是HPV高危亚型HPV16和HPV18等的感染。HPV感染在女性中很常见，但大多数为瞬时感染，在两年内可被免疫系统，只有持续性的HPV感染可引起机体内一系列基因结构和功能的改变，主要涉及HPV整合到宿主基因，导致抑癌基因E2的缺失，E6、E7基因的异常表达(王娇.P16、Ki67在宫颈脱落细胞中的诊断意义及在宫颈病变筛查中的价值研究[D].河北北方学院,2016.)。HPV感染引起宫颈癌的发生过程为初始HPV感染-持续性HPV感染-细胞分化-癌前病变(CIN)-原位癌-早期浸润癌。宫颈癌前病变的非典型性增生又称宫颈上皮内瘤变，分为I－III级，包括轻度上皮非典型增生CIN1级，中度非典型增生CIN2级和重度非典型增生CIN3级。CIN1又称低级别上皮内瘤变LSIL，CIN2/CIN3统称高级别上皮内瘤变HSIL。
目前临床上宫颈病变和宫颈癌的筛查方法有多种，HPV的检测是目前临床上用来筛查宫颈病变的重要手段，但HPV RCR检测手段往往会有假阳性的情况，因为HPV的感染是在暂时的，在1～2年内可以已被机体免疫系统清退，不太可能导致恶性转化。P16/Ki67联合标记的筛查也是目前临床上用于诊断宫颈癌前病变和宫颈癌的重要指标。P16(又称p16INK4a),是一种周期依赖性蛋白激酶抑制剂，通过抑制细胞周期依赖性蛋白激酶CPK的活性调节细胞周期。Ki67是一种核增殖标志物，反应细胞增殖活性。P16的表达情况在病理诊断上尤为重要。
但有研究报道，P16在几乎所有的HSIL/CIN2-3均能检测到，但在未成熟的化生和萎缩中则呈阴性或局灶染色，而且，在CIN1级病变中，P16的免疫组化染色情况也是可变的，在一些组织中呈阳性而另一些组织呈阴性。因此，P16作为宫颈低级别上皮内瘤变的标志物价值很低(Sagasta A,Castillo P,Saco A,et al.p16 staining has limited value inpredicting the outcome of histological low-grade squamous intraepitheliallesions of the cervix.Mod Pathol.2016；29(1):51-59.doi:10.1038/modpathol.2015.126)，并且Ki67在组织中的染色情况也是可变的，特异性也不高(SariAslani F,Safaei A,Pourjabali M,et al.Evaluation of Ki67,p16 and CK17 markersin differentiating cervical intraepithelial neoplasia and benign lesions.IranJ Med Sci 2013；38:15–21.)。因此临床上仍亟待发现具有高灵敏度和特异度的宫颈病变早期分子标志物，提高病变检出率，以实现宫颈病变的早期高效可靠的筛查与诊断。
发明内容
本发明的目的之一在于提供一种用于宫颈病变早期阶段筛查与诊断的分子标志物，具体为采用高迁移率蛋白HMGB3或其基因作为分子标志物用于宫颈病变早期阶段的筛查和诊断。
优选的，所述宫颈病变为宫颈上皮非典型增生或恶性增生。
所述宫颈上皮非典型增生的早期阶段为低级别上皮内瘤变。
本发明的目的之二在于提供一种用于宫颈病变早期阶段筛查与诊断的检测产品，所述检测产品利用高迁移率蛋白HMGB3或其基因作为检测标志物。
优选的，所述检测产品为试剂盒。
本发明所述高迁移率蛋白HMGB3包括四个亚型，相对应的，其基因包括四种序列，四个亚型的氨基酸序列表和DNA序列表参见说明书序列表部分。
与现有技术相比，本发明的有益效果在于：
本发明通过结合生物信息学的方法，在GEO数据库中筛选出宫颈病变各个阶段中的差异基因，根据对差异基因在病变组织中的表达情况作统计，筛选出优先级较高的基因HMGB3作后续验证。经免疫组化实验验证表明，高迁移率蛋白HMGB3在正常宫颈上皮中表达呈阴性，在宫颈病变和宫颈癌中的表达阳性。在CIN1级病变中，HMGB3的表达阳性率远高于现有诊断方法中使用的P16的阳性率，说明HMGB3作为标志物的灵敏度相对于现有诊断方法有显著优势，可替代P16用于宫颈病变的早期检测，筛查与诊断。
附图说明
图1是本发明实施例1中48个共同差异表达基因的韦恩图。图中展示在各种宫颈病变类型中的共同上下调差异基因，病变类型包括HSIL、CSCC、CIN1、CIN3等，图1中左侧A表示各组别上调基因，共有34个共同表达上调基因；图1中右侧B表示各组别下调基因，共有14个共同表达下调基因。
图2是本发明实施例2中在Human protein atlas数据库中统计所有差异基因编码蛋白的免疫组化情况图，根据在正常宫颈上皮细胞中HMGB3的染色情况以及在宫颈癌样本中染色的阳性率，按照从高到低的顺序筛选。图2中展示了前10位基因的排序结果。
图3A是本发明实施例3中高迁移率蛋白HMGB3和P16分别在正常宫颈上皮(宫颈炎样本)、CIN1、CIN2、CIN3、以及宫颈鳞状细胞癌组织中的HE染色表达情况，图中HE栏为对照。
图3B是图3A的彩色版本，图中金黄色细胞为阳性染色细胞，从图中可见高迁移率蛋白HMGB3在宫颈病变C1N1级中表达情况高于P16的表达。
图4是本发明实施例3中高迁移率蛋白HMGB3和现有技术中的P16在正常宫颈上皮(宫颈炎样本)、宫颈病变CIN1级、CIN2级、CIN3级、以及宫颈鳞状细胞癌组织(CSCC)中的表达情况统计。可以看出在正常宫颈上皮中高迁移率蛋白HMGB3和P16均无表达，在宫颈病变CIN2级、CIN3级、以及宫颈鳞状细胞癌组织(CSCC)中高表达，而高迁移率蛋白HMGB3在宫颈初级病变CIN1级表达阳性率显著高于P16。
具体实施方式
下面结合实施例对本发明的技术方案做出更为具体的说明，除非另有说明，本文中所使用的术语均具有本领域技术人员常规理解的含义。
实施例1：数据筛选与整合分析
实验方法：GEO数据库是由NCBI(美国国立生物技术信息中心)开发的功能基因组数据公开储存库，在GEO数据库中检索到两个基因表达谱：GSE7803(来自平台GPL96[HG-U133A]Affymetrix Human Genome U133A Array,包含10例正常宫颈鳞状上皮样本，7例HSIL样本和21例CSCC样本)；GSE51993(来自平台Illumina Human v2 MicroRNAexpression beadchip和平台Illumina HumanHT-12V4.0 expression beadchip，每个平台包含9个CIN1级宫颈病变样本，8个CIN3级宫颈病变样本和7个正常的宫颈鳞状上皮样本)。
GEO2R是一种网络分析工具，用以比较GEO数据集中的两组或两组以上样本，以识别出不同样本中的差异基因。利用GEO2R工具分别分析出数据集GSE7803中包含的HSIL、CSCC组和正常宫颈样本之间的差异基因以及数据集GSE51993中包含的CIN1、CIN3组样本与正常宫颈样本间的差异基因。对以上四组(HSIL、CSCC、CIN1、CIN3相对于正常宫颈上皮)的差异基因进行筛选，以|logFC|≧0.5、adjusted P value<0.05为阈值，在CSCC组发现1997个上调差异基因与1744个下调差异基因，HSIL组发现1651个上调和1643个下调差异基因，CIN3组发现388个上调和281个下调差异基因，CIN1组发现227个上调和281个下调差异基因。
Draw Venn Diagram在线网络工具用来分析筛选后的四组差异基因之间的共同差异基因。利用draw venn diagram在线工具，分别对以上四组的上调差异基因和下调差异基因分别取交集。
实验结果：如图1所示，共发现48个共同差异表达基因，其中34个共同上调差异基因和14个共同下调差异基因，这些共同差异表达基因名称在表1、表2中列出。
表一34个共同上调差异表达基因名称
Figure BDA0002753681580000041
Figure BDA0002753681580000051
表二14个共同下调差异表达基因名称
Figure BDA0002753681580000052
实施例2.共表达差异基因在组织中的蛋白表达情况统计
Human Protein Atlas(HPA)在线数据库提供了26000种人类蛋白质在组织和细胞的分布信息，通过免疫印迹、免疫荧光、和免疫组化技术检测每个蛋白在细胞系，正常组织和肿瘤组织中的表达情况。本发明对48个共同差异基因在正常宫颈组织和宫颈癌组织中的蛋白表达情况作统计，按照在正常宫颈上皮组织表达阴性而在宫颈癌组织中表达阳性的标准以筛选出宫颈病变潜在标志物。
为验证34个上调共表达差异基因和14个下调共表达差异基因在不同宫颈组织中的表达，在The human protein atlas蛋白数据库中分别统计实施例1中获得的34个共同上调基因和14个共同下调基因在宫颈组织细胞(正常鳞状上皮细胞、腺上皮细胞、宫颈癌细胞)中的表达情况，按照各个基因在正常宫颈组织和宫颈癌组织中表达的差异程度，以及各个基因在宫颈癌组织中通过抗体染色表达的阳性率高低，给各个基因进行优先级排序。最终在共同差异基因中，选取了前10个通过不同抗体染色显示癌组织阳性率较高而正常组织为阴性的基因。包括：HMGB3、CFB、IDO1、RBP1、SLC16A3等。
如图二所示，基于HPA公共数据库中已有图片的免疫组化结果，HMGB3、CFB、IDO1、RBP1、SLC16A3等在正常鳞状上皮细胞中不表达，而在宫颈癌组织中呈高度或中度表达。其中排在第一位的HMGB3在正常宫颈鳞状上皮细胞不表达，在宫颈癌中呈现阳性率超过90％的中度至高度表达。
实施例3.组织样本筛选与免疫组化验证
1.组织的HE染色和免疫组化实验
实验方法：选取中国科学院合肥肿瘤医院病理科近年来病人组织活检的组织蜡块，这些蜡块由病人新鲜组织经过脱水、透明、浸蜡、包埋处理。包括宫颈炎(6例)、宫颈CIN1级病变(15例)、CIN2级病变(8例)、CIN3级病变(5例)，以及宫颈癌样本(5例)。
S1.将各组织蜡块切成2μm厚的组织切片，附于免疫组化专用的黏附载玻片，60度过夜烤片。
S2.脱蜡及水化：烤好的组织切片依次放入二甲苯Ⅰ(10min)→二甲苯Ⅱ(10min)→100％乙醇5min→95％乙醇5min→90％乙醇5min→80％乙醇5min→流水冲洗10min。
S3.抗原修复：PBS冲洗脱蜡及水化后的组织切片3min×3遍，再置于柠檬酸中高压蒸汽90s，流水冲洗降温。
S4.封闭：降温后的组织切片用3％过氧化氢浸泡10min，使内源性过氧化物酶猝灭→PBS冲洗5min×3遍，PBST冲洗5min→10％山羊血清室温封闭1h，以封闭组织中的非特异抗原，减少非特异染色。
S5.一抗孵育：PBST冲洗5min×3遍，一抗室温孵育3h(美国BOSTER生物工程有限公司的Anti-HMGB3兔多克隆抗体，稀释比1:300)或4℃过夜孵育(ATLAS ANTIBODIES公司的Anti-HMGB3兔多克隆抗体，稀释比1:1000)。
S6.二抗孵育：PBST冲洗5min×3遍，加入二抗(迈新生物技术公司的TM HRP-Polymer anti-Mouse/Rabbit免疫组化试剂盒)室温孵育30min。
S7.DAB显色和苏木素(HE)复染：PBS冲洗5min×3遍→滴加DAB显色液显色2min→PBS冲洗→苏木素复染1min→流水返蓝10min。
S8.脱水透明：75％乙醇浸泡5min→85％乙醇浸泡5min→95％乙醇5min→无水乙醇5min→二甲苯Ⅰ10min→二甲苯Ⅱ10min。
S9.中性树脂封片。
2.实验结果：HMGB3作为一个特异性和灵敏性较高的宫颈病变早期阶段分子标志物的验证
根据前述1中的组织处理过程和免疫组化染色检测各组织样本中的HMGB3的表达情况。
如图3所示，实验结果表明，HMGB3在宫颈炎中的表达阳性率为0(0/6)、CIN1中表达的阳性率为93.3％(14/15)、CIN2中表达的阳性率为100％(8/8)、CIN3中表达的阳性率为100％(5/5)、宫颈癌中表达的阳性率为100％(5/5)。而在这些组织样本中，同时用免疫组化法检测P16表达的阳性率为宫颈炎中0(0/6)、CIN1中66.7％(10/15)、CIN2中100％(8/8)、CIN3中100％(5/5)、宫颈癌中100％(5/5)。
以上结果显示，HMGB3在正常宫颈上皮中无阳性表达，而在宫颈病变上皮组织和宫颈癌中均稳定表达，表明HMGB3可作为宫颈病变及宫颈癌的进展性标志物。特别是在低级别上皮内瘤变中，如图4所示，HMGB3的表达阳性率为93.3％，P16的阳性率66.7％，表明以HMGB3作为标志物检出低级别上皮内瘤变的灵敏度显著高于P16染色，HMGB3可应用于宫颈病变和宫颈癌的检测中。
上述实施例中筛选的HMGB3即高迁移率蛋白HMGB3，又称HMG-4和HMG-2a，是一种约23kDa的核蛋白。它属于一个蛋白质家族的成员，该家族包含一个或多个高迁移率组DNA结合序列(HMG box)。人的HMGB3的主要亚型1被合成为200个氨基酸(aa)前体，该前体被去甲基化产生199aa成熟链。该蛋白含有两个HMG box DNA结合区域和一个酸性的富含Asp/glu的区域。人HMGB3是一种在不同细胞内具有不同作用的多功能蛋白，本发明中高迁移率蛋白HMGB3在宫颈病变早期阶段的筛查和诊断中的应用属于首次提出。
本发明中高迁移率蛋白HMGB3及其基因在宫颈病变早期阶段的筛查和诊断中具有突出优势，但应当理解，根据实验结果，其在宫颈病变的宫颈上皮非典型增生的全部阶段和宫颈癌的筛查与诊断中同样具有检测效力，倘若出现一种利用高迁移率蛋白HMGB3或其基因作为宫颈上皮非典型增生任一阶段或宫颈癌的筛查与诊断的分子标志物的应用，同样视为本发明创造的精神和原则之内。
以上实施方式仅用以说明本发明的技术方案，而并非对本发明的限制；尽管参照前述实施方式对本发明进行了详细的说明，本领域的普通技术人员应当理解：凡在本发明创造的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明创造的保护范围之内。
Figure BDA0002753681580000081
Figure BDA0002753681580000091
Figure BDA0002753681580000101
Figure BDA0002753681580000111
Figure BDA0002753681580000121
Figure BDA0002753681580000131
Figure BDA0002753681580000141
Figure BDA0002753681580000151
序列表
<110> 中国科学院合肥物质科学研究院/中国科学院合肥肿瘤医院
<120> 一种宫颈病变早期筛查和诊断的分子标志物及其应用
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Claims (6)

1.高迁移率蛋白HMGB3作为宫颈病变早期阶段的筛查与诊断的分子标志物的应用。
2.根据权利要求1所述的应用，其特征在于，所述宫颈病变为宫颈上皮非典型增生或恶性增生。
3.高迁移率蛋白HMGB3的基因作为宫颈病变早期阶段的筛查与诊断的分子标志物的应用。
4.根据权利要求3所述的应用，其特征在于，所述宫颈病变为宫颈上皮非典型增生或恶性增生。
5.高迁移率蛋白HMGB3作为分子标志物在制备宫颈病变早期阶段的筛查与诊断的检测产品中的应用。
6.高迁移率蛋白HMGB3的基因作为分子标志物在制备宫颈病变早期阶段的筛查与诊断的检测产品中的应用。
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