CN110734977A - 以sf3b1为靶点在制备预防或治疗乳腺癌的药物中的应用 - Google Patents
以sf3b1为靶点在制备预防或治疗乳腺癌的药物中的应用 Download PDFInfo
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Abstract
本发明属于生物医药技术领域,提供新的预防或治疗乳腺癌的分子靶点SF3B1,以及该靶点在制备预防或治疗乳腺癌的药物中的应用。其中以SF3B1基因为靶点包括采用基因敲除、基因敲减或者化学药物降低SF3B1基因的表达。SF3B1在乳腺癌细胞中的表达与正常组织细胞相比显著上调。SF3B1表达水平与淋巴结转移相关。乳腺癌中SF3B1基因敲除可诱导细胞凋亡和细胞周期阻滞。SF3B1基因敲除可降低细胞增殖、集落形成、侵袭和迁移能力。外显子跳越是SF3B1敲除后最常见的剪接事件。研究表明SF3B1可能是乳腺癌治疗的重要分子靶点。
Description
技术领域
本发明属于生物医药技术领域,具体涉及以SF3B1为靶点在制备预防或治疗乳腺癌的药物中的应用。
背景技术
异常剪接是癌症相关表型的特征之一,并且肿瘤的发生通常伴随异常RNA剪接。众所周知,选择性剪接(AS)是真核生物的一个重要特征,由小核RNA(snRNA)和各种蛋白质组成的剪接体控制。剪接因子基因的突变和表达水平的改变可以导致外显子丢失、内含子保留、剪接位点的错误识别、开放阅读框的改变,从而影响大多数参与细胞周期控制、血管生成、凋亡、转移等基因的转录后加工和表达。癌症有许多在正常样本中不存在的选择性剪接事件。因此,靶向剪接去调控可能代表一种新的癌症治疗方法。
全基因组测序的应用揭示了多种癌症中剪接因子的显著体细胞突变。其中,SF3B1突变发生在乳腺癌,前列腺癌,子宫内膜癌,葡萄膜黑色素瘤和几种类型的血液系统恶性肿瘤。对892例和510例乳腺癌样本的全基因组水平的测序显示,特别是在雌激素受体(ER)阳性的乳腺癌患者中,SF3B1的突变显著且频繁。在这些突变中,错义突变占主导地位,大多数错义突变位于K700E和Q534P等突变热点上。这些突变可能在正向选择的压力下赋予癌细胞生长优势,并可能在乳腺癌的进化中发挥关键作用。
SF3B1基因位于染色体2q33.1,编码剪接因子3b的亚基1,它是U2小核核糖核蛋白复合体的核心部分。SF3B1对于识别和结合靠近3’剪接位点的分支点序列是必需的,并且在从前mRNA中精确切除内含子以形成成熟mRNA中发挥重要作用。SF3B1突变可能是肿瘤发生过程中的驱动事件,不仅导致转录偶联中的剪接异常,而且还影响基因组不稳定和干细胞的分化。
乳腺癌是对妇女健康的主要威胁之一。众所周知,乳腺癌是一种由大量分子异常驱动的异质性疾病,这些分子异常导致了其多样化的病理临床表现。尽管靶向治疗方法在乳腺癌治疗方面取得了成功,但耐药仍然是一个非常严重的问题,乳腺癌的分子机制仍需澄清。虽然SF3B1突变在乳腺癌中经常被报道,但SF3B1在乳腺癌中的表达状态、功能和分子后果尚未见报道。目前尚不清楚SF3B1是否可能成为乳腺癌的分子靶点。
发明内容
本发明的目的是提供新的预防或治疗乳腺癌的分子靶点SF3B1,以及该靶点在制备预防或治疗乳腺癌的药物中的应用。其中以SF3B1基因为靶点包括采用基因敲除、基因敲减或者化学药物降低SF3B1基因的表达。
需要说明的是,本申请经过研究发现,乳腺癌中SF3B1基因敲除可诱导乳腺癌细胞凋亡和细胞周期阻滞。SF3B1基因敲除可降低乳腺细胞增殖、集落形成、侵袭和迁移能力。SF3B1抑制剂可明显抑制乳腺癌细胞生长。具有预防肿瘤的效果;并且能够治疗并抑制已经存在的肿瘤细胞;因此,SF3B1基因缺失能够用于制备预防或治疗乳腺癌的药物。其中SF3B1基因缺失可以通过基因敲除、基因敲减或者化学药物降低SF3B1基因的表达实现。
本发明还提供了一种试剂盒,用于所述的以SF3B1基因为靶点采用基因敲除降低SF3B1基因的表达;所述试剂盒中包含:两个慢病毒shRNA序列:命名为SF3B1-sh1:5’-ccggGAGCACAGACCTCCAAAGATTctcgagAATCTTTGGAGGTCTGTGCTCtttttg-3’;命名为SF3B1-sh2:5’-ccggTGCAACTGAACTTGAAGATGActcgagTCATCTT CAAGTTCAGTTGCAtttttg-3’。
本发明为了进一步研究SF3B1在乳腺癌中的作用,将ER阳性的ZR-75-30细胞和代表三阴性乳腺癌(TNBC)的MDA-MB-231细胞中的SF3B1基因敲除,观察其生物学效应。结果表明,SF3B1的敲除抑制了两种细胞的增殖、集落形成、侵袭和迁移,并促进了细胞凋亡。SF3B1突变、表达失调、丝氨酸合成与乳腺癌亚型之间错综复杂的关系还有待进一步研究。
此外,还鉴定了与SF3B1相关的重要信号通路,包括Ras信号通路、MAPK信号通路、甘氨酸、丝氨酸和苏氨酸代谢等。在SF3B1抑制或敲除后的差异剪接事件中,外显子跳越是最常见的分子后果。
最后,应用SF3B1抑制剂处理ZR-75-30和MDA-MB-231乳腺癌细胞,可明显观察到乳腺癌细胞生长受抑制,其抑制效应具有明显的时间依赖性及浓度依赖性,其最适的作用浓度仅在nmol/L级别。
总之,研究表明SF3B1在乳腺癌细胞中的表达与正常组织细胞相比显著上调。SF3B1表达水平与淋巴结转移相关。乳腺癌中SF3B1基因敲除可诱导细胞凋亡和细胞周期阻滞。SF3B1基因敲除可降低细胞增殖、集落形成、侵袭和迁移能力。外显子跳越是SF3B1敲除后最常见的剪接事件。SF3B1抑制剂可明显抑制乳腺癌细胞的生长。研究表明SF3B1可能是乳腺癌治疗的重要分子靶点。
附图说明
图1为与邻近的非肿瘤组织相比,SF3B1在乳腺癌组织中表达上调;
图2为ROC曲线筛选乳腺癌组织中SF3B1表达阈值图,AUC代表曲线下面积;
图3为乳腺癌细胞中SF3B1敲除效率检测; (A)qPCR法检测SF3B1的敲除效率;(B)Western blot法检测SF3B1的敲除效率;
图4为SF3B1敲低后抑制ZR-75-30(左)和MDA-MB-231(右)乳腺癌细胞的增殖;
图5为SF3B1敲低后抑制ZR-75-30和MDA-MB-231乳腺癌细胞的克隆形成能力;
图6为SF3B1敲低后影响ZR-75-30和MDA-MB-231乳腺癌细胞的细胞周期;
图7为SF3B1敲低后促进ZR-75-30和MDA-MB-231乳腺癌细胞的凋亡;
图8为SF3B1敲低后明显抑制ZR-75-30和MDA-MB-231细胞的侵袭;
图9为SF3B1敲低后明显抑制ZR-75-30和MDA-MB-231细胞的迁移;
图10为RNA测序方法分析出SF3B1敲低细胞与相应对照细胞相比,差异表达基因、癌症关键通路。(A)前100个差异表达基因的热图。每一行代表一个基因,每一列代表一个重复。(B)差异表达基因的通路富集分析;
图11为分析SF3B1敲低后的不同剪接事件。(A)不同剪接事件的原理图说明。(B)SF3B1敲低后,MDA-MB-231细胞中以外显子跳越事件为主;
图12为SF3B1抑制剂抑制ZR-75-30和MDA-MB-231乳腺癌细胞的增殖。
具体实施方式
根据当地伦理委员会的指导方针,从山西医科大学第一附属医院获得110例确诊为乳腺癌的患者的癌组织样本,其中40例有非肿瘤对照组织相匹配。分子亚型包括LuminalA(38),Luminal B(38),HER2富集(14)和三阴性/基底样乳腺癌(20)。手术前没有患者接受放疗或化疗。
免疫组织化学:石蜡包埋福尔马林固定组织样本用于免疫组织化学检测。将标本切成4μm厚的切片。使用梯度二甲苯和乙醇对切片进行脱蜡和再水化。然后将样品用3%H2O2孵育10-15分钟,用蒸馏水冲洗三次。在柠檬酸钠缓冲液(pH6.0)中提取抗原2min并在山羊血清中封闭后,将切片与特异性抗SF3B1抗体(Abcam)在4℃下1:200稀释孵育过夜。用PBS洗涤后,将切片与第二抗体(Maixin)在37˚C下孵育20min,然后使用DAB检测试剂盒(Maixin)进行检测。通过免疫组织化学方法用Aperio Nuclear v.9软件分析SF3B1的表达水平和定位。
细胞培养:MDA-MB-231和ZR-75-30乳腺癌细胞株在L-15和RPMI-1640培养基中培养,培养基中添加10%胎牛血清和1%青霉素/链霉素组成的抗生素。将培养物保存在37˚C的由95%空气和5%CO2构成的湿润环境中。
乳腺癌细胞系中SF3B1基因的敲除:为了敲除内源性SF3B1,使用两个慢病毒shRNA序列:1.命名为SF3B1-sh1:5’-ccggGAGCACAGACCT CCAAAGATTctcgagAATCTTTGGAGGTCTGTGCTCtttttg-3’;2.命名为SF3B1-sh2:5’-ccggTGCAACTGAACTTGAAGATGActcgagTCATCTTCAAGTTCAGTTGCA tttttg-3’。
利用Lipofectamine 2000试剂(Invitrogen)将shRNA序列克隆到GV248载体(hU6-MCS-泛素- EGFP- IRE -嘌呤霉素)中,并与包装质粒共转染293T细胞。慢病毒转导:将乳腺癌细胞以30%-40%的融合率接种,以适当的病毒滴度感染,过夜孵育。然后用新鲜培养基替代病毒上清液,48小时后用嘌呤霉素筛选感染细胞群体。
蛋白质印迹:用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分离蛋白质。将蛋白质转移到硝化纤维滤膜上,并将该膜与5%脱脂牛奶在室温下孵育1h。然后将膜与一抗(Abcam)在4℃下孵育过夜。用辣根过氧化物酶标记的二抗(Sigma)检测印迹,用LAS4000装置(富士)检测化学发光。
实时定量PCR (qPCR):用Trizol试剂从细胞系中提取总RNA。然后按照TaqMan RT试剂盒说明,用TaqMan RT试剂盒将总RNA反转录为cDNA。使用SYBR-Green PCR Master Mix通过20μl反应体系的PCR扩增cDNA。使用SF3B1引物(F:5’-GAGAAATTCAAGGCAAGAAGG-3’;R:5’-GACCAAGCAAACTC GTAGATG-3’);GAPDH(F:5’-TGACTTCAACAGCGACACCCA-3’;R:5’-CACCCTGTTGCTGT AGCCAAA-3’)。所有RT-qPCR反应均重复三次。
比色法:将细胞以4×103个/孔的密度接种到96孔板中,并在正常条件下培养。在指定的时间,每孔加入20μl MTT液(5 mg/ml),并将细胞孵育4小时直至形成晶体。然后取出培养基并用200μlDMSO代替。振荡15分钟直至晶体溶解后,用酶标仪(Bio-Rad)在490nm处测定光密度。
细胞集落形成、迁移和侵袭试验:在含有完全培养基的6孔板中每孔接种500-800个细胞,并在标准条件下培养。第14天,细胞用4%多聚甲醛固定15min,1%结晶紫染色20min。然后用流水慢慢冲洗样品。Transwell试验用于评估乳腺癌的迁移和侵袭能力。使用有或无Matrigel基底膜基质 (BD)的transwell室底膜进行入侵和迁移检测。将无血清培养基中的2~3×104个细胞接种于上腔室,下腔室加入含血清培养基。培养48h后,通过膜的细胞用4%甲醛固定20min,1%结晶紫染色20min,并在显微镜下计数。
细胞凋亡与细胞周期测定:使用4˚C预冷D-Hanks和1×结合缓冲液洗涤6孔板中收集的细胞沉淀。将细胞沉淀重悬于200μl的1x结合缓冲液中,并在室温下于黑暗中使用10μl膜联蛋白V-APC染色10-15分钟,并通过流式细胞仪检测。将6cm培养皿中的细胞收集在5ml管中,在1300rmp下离心5min。丢弃上清液,然后加入4°C预冷的D-Hanks。离心后丢弃上清液,用预冷的70%乙醇在4 ℃下过夜固定细胞。洗涤后加入含核糖核酸酶的PI溶液,在黑暗中孵育30min 。最后,过滤细胞并用流式细胞仪(BD Biosciences,San Diego,CA,USA)进行分析。
测序:在总RNA合格后,用含Oligo(dT)的磁珠富集mRNA。以提取的mRNA随机片段作为模板,用随机引物合成cDNA,然后用缓冲液、dNTPs、RNaseH和DNA聚合酶合成双链cDNA。用AMPure XP珠子纯化产物。然后,用T4 DNA聚合酶和Klenow DNA聚合酶修复DNA的粘性末端至平端,然后在3‘端添加碱基A和接头。最后进行PCR扩增,得到最终测序库。测序库合格后,用Illumina Hiseq4000进行测序,测序读数长度为双端2×150bp(PE150)。
统计分析:采用秩和卡方检验(χ2)比较SF3B1表达与临床病理参数的差异。测量数据用平均值±标准差表示。使用SPSS.20统计软件进行分析。t检验用于比较两组之间的差异。采用单因素或双因素方差分析比较三组或三组以上的组之间差异。p值小于0.05被认为具有统计学意义。
结果:
1.SF3B1表达与临床病理特征的关系:首先,测量了110例乳腺癌组织中的SF3B1蛋白水平。免疫组化结果如图1所示,显示SF3B1在乳腺癌细胞的细胞核中表达。乳腺癌组织中SF3B1蛋白的H-Score范围为1.38278~196.3014,中位数为162.7205。SF3B1蛋白在癌组织中的表达明显高于非肿瘤组织(p<0.01) (图1)。此外,根据ROC曲线分析(图2),选择具有较高敏感性和特异性的SF3B1表达水平的最佳切点值71.2758,将所有乳腺癌病例分为两组:SF3B1-低表达(H-Score<71.2758)和SF3B1-高表达(H-Score≥71.2758)。分析显示SF3B1的表达水平与淋巴结转移有关(p<0.05),而在不同年龄、临床阶段、肿瘤大小、雌激素、孕激素、HER-2、Ki-67水平的患者中未发现明显的相关性(表1)。
基因敲除对细胞增殖和集落形成的影响:选择内源性SF3B1高表达的MDA-MB-231和ZR-75-30乳腺癌细胞系用于敲除实验。
如图3所示,通过RT-qPCR和western blot证实了ZR-75-30和MDA-MB-231细胞系中的敲除效率。然后用MTT法分析SF3B1基因敲除与细胞增殖的关系。分别于24,48,72,96和120小时测定吸光度值。在72,96和120小时,ZR-75-30和MDA-MB-231细胞中SF3B1-sh1和sh2的吸光度值与相应的阴性对照组相比显著降低(p<0.05) (图4)。此外,如集落形成试验所示,shRNA处理的ZR-75-30和MDA-MB-231细胞系的集落形成效率也降低(p<0.01)(图5)。
基因敲除对细胞周期和凋亡的影响:流式细胞仪检测SF3B1基因敲除对ZR-75-30和MDA-MB-231细胞周期分布的影响。受影响的细胞周期进程在这两种细胞中是不同的。对于ZR-75-30细胞系,阴性对照组和SF3B1 敲除组之间没有明显差异(图6)。对于MDA-MB-231细胞系,DNA倍性检测表明,与阴性对照组相比,SF3B1敲除组G1和S期细胞的比率明显降低(p<0.05)(图6)。然而,SF3B1敲除组的G2/M期细胞的比率与阴性对照组相比显著增加(p<0.01) (图6)。结果表明,SF3B1敲除抑制DNA复制,并在G2/M期阻滞中起重要作用。ZR-75-30和MDA-MB-231细胞中SF3B1基因敲除后对细胞周期进程的不同结果可能与遗传背景有关,其中ZR-75-30代表管腔B型乳腺癌细胞株,MDA-MB-231代表基底细胞型乳腺癌细胞株。
研究SF3B1敲除对细胞凋亡的影响。膜联蛋白V-APC检测显示,ZR-75-30细胞中SF3B1敲除组的凋亡率(9.75%±0.27%或9.95%±0.47%)明显高于阴性对照组(4.30%±0.22%)(p<0.01) (图7。在MDA-MB-231细胞中,SF3B1敲除的促凋亡作用更为明显,其中敲除组的凋亡率显著高于阴性对照组(22.90%±0.79%或24.90%±0.89%vs1.43%±0.21%)(p<0.001)(图7)。
基因敲除对细胞迁移和侵袭的影响:接下来,使用涂有基质胶的transwell小室检测SF3B1敲除对乳腺癌细胞侵袭的影响。如图8所示,与阴性对照组相比,SF3B1敲除组细胞的细胞侵袭能力显著降低。ZR-75-30细胞中,SF3B1基因敲低组细胞的相对侵袭率为48.00±4.91或40.1±3.92,而阴性对照细胞的相对侵袭率为155.10±2.31(p<0.01)。MDA-MB-231细胞中,SF3B1敲除细胞的相对侵袭率较阴性对照组更显著降低(23.21±0.17或32.00±1.15 vs.170±12.45,p<0.01)(图8)。
Transwell小室试验也用于检测迁移能力。在两种细胞系中,SF3B1敲除组细胞中渗透的细胞数明显低于对照组细胞(ZR-75-30细胞:107.00± 0.85或99.10±2.88vs.153.00±6.37;MDA-MB-231细胞:3.01±0.26或2.13±0.16vs.110.00±1.44)(图 9)。
观察SF3B1敲除后的下游途径和受影响的选择性剪接事件:为了检测乳腺癌中SF3B1敲除对整个转录组的影响,我们提取了MDA-MB-231细胞中SF3B1敲除和阴性对照组细胞的RNA,并进行了RNA测序。进行了三个独立的生物学重复。平均每个样品获得5200万个配对末端150bp的测序读数。使用两倍变化的显着差异阈值的差异表达分析显示,与阴性对照组相比,SF3B1敲除组中有860个基因显著上调,776个基因显著下调。图10A热图显示了前100个差异表达基因并根据表达变化对3个重复序列进行聚类。通路富集分析表明差异表达基因富集在信号通路中,包括RAS信号通路;细胞因子-细胞因子受体相互作用;紧密连接;MAPK信号通路;甘氨酸,丝氨酸和苏氨酸代谢等,表明SF3B1可能通过这些通路促进乳腺癌肿瘤的发生(p<0.05)(图10B)。
应用ASprofile软件于RNA测序数据,以研究SF3B1敲除对剪接的全局影响。将选择性剪接事件分为七类(图11A),并分析SF3B1敲除时剪接模式变化的频率。将SF3B1基因敲除组的选择性剪接事件作为改变后的剪接模式,剪接次数多于或少于对照组。分析表明,外显子跳跃(SKIP)和盒式外显子(MSKIP)是最常见的分子效应(p<0.05)(图11B)。此外,SF3B1敲除几乎不影响其他选择性剪接模式,包括保留单个(IR)、多个(MIR)内含子、选择性外显子末端(AE)、选择性转录起始位点(TSS)和选择性转录终止位点(TTS)。
观察SF3B1抑制剂对乳腺癌细胞增殖的影响:应用SF3B1抑制剂处理ZR-75-30和MDA-MB-231乳腺癌细胞,可明显观察到乳腺癌细胞生长受抑制,其抑制效应具有明显的时间依赖性及浓度依赖性,其最适的作用浓度仅在nmol/L级别。
据报道,选择性剪接的解除与癌症的发展和侵袭性有关。剪接体成分的突变和异常的剪接活性已成为癌症表型的标志之一。在乳腺癌中,SF3B1突变已被证明是ER阳性患者的优先驱动事件。值得注意的是,尽管SF3B1在乳腺癌中存在明显的突变,但SF3B1在乳房中的表达模式以及功能和分子后果仍然不清楚。
结果表明,SF3B1表达失调可能参与了乳腺癌的发生发展,并且SF3B1可能代表了一种新的治疗靶点。遗憾的是,由于本研究中招募的乳腺癌患者均为近几年确诊的,随访时间很短,我们没有可靠的生存分析数据。
为了进一步研究SF3B1在乳腺癌中的作用,我们将ER阳性的ZR-75-30细胞和代表三阴性乳腺癌(TNBC)的MDA-MB-231细胞中的SF3B1基因敲除,观察其生物学效应。结果表明,SF3B1的敲除抑制了两种细胞的增殖、集落形成、侵袭和迁移,并促进了细胞凋亡。SF3B1突变、表达失调、丝氨酸合成与乳腺癌亚型之间错综复杂的关系还有待进一步研究。
此外,我们还鉴定了与SF3B1相关的重要信号通路,包括Ras信号通路、MAPK信号通路、甘氨酸、丝氨酸和苏氨酸代谢等。在SF3B1抑制或敲除后的差异剪接事件中,外显子跳越是最常见的分子后果。
总之,研究表明SF3B1在乳腺癌细胞中的表达与正常组织细胞相比显著上调。SF3B1表达水平与淋巴结转移相关。乳腺癌中SF3B1基因敲除可诱导细胞凋亡和细胞周期阻滞。SF3B1基因敲除可降低细胞增殖、集落形成、侵袭和迁移能力。外显子跳越是SF3B1敲除后最常见的剪接事件。SF3B1抑制剂可明显抑制乳腺癌细胞生长。研究表明SF3B1可能是乳腺癌治疗的重要分子靶点。
在本研究中,评估SF3B1在乳腺癌中的表达水平及其与临床和病理特征的关系。此外,探索了SF3B1敲除后乳腺癌细胞的生物学效应和下游分子事件。剪接因子3b亚基1(SF3B1)在乳腺癌中频繁发生突变。
Claims (6)
1.以SF3B1基因为靶点在制备预防或治疗乳腺癌的药物中的应用,其特征在于:所述以SF3B1基因为靶点包括采用基因敲除、基因敲减或者化学药物降低SF3B1基因的表达。
2.根据权利要求1所述的应用,其特征在于:所述以SF3B1基因为靶点为采用基因敲除降低SF3B1基因的表达。
3.一种试剂盒,用于权利要求2所述的以SF3B1基因为靶点为采用基因敲除降低SF3B1基因的表达;其特征在于:所述试剂盒中包含:两个慢病毒shRNA序列:命名为SF3B1-sh1:5’-ccggGAGC ACAGACCTCC AAAGATTctcgagAATCTTTGGAGGTCTGTGCTCtttttg-3’;命名为SF3B1-sh2:5’-ccggTGCAACTGAACTTGAAGATGActcgagTCATC TTCAAGTTCAGTTGCAtttttg-3’。
4.根据权利要求1所述的应用,其特征在于:所述以SF3B1基因为靶点为化学药物降低SF3B1基因的表达。
5.根据权利要求1所述的应用,其特征在于:所述以SF3B1基因为靶点为采用SF3B1抑制剂抑制SF3B1基因活性。
6.根据权利要求4所述的应用,其特征在于:所述化学药物为抑制Ras信号通路、MAPK信号通路、甘氨酸、丝氨酸和苏氨酸代谢通路的抑制剂。
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