CN114561469B - Er阳性乳腺癌分子标志物及其应用 - Google Patents
Er阳性乳腺癌分子标志物及其应用 Download PDFInfo
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Abstract
本发明属于生物技术领域，尤其涉及ER阳性乳腺癌分子标志物及其应用。所述分子标志物包括以下任一种或其组合：1）RBCK1基因，其具有SEQ ID NO.1所示的DNA序列；2）RBCK1基因的表达产物。本发明运用RT‑PCR、q‑PCR、Western Blot、划痕实验和Transwell小室模型等现代分子生物学技术，在细胞水平研究干扰RBCK1基因后对ER阳性乳腺癌细胞迁移能力的影响，首次发现了RBCK1基因表达与ER阳性乳腺癌细胞系迁移能力有关，ER阳性乳腺癌细胞迁移能力在一定程度上可反映ER阳性乳腺癌患者发生转移的可能性大小，为ER阳性乳腺癌的治疗提供了新的思路和新的方案。
Description
技术领域
本发明属于生物技术领域，尤其涉及ER阳性乳腺癌分子标志物及其应用。
背景技术
乳腺癌是世界范围内发病率第一位的女性恶性肿瘤。根据2018年全球流行病学数据统计，乳腺癌占女性肿瘤发病率的24.2％；占肿瘤相关死亡的15.1％。乳腺癌的发病与年龄>50岁、绝经晚、月经初潮早、胸部放疗史、乳腺癌家族史、己烯雌酚服药史、酒精摄入等因素有关。此外，乳腺小叶不典型增生和乳腺上皮内瘤变也是乳腺癌的癌前病变。少部分乳腺癌有家族遗传倾向，如BRCA1、BRCA2与ATM等基因突变可以显著增加乳腺癌的发病概率。
雌激素受体(ER)阳性/人表皮生长因子受体2(HER2)阴性乳腺癌是最常见的乳腺癌亚型，占年龄＜50岁患者的65％，占老年患者的75％。雌激素与ER结合后会激活下游的信号通路，最终促进肿瘤细胞生长与增殖。激素治疗为基础的方案可减少ER+乳腺癌患者雌激素的生成、阻断ER信号通路、降解ER或改变ER调控的信号通路或增殖通路。由于ER阳性乳腺癌的复发潜伏期较长，受到多种因素的影响，因此对于其远期复发的预测一直是一个难题。
含RANBP2型和C3HC4型锌指的蛋白(RBCK1)是一个58kDa的蛋白，包含N端泛素样(UBL)域，Npl4型锌指(NZF)域和催化碳终端RBR域,是一种泛素蛋白。Oncomine数据库确定了RBCK1在乳腺癌中的mRNA表达高于正常乳腺组织，且与ERα阴性乳腺癌组织相比，ERα阳性乳腺癌组织中RBCK1 mRNA水平升高。有报道称，RBCK1对ERα的招募与乳腺癌细胞的表达、雌激素信号和细胞增殖呈正相关。RBCK1也被认为通过负性调节TAB2/TAB3并靶向它们进行蛋白酶体降解，从而抑制NF-κB的激活。由于NF-κB调节了500多个涉及炎症、细胞转化、存活、增殖、血管生成、侵袭和转移的基因，因此在乳腺癌中经常观察到异常NF-κB活化是癌症发生的关键。因此RBCK1通过在乳腺癌中的各种调节机制，从而对雌激素受体信号传导、细胞增殖和内分泌治疗反应具有重要意义。
发明内容
针对现有技术存在的问题，本发明提供了ER阳性乳腺癌分子标志物及其应用。
本发明的第一个方面，提供ER阳性乳腺癌分子标志物，所述分子标志物包括以下任一种或其组合：
1)RBCK1基因，其具有SEQ ID NO.1所示的DNA序列；
2)RBCK1基因的表达产物。
进一步的，所述RBCK1基因的表达产物包括RBCK1 mRNA和/或RBCK1蛋白。
更进一步的，所述RBCK1蛋白具有SEQ ID NO.2所示的氨基酸序列。
本发明的第二个方面，提供所述ER阳性乳腺癌分子标志物的检测试剂在制备诊断ER阳性乳腺癌的工具中应用。
进一步的，所述检测试剂包括特异扩增RBCK1基因的引物，所述特异扩增RBCK1基因的引物序列如SEQ ID NO.7和SEQ ID NO.8所示。
进一步的，所述检测试剂包括RBCK1蛋白的免疫检测产品，所述RBCK1蛋白的免疫检测产品包括与RBCK1蛋白特异性结合的抗体。
本发明的第三个方面，提供一种筛选抑制ER乳腺癌细胞迁徙的候选药物的方法，所述方法包括：
用待测物质处理表达所述ER阳性乳腺癌分子标志物的体系；和
检测体系中所述ER阳性乳腺癌分子标志物的表达水平；
其中，若所述待筛选物质可以降低所述ER阳性乳腺癌分子标志物，则表明该待筛选物质是抑制ER乳腺癌细胞迁徙的候选药物。
本发明的第四个方面，提供所述ER阳性乳腺癌分子标志物的抑制剂在制备抑制ER乳腺癌细胞迁徙的药物中的应用。
进一步的，所述抑制剂包括靶向RBCK1基因的siRNA。
更进一步的，所述siRNA的序列如SEQ ID NO.3或SEQ ID NO.5所示。
本发明具有如下有益效果：
本发明中通过迁移和侵袭实验发现，将RBCK1基因沉默后ER阳性乳腺癌细胞系的迁移和侵袭能力明显减弱。研究ER阳性乳腺癌发生、发展以及转移过程的分子机制，可为ER阳性乳腺癌患者的治疗提供新的思路。因此RBCK1基因可作为一种检测ER阳性乳腺癌迁移和侵袭能力的一种分子标志物。
与现有技术相比，本发明运用RT-PCR、q-PCR、Western Blot、划痕实验和Transwell小室模型等现代分子生物学技术，成功构建了靶向RBCK1基因干扰siRNA，在细胞水平研究干扰RBCK1基因后对ER阳性乳腺癌细胞迁移能力的影响，首次发现了RBCK1基因表达与ER阳性乳腺癌细胞系迁移能力有关，ER阳性乳腺癌细胞迁移能力在一定程度上可反映ER阳性乳腺癌患者发生转移的可能性大小，为ER阳性乳腺癌的治疗提供了新的思路和新的方案。
附图说明
图1是本发明实施例1中检测干扰siRNA在蛋白水平上对RBCK1基因的沉默效果。
图2是本发明实施例2中检测干扰siRNA在mRNA水平上对RBCK1基因的沉默效果。
图3是本发明实施例3中利用划痕实验检测不同组ER阳性乳腺癌细胞系迁移能力的影响结果图，图A为培养不同时间的细胞迁移形态，图B为伤口愈合程度统计。
图4是本发明实施例4中利用Transwell小室模型实验检测不同组ER阳性乳腺癌细胞系迁移和侵袭能力的影响结果图，图A为培养不同时间细胞侵袭形态，图B为侵袭细胞数统计。
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，但不应理解为本发明的限制。如未特殊说明，下述实施例中所用的技术手段为本领域技术人员所熟知的常规手段，下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
本发明披露了ER阳性乳腺癌分子标志物及其应用。本发明中所涉及的RBCK1基因的编码序列具有SEQ ID NO.1所示的DNA序列，其中，该序列后三个碱基不翻译氨基酸；RBCK1蛋白的氨基酸序列为SEQ ID NO.2所示的序列；ER阳性乳腺癌细胞系为T47D细胞株，购于ATCC；RBCK1抗体购自proteintech；细胞蛋白的提取按配制好的1×loading buffer进行，Western blot方法检测蛋白表达；实时定量PCR方法参照Trizol说明书提取细胞总RNA，测浓度后，按TaKaRa公司PrimeScript RT Master Mix试剂盒进行反转录，按Appliedbiosystems公司SYBR Select Master Mix试剂盒检测相关基因表达(36B4为内参)；RBCK1-siRNA及其末端悬垂直接由sigma公司购买，具体序列见表1：
表1 RBCK1-siRNA及其末端悬垂序列
| siRBCK1#1 | 5'–GCCUCAGCUACCAUGCATT-3'，如SEQ ID NO.3所示 |
| dTdT | 5'-UGCAAUGGUAGCUGAAGGCTT-3'，如SEQ ID NO.4所示 |
| siRBCK1#2 | 5'-CACACCUUCUGCAGGGAGUTT-3'，如SEQ ID NO.5所示 |
| dTdT | 5'-ACUCCCUGCAGAAGGUGUGTT-3'，如SEQ ID NO.6所示 |
转染方法按Invitrogen公司Lipofectamine RNAiMAX Reagent转染试剂说明书进行，q-PCR及Western blot方法检测表达及干扰效果；细胞迁移实验Transwell(8mm孔径)小室购自Cosar公司，事先进行饥饿培养；细胞划痕实验所使用12孔板购自Cosar公司。
实施例1：细胞培养、转染及蛋白免疫印迹法(Western Blot)检测细胞株中RBCK1蛋白的干扰效果
1、细胞复苏：从-80℃冰箱取出T47D细胞，立即放入37℃水浴箱中快速融化，使细胞在最短时间内完全解冻。用75％酒精擦拭消毒冻存管表面后，转移至事先准备好的5mLEP管(已事先加入1mL DMEM+10％FBS)中，1000rpm离心5min。弃上清，用1mL DMEM完全培养基(DMEM+10％FBS)重悬，混匀，取5mL DMEM完全培养基(DMEM+10％FBS)加入培养瓶中，将重悬细胞悬液转移至培养瓶，放于37℃，5％CO2培养箱中培养，次日更换一次培养基，继续培养，待细胞密度80％-90％左右时，开始传代。
2、细胞传代培养：将细胞密度约80％-90％的培养瓶中的旧培养液弃去，用预热的PBS冲洗1-2次，再向培养瓶内加入胰蛋白酶消化液(0.25％胰酶+0.02％EDTA)1mL，37℃消化2-3min，显微镜下观察细胞状态，当细胞体积缩小变圆，间隙变大时，加入1mL DMEM完全培养基(DMEM+10％FBS)终止消化，用移液枪轻柔反复吹打瓶底。将吹打好的细胞悬液收集至5mL离心管内以每分钟1000rpm，离心5min，弃去上清，用1mL DMEM完全培养基(DMEM+10％FBS)重悬，混匀，取5mL DMEM完全培养基(DMEM+10％FBS)加入新的培养瓶中，将适量重悬细胞悬液转移至培养瓶，放于37℃，5％CO2培养箱中培养。待细胞达80％-90％密度后，按上述传代方法将F2细胞接种到12孔板培养。
3、细胞转染：12孔板细胞密度约30％-50％时，将si RBCK1#1和si RBCK1#2作为实验组，si-control作为对照组，取4个EP管，标记A1、B1、A2、B2，在A1中加入50μL减血清培养基(Opti-MEM)及2μL si-RBCK1#1(购自sigma)，在B1中加入50μL减血清培养基(Opti-MEM)及2ul Lipofectamine RNAiMAX Reagent(购自Invitrogen公司)，在A2中加入50μL无血清培养基(Opti-MEM)及2μL si-RBCK1#2(购自sigma)，静置5分钟，将B液分别加入A液中，轻柔混匀，室温静置15-20分钟；将复合物按顺序分别加入上述步骤中已接种培养细胞的12孔板的培养基中，每孔100μL，放于37℃，5％CO2培养箱中继续培养，转染后4-6h换液，继续培养24或48h后，进行转染后的其它检测步骤。
4、Western Blot
(1)总蛋白的提取：将转染后24h的12孔板的细胞中旧培养基弃去，加入1×loading buffer 100uL，用减去枪头尖的200ul均匀刮每个孔，将得到的蛋白液移至标记好的1.5mL离心管中，100℃金属浴中煮10min，-80℃保存备用。
(2)配聚丙烯酰胺凝胶：根据蛋白分子量大小配制10％的聚丙烯酰胺凝胶。清洗晾干两块用于配胶的1.5mm玻璃板，然后对齐放入夹中卡紧，垂直卡在架子上，按说明书准备配胶试剂，先加分离胶，先快后慢，避免产生气泡，加至距上端1.5cm处时加无水乙醇密封，静置20分钟后弃去无水乙醇，用离子水清洗3次后加浓缩胶至顶端，插入梳子，注意不能有气泡产生，静置10分钟左右，可进行上样。
(3)上样电泳：将制备好的样本按照一定顺序，每孔加20ul，每一组实验样本周边用预染蛋白质Marker隔开，起始电压为100V，约40min，待溴酚蓝进入分离胶以后，电压改为200v，约40min,当小分子蛋白拉开距离以后，停止电泳。
(4)转膜：先将需要的PVDF膜放在甲醇中激活1min左右，再把PVDF膜覆盖于电泳后的凝胶上，PVDF膜和凝胶的非接触面均覆盖滤纸和海绵，至于电转槽中，恒流300mA，90min。
(5)封闭：电转后，取出PVDF膜，放入现配的封闭液(含5％的脱脂牛奶的1×TBST)中，摇床上4℃过夜或室温2h。
(6)封一抗：封闭结束后，配制含5％的脱脂牛奶的1×TBST稀释的一抗(抗RBCK1抗体稀释5000倍，抗β-Actin抗体稀释10000倍，各稀释2mL放EP管中)，在相应蛋白所在位置剪开，在封抗体盒子中分别加2mL稀释后的相应一抗，摇床上4℃过夜或室温2h。
(7)洗膜，孵二抗，洗二抗：取出PVDF膜，1×TBST摇床上洗3次，每次15min，将洗好的膜分别放在封抗体盒子中，配制含5％的脱脂牛奶的1×TBST稀释的二抗(二抗稀释5000倍，稀释2mL放离心管中)在封抗体盒子中分别加2mL稀释后的相应二抗，摇床上室温1h，摇床上用1×TBST洗3次，每次15min。
(8)显影：按化学发光试剂盒说明书将ECL发光液A、B液等比例混合(使用前配制)，将洗好的膜用吸水纸吸干多余的1×TBST，将膜的正面向上放置在发光板上，均匀的滴加显影液，凝胶成像系统拍照、保存。
结果如图1所示，结果显示，Western Blot检测细胞株中干扰后的RBCK1蛋白表达水平较对照组明显减低。
实施例2：通过实时定量PCR检测细胞株中RBCK1的mRNA干扰效果
1、细胞传代培养：同实施例1。
2、细胞转染：同实施例1。
3、总RNA的提取：收集细胞后，参照Trizol说明书提取细胞总RNA，在该操作过程中需遵循无RNA酶操作，佩戴无菌手套和口罩。具体步骤如下：
1)首先将要提取RNA的12孔板细胞取出放置冰上，(以下实验步骤全程冰上进行)吸弃培液，PBS洗一遍，然后每孔加入1ml Trizol，冰上裂解5分钟。
2)收集细胞裂解液至1.5ml无核酶EP管中，标记清楚相对应的样品名称
3)向裂解液中加入200ul氯仿，用力甩30s，冰上静置3min，4℃12000rpm，15min。
4)沿没有DNA薄膜的侧壁轻轻吸取上清约400ul至相对应的干净无核酶EP管中，加入等体积异丙醇，用力甩几下，冰上10min。
5)4℃，13000rpm，15min，后弃上清。
6)每管加入1ml75％无水乙醇，4℃，7500rpm，5min。
7)沿没有RNA白色沉淀的那一侧，用真空泵吸弃上清。
8)开盖置于通风橱中，静置5-10min，待RNA沉淀由白色变成无色透明状即可盖上盖子取出至冰上。
9)取20微升DEPC水溶解，冰上静置5min，得到RNA溶液，测量浓度并标记，即可用于后续实验或者-80℃保存防止RNA降解。
紫外分光光度仪测定提取RNA的浓度(单位μg/u)和纯度，用DEPC水调零，OD260/OD280在1.8-2.0之间，表明提取的RNA纯度好。
逆转录为cDNA(reverse transcriptas，RT):反应体系为:按TaKaRa公司PrimeScript RT Master Mix试剂盒进行反转录：5×PrimeScript RT Master MIX：2μL，所提总RNA：1μg；加ddH2O至总体积为10μL，置于PCR仪中进行逆转录，反应体系为37℃15分钟，85℃5秒，4℃保存备用。
实时定量PCR方法按Appliedbiosystems公司SYBR Select Master Mix试剂盒检测相关基因的表达(36B4为内参)的反应体系为：17μL SYBR，14μL去RNA酶水，1μL Forwardprimer(5’-TGCTCAGATGCACACCGTC-3’，SEQ ID NO.7)，1μL Reverse primer(5’-CAAGACTGGTGGGAAGCCATA-3’，SEQ ID NO.8)，1μL cDNA。反应程序为Holding Stage：50℃2min，95℃10min，Cycling Stage：95℃，15s，50℃，1min，Number of Cycles:40。计算CT值，结果以柱状图呈现。
结果如图2所示，结果显示，q-PCR检测细胞株中干扰后的RBCK1 mRNA表达水平较对照组明显减低。
实施例3：利用划痕实验检测转染后ER阳性乳腺癌细胞系迁移能力的影响
1、细胞传代培养：同实施例1。
2、细胞转染：同实施例1。
3、划痕实验：12孔板转染后的细胞株待密度达100％时，用无血清DMEM培养基饥饿处理12h；100ul枪头在12孔板各实验孔内划十字痕迹，创造划痕，PBS洗去刮下细胞，洗3次，加1mL DMEM完全培养基(DMEM+10％FBS)，显微镜下拍照，记录划痕宽度。继续培养24、48、72h，显微镜下拍照(每次拍照前用PBS清洗并更换新鲜1mL DMEM完全培养基(DMEM+10％FBS)),记录划痕宽度，用Image J计算细胞愈合面积。
结果如图3所示，结果RBCK1基因干扰后，T47D细胞系的迁移能力显著下降，较对照组明显降低。
实施例4：Transwel小室观察转染前后细胞迁移和侵袭能力的改变
1、细胞传代培养：同实施例1。
2、细胞转染：同实施例1。
3、侵袭实验：取转染后的实验组细胞、阴性对照组细胞，分别用0.25％胰蛋白酶消化后，细胞计数后用无血清DMEM培养基重悬，调整细胞密度为5×105/mL。在上室加入200μL细胞悬液，细胞数为1×105个，下室为含20％胎牛血清的DMEM培养液500L，37℃、5％CO2培养箱中孵育18h。孵育结束后，取出小室，用PBS洗两遍，棉签轻轻擦拭上室滤膜内侧面贴壁细胞，PBS洗两遍。将小室滤膜用4％多聚甲醛固定10分钟，吸去固定液，每孔加入500μL结晶紫染液，染色20min，吸弃染色液，PBS洗三遍，将上室取出，于倒置显微镜下拍照并计数。
Transwell侵袭实验与迁移实验不同的是：Transwell小室上室表面需要提前均匀铺上用无血清培养基：Matrigel胶＝8:1稀释过的Matrigel胶，于37℃细胞培养箱2-3小时使胶体凝固，模拟血管基底膜。
结果如图4所示，RBCK1基因干扰后，T47D细胞系的侵袭能力显著下降，较对照组明显降低，差异有统计学意义(P<0.01)。
尽管已描述了本发明的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。
序列表
<110> 新乡医学院
<120> ER阳性乳腺癌分子标志物及其应用
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atggggtgcg gcagaatggg gacagtgcct acctctatct gctgtcagcc cgcaacacct 60
ccctcaaccc tcaggagctg cagcgggagc ggcagctgcg gatgctggaa gatctgggct 120
tcaaggacct cacgctgcag ccgcggggcc ctctggagcc aggcccccca aagcccgggg 180
tcccccagga acccggacgg gggcagccag atgcagtgcc tgagccccca ccggtgggct 240
ggcagtgccc cgggtgcacc ttcatcaaca agcccacgcg gcctggctgt gagatgtgct 300
gccgggcgcg ccccgaggcc taccaggtcc ccgcctcata ccagcccgac gaggaggagc 360
gagcgcgcct ggcgggcgag gaggaggcgc tgcgtcagta ccagcaggga gtgcctgcag 420
ggcaccatcc gcaacagcca ggaggcggag gtctcctgcc ccttcattga caacacctac 480
tcgtgctcgg gcaagctgct ggagagggag atcaaggcgc tcctgacccc tgaggattac 540
cagcgatttc tagacctggg catctccatt gctgaaaacc gcagtgcctt cagctaccat 600
tgcaagaccc cagattgcaa gggatggtgc ttctttgagg atgatgtcaa tgagttcacc 660
tgccctgtgt gtttccacgt caactgcctg ctctgcaagg ccatccatga gcagatgaac 720
tgcaaggagt atcaggagga cctggccctg cgggctcaga acgatgtggc tgcccggcag 780
acgacagaga tgctgaaggt gatgctgcag cagggcgagg ccatgcgctg cccccagtgc 840
cagatcgtgg tacagaagaa ggacggctgc gactggatcc gctgcaccgt ctgccacacc 900
gagatctgct gggtcaccaa gggcccacgc tggggccctg ggggcccagg agacaccagc 960
gggggctgcc gctgcagggt aaatgggatt ccttgccacc caagctgtca gaactgccac 1020
tga 1023
<210> 2
<211> 340
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Pro Ala Thr Pro Pro Ser Thr Leu Arg Ser Cys Ser Gly Ser Gly Ser
20 25 30
Cys Gly Cys Trp Lys Ile Trp Ala Ser Arg Thr Ser Arg Cys Ser Arg
35 40 45
Gly Ala Leu Trp Ser Gln Ala Pro Gln Ser Pro Gly Ser Pro Arg Asn
50 55 60
Pro Asp Gly Gly Ser Gln Met Gln Cys Leu Ser Pro His Arg Trp Ala
65 70 75 80
Gly Ser Ala Pro Gly Ala Pro Ser Ser Thr Ser Pro Arg Gly Leu Ala
85 90 95
Val Arg Cys Ala Ala Gly Arg Ala Pro Arg Pro Thr Arg Ser Pro Pro
100 105 110
His Thr Ser Pro Thr Arg Arg Ser Glu Arg Ala Trp Arg Ala Arg Arg
115 120 125
Arg Arg Cys Val Ser Thr Ser Arg Glu Cys Leu Gln Gly Thr Ile Arg
130 135 140
Asn Ser Gln Glu Ala Glu Val Ser Cys Pro Phe Ile Asp Asn Thr Tyr
145 150 155 160
Ser Cys Ser Gly Lys Leu Leu Glu Arg Glu Ile Lys Ala Leu Leu Thr
165 170 175
Pro Glu Asp Tyr Gln Arg Phe Leu Asp Leu Gly Ile Ser Ile Ala Glu
180 185 190
Asn Arg Ser Ala Phe Ser Tyr His Cys Lys Thr Pro Asp Cys Lys Gly
195 200 205
Trp Cys Phe Phe Glu Asp Asp Val Asn Glu Phe Thr Cys Pro Val Cys
210 215 220
Phe His Val Asn Cys Leu Leu Cys Lys Ala Ile His Glu Gln Met Asn
225 230 235 240
Cys Lys Glu Tyr Gln Glu Asp Leu Ala Leu Arg Ala Gln Asn Asp Val
245 250 255
Ala Ala Arg Gln Thr Thr Glu Met Leu Lys Val Met Leu Gln Gln Gly
260 265 270
Glu Ala Met Arg Cys Pro Gln Cys Gln Ile Val Val Gln Lys Lys Asp
275 280 285
Gly Cys Asp Trp Ile Arg Cys Thr Val Cys His Thr Glu Ile Cys Trp
290 295 300
Val Thr Lys Gly Pro Arg Trp Gly Pro Gly Gly Pro Gly Asp Thr Ser
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Gly Gly Cys Arg Cys Arg Val Asn Gly Ile Pro Cys His Pro Ser Cys
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Gln Asn Cys His
340
<210> 3
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gccucagcua ccaugcatt 19
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ugcaauggua gcugaaggct t 21
<210> 5
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cacaccuucu gcagggagut t 21
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acucccugca gaaggugugt t 21
<210> 7
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tgctcagatg cacaccgtc 19
<210> 8
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<212> DNA
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<400> 8
caagactggt gggaagccat a 21
Claims (1)
1.RBCK1基因的抑制剂在制备抑制ER乳腺癌细胞迁移和侵袭的药物中的应用，其特征在于，所述RBCK1基因的DNA序列如SEQ ID NO.1所示，所述抑制剂为靶向RBCK1基因的siRNA，所述siRNA的序列如SEQ ID NO.5所示。
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| CN110592225A (zh) * | 2019-11-05 | 2019-12-20 | 新乡医学院 | 一种三阴性乳腺癌分子标志物及其应用 |
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| CN110592225A (zh) * | 2019-11-05 | 2019-12-20 | 新乡医学院 | 一种三阴性乳腺癌分子标志物及其应用 |
| CN111154873A (zh) * | 2020-01-07 | 2020-05-15 | 新乡医学院 | 一种三阴性乳腺癌细胞迁移和侵袭能力检测分子标志物及其应用 |
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