CN117144014A - 一种用于乳腺癌转移风险监测和评估的分子标志物及其应用 - Google Patents
一种用于乳腺癌转移风险监测和评估的分子标志物及其应用 Download PDFInfo
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Abstract
本发明公开了一种用于乳腺癌转移风险监测和评估的分子标志物及其应用，属于医学检测技术领域。本发明通过对临床患者的乳腺癌组织进行测序，发现GAT1在转移性乳腺癌组织中低表达，且通过TCGA数据库分析及乳腺癌旁与转移性乳腺癌组织标本免疫组化证明GAT1可作为乳腺癌转移检测的分子标志物，通过H‑score值进行评分，评估乳腺癌将发生转移的风险，若患者GAT1蛋白H‑score值低于150时，则显示乳腺癌将发生转移风险。本发明可有效检测乳腺癌转移情况，能够延长这部分乳腺癌患者的生存期。
Description
技术领域
本发明属于医学检测技术领域，具体涉及一种用于乳腺癌转移风险监测和评估的分子标志物及其应用。
背景技术
乳腺癌是女性最常见的恶性肿瘤，占女性癌症死亡第二位，其发病率呈逐年上升和年轻化趋势，严重影响女性健康。约25-30％乳腺癌患者在治疗晚期出现耐药、转移等情况，患者预后较差。目前，临床上确认肿瘤转移的指标仍是通过影像学检测联合病理检测，但是通常在影像学(乳房x线照相、超声检查、乳腺MRI检查)上监测到时，肿瘤已经发生转移。肿瘤标志物(生物标志物)是肿瘤产生的或身体对肿瘤起反应而产生的蛋白质，可追踪转移性乳腺癌的进展。
目前临床上常见的肿瘤标志物有：肿瘤抗原15-3(CA 15-3)：乳腺肿瘤细胞生成的蛋白质，50％-90％的转移性乳腺癌患者会升高，携带它的早期乳腺癌患者只有30％，在骨或肝转移患者身上，它的水平一般会很高。这也是肿瘤临床疗效评价指标，但肿瘤对治疗产生应答或进展后要等数周(通常四到六周)才会看到它的水平变化。肿瘤抗原27.29(CA27.29)：MUC-1基因生成的蛋白质(单克隆抗体)，它位于乳腺肿瘤细胞表面，与其他标志物相比，这是唯一能表明乳腺肿瘤细胞存在的蛋白质。但目前并不能用于监测早期乳腺癌女性患者的复发情况，有人认为在患者意识到癌症复发之前五个月左右，它的水平可能升高。如同CA 15-3，它的水平升高可能是乳腺癌以外的原因导致的，在癌症得到有效的治疗后它的水平仍保持高位很长一段时间(两到三个月)。因此，深入研究乳腺癌发生、发展的新型分子机制，发现新的生物分子标志物，预测乳腺癌患者是否会发生转移，及时监测乳腺癌是否发生转移，改善临床现有的治疗对于攻克乳腺癌治疗尤为重要。
GABA转运体1(GAT1)负责将突触间隙的GABA回收至突触前膜神经元，从而调控下游受体的激活状态，GAT1维持着神经系统抑制微环路的稳定，其功能异常会导致多种神经类疾病，比如癫痫、精神分裂症，GAT1是重要的抗癫痫靶点，通过阻断GAT1的功能可以抑制突触间隙中GABA的清除，进而减少神经冲动以减轻癫痫症状。然而，将GAT1用于乳腺癌转移风险监测和评估方面的作用尚未见任何的报道。
发明内容
针对现有临床上治疗晚期乳腺癌患者诊治过程中，对于乳腺癌是否发生转移以及乳腺癌转移检查和预警，缺乏特异性靶点及规范的治疗措施，导致乳腺癌转移的患者发现时已经处于晚期，治疗效果较差等技术问题，本发明的目的是提供了一种用于乳腺癌转移风险监测和评估的分子标志物及其应用。
本发明通过对临床患者的乳腺癌组织进行测序，发现GAT1在转移性乳腺癌组织中低表达，且通过TCGA数据库分析及乳腺癌旁与转移性乳腺癌组织标本免疫组化证明GAT1可作为乳腺癌转移检测的分子标志物，可有效检测乳腺癌转移情况，能够延长这部分乳腺癌患者的生存期。
本发明目的是通过以下方式实现：
本发明提供一种用于乳腺癌转移风险监测和评估的分子标志物，所述的分子标志物为GAT1。
本发明还提供上述的分子标志物在制备乳腺癌转移风险监测和评估试剂或试剂盒中的应用。
基于上述技术方案，进一步地，所述乳腺癌包括三阴乳腺癌和非三阴乳腺癌。
基于上述技术方案，进一步地，所述的GAT1通过检测GAT1的mRNA和/或GAT1蛋白的试剂进行检测。
基于上述技术方案，进一步地，检测GAT1蛋白的方法包括酶联免疫吸附、免疫组织化学和蛋白质免疫印迹。
基于上述技术方案，进一步地，检测GAT1的mRNA的方法包括实时荧光定量PCR。
基于上述技术方案，进一步地，所述检测的病理样本为乳腺癌病理组织。
基于上述技术方案，进一步地，通过H-score值进行评分，评估乳腺癌将发生转移的风险。
基于上述技术方案，进一步地，若患者GAT1蛋白H-score值低于150时，则显示乳腺癌将发生转移风险。
本发明相对于现有技术具有的有益效果如下：
1.本发明通过对乳腺癌患者的病理组织标本进行检测，发现GAT1可作为乳腺癌转移的分子标志物，在临床检测中，只需要对患者的组织进行免疫组织化学染色，即可对人群进行区分，操作简单易行，可弥补传统TNM临床分型的不足，能有效提高这部分乳腺癌患者的治疗效果，具有巨大的临床应用潜力。
2.目前在临床上已经收集并跟踪了500余例乳腺癌患者，本发明的分子标志物对乳腺癌转移风险预测效果良好，根据预测结果及时给与药物治疗，能够预防后期发生转移，将有效延长乳腺癌患者的生存期。
附图说明
为了更清楚地说明本发明实施例，下面将对实施例涉及的附图进行简单地介绍。
图1为实施例1中统计4000余例乳腺癌患者GAT1的表达检测结果与转移性乳腺癌患者预后的关系图。
图2为转移性乳腺癌患者和非转移性乳腺癌患者GAT1的表达情况对比图，其中，A为未转移乳腺癌患者和发生转移乳腺癌患者中的GAT1表达量统计图，B为GAT1在不同病理期乳腺癌中的表达量对比图。
图3为细胞层面验证GAT1对乳腺癌细胞的增殖能力结果。
图4为细胞层面验证GAT1对乳腺癌细胞的转移能力结果。
图5为临床组织标本检测GAT1表达与乳腺癌转移的验证。
具体实施方式
下面结合实施例对本发明进行详细的说明，但本发明的实施方式不限于此，显而易见地，下面描述中的实施例仅是本发明的部分实施例，对于本领域技术人员来讲，在不付出创造性劳动性的前提下，获得其他的类似的实施例均落入本发明的保护范围。
实施例1
GAT1与转移性乳腺癌患者的预后的情况：通过对4000余例乳腺癌患者GAT1的表达情况进行检测，并收集患者的预后信息，具体检测过程如下：
利用癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库中的转移性乳腺癌患者转录组数据和临床相关数据，结合有效的生物信息学分析方法，根据Kaplan-Meier法得到GAT 1与转移性乳腺癌患者预后的关系。
结果如图1所示，统计证明转移性乳腺癌患者的乳腺癌病理组织中GAT1表达较低的患者生存时间更短，预后较差，P值小于0.05，具有统计学差异。
实施例2
GAT1与乳腺癌不同病理分期患者的预后的情况：通过对上述4000余例乳腺癌患者进行分层分析，结果如图2所示，统计不同疾病进展患者的GAT1的表达情况，证明随着乳腺癌疾病进展，发生转移后(Stage2、Stage3、Stage4)的患者，检测乳腺癌病理组织中GAT1表达水平，随着转移发生，GAT1表达更低，生存时间更长，P值小于0.05，具有统计学差异。
实施例3
细胞层面验证GAT1对乳腺癌细胞的增殖能力：通过转染siRNA，在TNBC细胞MDA-MB-231和MCF7中使GAT1低表达，考察GAT1低表达对乳腺癌细胞的增殖能力的影响，具体过程如下：
转染前一天铺板，一个6孔板的单孔对应一个板，具体根据细胞量调整，细胞汇合70％-90％，将400μL无血清培养基分为两份，分别加入5μL siRNA(siRNA-1或siRNA-2)和5μL lipo2000；静置五分钟后混合均匀，将混合物静置20分钟后加入已经种好的培养板，并于6-8h后换成正常的培养基，孵育24h后消化重悬细胞，铺于96孔板用于后续实验检测。
siRNA-1序列(5'-3')：GCUAUCAUUCUGGCUGAACAUTT；
siRNA-2序列(5'-3')：CUUCUACAUCACACCCAACUUTT。
通过转染Flag-GAT1过表达质粒使乳腺癌细胞MDA-MB-231和MCF7中的GAT1高表达，考察GAT1高表达对乳腺癌细胞的增殖能力的影响，具体过程如下：
转染前一天每孔0.5-2×105个细胞接种于6孔板单孔中，添加1500μL完全培养基中，在转染时细胞可长至90-95％汇合度。转染体系配制，每孔细胞用量如下：A.用125μL无血清培养基稀释2ug Flag-GAT1过表达质粒DNA，轻轻混匀。B.取6μL的PEI转染试剂在125μL无血清培养基中稀释，室温静置孵育5min。C.将上述稀释配好的质粒DNA和PEI混合，轻轻混匀，室温放置15min。在每孔细胞中加入混合后的转染液，轻轻摇匀。贴壁细胞可在转染4-6h后更换成完全培养基，孵育24h后消化重悬细胞，铺于96孔板用于后续实验检测。
Flag-GAT1的载体为pLVX-IRES-Puro-3xFlag，Flag-GAT1质粒的构建过程如下：首先根据Ensembl网站上查询到GAT1基因信息，确定表达载体的酶切位点，设计正向和反向引物进行PCR扩增，并用同源重组法进行质粒连接。取5μL重组产物加入到50μL感受态细胞中，冰上静置30min。.42℃水浴热激45sec后，立即置于冰上冷却5-10min。加入500μL LB培养基，37℃摇菌，弃上清。用剩余培养基(约200μL)将菌体重悬，取100μL用无菌涂布棒在含有正确抗性的平板上轻轻涂匀，37℃培养箱中倒置培养12-16h。用小枪头挑取重组反应转化平板上若干个单克隆菌落，溶于20μL ddH2O中，取1μL作为模板进行菌落PCR鉴定，选择菌落PCR鉴定为阳性的菌落，将剩余菌液接种至含有适当抗生素的3mL液体LB培养基中摇菌6-8小时，再从3mL菌液中取1mL，接种至含有适当抗生素的30mL(后续中提)/200mL(后续大提)液体LB培养基中扩大摇菌12-14小时，取1mL新鲜菌液于1.5mL离心管中，封口膜密封后标记送测序，测序完成后进行序列比对，序列正确无突变进行质粒提取。
质粒提取：取30mL过夜培养的菌液加入离心管中，尽量吸除上清，根据天根质粒中提试剂盒说明书进行质粒提取，并检测质粒浓度，提取的质粒可在细胞房4℃保存。
乳腺癌细胞的增殖能力通过CCK8实验检测，具体过程如下：
观察细胞的生长情况，选择处于对数生长期的细胞，弃去上清，胰酶消化，800rpm离心5min，添加完全培养基吹打混匀细胞沉淀，制成单细胞悬液，使用细胞计数板计数细胞数量，取5000个/孔接种于96孔板中，放入恒温孵箱中培养，每组设置3个复孔；按需培养相应的时间后，更换为CCK8溶液，孵育1-2h后，于450nm处测吸光值，考虑细胞增殖能力。
结果如图3所示，图中GAT1-SI1/SI2代表在细胞内特异性敲低GAT1基因的表达，NC为对照，Flag-GAT1代表在细胞内过表达GAT1基因，EV为对照。结果证明敲低乳腺癌细胞中GAT1基因，通过CCK8实验检测乳腺癌细胞的增殖能力(若乳腺癌细胞活性高，则450nm OD值较高)，结果发现敲低GAT1(GAT1-SI1、GAT1-SI2)能够有效促进乳腺癌细胞的增殖能力；使乳腺癌细胞中GAT1基因过表达(Flag-GAT1)，能够明显抑制乳腺癌细胞的增殖能力。
实施例4
细胞层面验证GAT1对乳腺癌细胞的转移能力：通过Transwell实验验证GAT1对乳腺癌细胞的转移能力，选用实施例3中的高表达GAT1的MDA-MB-231和MCF7，具体过程如下：
取出基质胶融化为液体状态，加入无血清培养基吹打混匀，在上室加入70μL混合物放置于24孔板中，等待基质胶凝固成胶状。消化细胞，用不含胎牛血清的培养基，每个孔加入8*104-105个细胞，在24孔板中(下室)孵育48小时后，使用棉签轻轻蘸去上室残留的细胞悬液，防止刮伤到膜上的细胞。PBS清洗小室，加入甲醇，固定15min，PBS冲洗，加入600μL结晶紫，显微镜拍摄，计数穿膜细胞数。
结果如图4所示，结果显示使乳腺癌细胞中GAT1基因过表达(Flag-GAT1)，能明显抑制乳腺癌细胞MDA-MB-231和MCF7的转移能力(侵袭、迁移能力)。
实施例5
临床组织标本检测GAT1表达与乳腺癌转移的验证：对来院治疗的500余例乳腺癌患者进行局部病理组织的穿刺活检，通过免疫组化检测GAT1蛋白表达情况，用GAT1抗体(Proteintech，1：1000稀释)，通过H-score值进行评分，考察临床组织标本中GAT1的低表达与乳腺癌转移的对应关系。
免疫组化方法具体过程如下：取部分乳腺癌患者的病理组织，切片置于二甲苯浸泡2次(20min/次)—无水乙醇2次(5min/次)—75％乙醇1次(5min/次)—蒸馏水冲洗；将切片浸入枸橼酸缓冲液，微波加热至沸腾，冷却(约5-10min)，反复两次；室温自然冷却，PBS洗涤，5％ BSA封闭，室温20min，甩去多余液体；滴加一抗(GAT1)，4℃过夜，PBS清洗3次，滴加二抗，置于室温孵育15-30min，滴加SABC，室温30min，滴加显色剂，苏木精复染，梯度酒精脱水处理，封片，显微镜下观察，并进行H-score值计算。
组织化学评分(histochemistry score)即H-SCORE，处理免疫组化结果的一种组织学评分方法，将每张切片内阳性的细胞数量及其染色强度转化为相应的数值，达到对组织染色半定量的目的。
部分患者的病理组织检测结果如图5所示，结果表明若患者GAT1蛋白H-score值降低至150时，则显示乳腺癌将发生转移风险，预测率接近100％。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例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：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (9)
1.一种用于乳腺癌转移风险监测和评估的分子标志物，其特征在于，所述的分子标志物为GAT1。
2.权利要求1所述的分子标志物在制备乳腺癌转移风险监测和评估试剂或试剂盒中的应用。
3.根据权利要求2所述的应用，其特征在于，所述乳腺癌包括三阴乳腺癌和非三阴乳腺癌。
4.根据权利要求2所述的应用，其特征在于，所述的GAT1通过检测GAT1的mRNA和/或GAT1蛋白的试剂进行检测。
5.根据权利要求4所述的应用，其特征在于，检测GAT1蛋白的方法包括酶联免疫吸附、免疫组织化学和蛋白质免疫印迹。
6.根据权利要求4所述的应用，其特征在于，检测GAT1的mRNA的方法包括实时荧光定量PCR。
7.根据权利要求2所述的应用，其特征在于，所述检测的病理样本为乳腺癌病理组织。
8.根据权利要求2所述的应用，其特征在于，通过H-score值进行评分，评估乳腺癌将发生转移的风险。
9.根据权利要求8所述的应用，其特征在于，若患者GAT1蛋白H-score值低于150时，则显示乳腺癌将发生转移风险。
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| US20160078167A1 (en) * | 2013-05-28 | 2016-03-17 | The University Of Chicago | Prognostic and predictive breast cancer signature |
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