CN117327661B - 一种抗癌药物敏感性增强的fact突变体细胞系及构建方法和应用 - Google Patents
一种抗癌药物敏感性增强的fact突变体细胞系及构建方法和应用 Download PDFInfo
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Abstract
本发明公开了一种抗癌药物敏感性增强的FACT突变体细胞系及构建方法和应用，通过改变Ssrp1与组蛋白结合表面的氨基酸电荷属性，得到了影响FACT与组蛋白结合的突变位点Q265K，进而建立FACT突变体细胞系。本发明构建了与Curaxins抑癌作用机制高度相似的FACT突变体细胞系，该细胞系可为未来靶向药物的研制提供作用模型和热点基因。本发明筛选得到的FACT‑Q265K突变体能够提高细胞对药物的敏感性，该突变位点的发现为解决药物耐受提供了重要参考。
Description
技术领域
本发明涉及抗癌药物研发技术领域，特别是涉及一种抗癌药物敏感性增强的FACT突变体细胞系及构建方法和应用。
背景技术
真核生物细胞中， DNA与组蛋白共同形成染色质，染色质的基本单位是核小体，每个核小体是由147对碱基长度的DNA片段缠绕一个组蛋白八聚体核心构成。染色质的结构将影响DNA复制、基因转录以及DNA损伤修复等过程。在染色质结构的动态变化过程中，核小体需要发生解离与重新组装，该过程的对于维持表观遗传信息的稳定传递非常重要。
目前，已有研究表明多种组蛋白分子伴侣参与染色质的解离与组装过程，组蛋白伴侣与组蛋白结合并调控组蛋白的动态变化，包括折叠、翻译后修饰、入核、组装、解组装以及定位等。已有前期研究工作表明，ASF1a、ASF1b以及CAF-1等组蛋白分子伴侣的表达水平在多种癌细胞中上升。在哺乳动物中，FACT复合物由SUPT16和SSRP1两个亚基组成，FACT复合物参与DNA复制、RNA转录以及DNA损伤修复等染色质动态变化过程。已有前期研究报道FACT的表达水平在乳腺癌、膀胱癌、黑色素瘤以及胶质瘤中显著上升，抗癌小分子药物Curaxins的作用机制是提升FACT与染色质的结合，进而导致基因表达调控紊乱，促进癌细胞死亡。目前，组蛋白分子伴侣异常表达在疾病发生与发展过程中的作用机制尚不完全明确，针对组蛋白分子伴侣的癌症治疗方法仍在初期阶段。
发明内容
本发明的目的是提供一种抗癌药物敏感性增强的FACT突变体细胞系及构建方法和应用。
本发明通过前期实验研究筛选到一种位于Ssrp1 M domain的突变体FACT-Q265K，Q265K突变提高了FACT与染色质的结合，尤其是该突变显著提升了癌细胞对于药物Curaxins的敏感性。FACT参与多种染色质动态变化过程，作用机制尚未阐明，与此同时，目前暂时未发现与疾病直接相关的突变位点，缺乏应用于抑癌研究的切入点，而本发明筛选得到的FACT突变位点，位于Ssrp1 M domain，直接影响FACT与染色质的结合。该突变位点的发现能够提升抗癌药物的作用，并为抗癌药物提供新的潜在靶点。
具体而言，本发明采用的技术方案具体如下：
一种抗癌药物敏感性增强的FACT突变体细胞系的构建方法，具体为：通过改变Ssrp1与组蛋白结合表面的氨基酸电荷属性，得到了影响FACT与组蛋白结合的突变位点Q265K，进而建立FACT突变体细胞系FACT-Q265K。
采用本发明的上述构建方法得到了抗癌药物敏感性增强的FACT突变体细胞系。
本发明构建的抗癌药物敏感性增强的FACT突变体细胞系可以在FACT功能机制研究、筛选和制备抗癌靶向药物中得到应用。
同现有技术相比，本发明的突出效果在于：
（1）本发明构建了携带突变的胚胎干细胞细胞系，可用于深入研究FACT在疾病发生过程中的调控机制，同时为药物筛选研发提供了参考。
（2）药物Curaxins的抑癌作用机制是加强FACT复合物与染色质的结合。本发明构建了与Curaxins抑癌作用机制高度相似的FACT突变体细胞系，该细胞系可为未来靶向药物的研制提供作用模型和热点基因。
（3）在癌症治疗过程中，药物耐受是影响治疗效果的重要问题，本发明筛选得到的FACT-Q265K突变体能够提高细胞对药物的敏感性，该突变位点的发现为解决药物耐受提供了重要参考；已有越来越多的实验证明，FACT的含量与癌症病人的预后生存率密切相关，因此，FACT-Q265K突变体与抑癌药物的协同作用，对于提高Curaxins等药物的抗癌效果将具有积极意义。
下面结合附图说明和具体实施例对本发明所述的抗癌药物敏感性增强的FACT突变体细胞系及构建方法和应用作进一步说明。
附图说明
图1为FACT复合物结构示意图。
图2为测序结果。
图3为Q265K突变提高了FACT结合组蛋白的能力；其中，no-Tag的细胞系为野生型，“Input”代表全细胞提取物，“IP”代表经过亲和纯化得到的蛋白复合物。
图4为Q265K导致FACT结合组蛋白水平上升。
图5为Q265K突变型显著提高细胞对Curaxins的敏感度。
具体实施方式
实施例1
1. FACT复合物
在哺乳动物细胞中，FACT复合物包括两个亚基组成，分别是SUPT16和SSRP1，两个亚基通过DD结构域(Dimerization Domain)形成异源二聚体，SSRP1 MD 包含串联的PH（pleckstrin homology）结构域（如图1所示，Zhou, K.D., Liu, Y., and Luger, K.(2020). Histone chaperone FACT Facilitates Chromatin Transcription:mechanistic and structural insights. Curr Opin Struc Biol 65, 26-32.），参与FACT结合组蛋白的过程。
2. FACT突变体细胞系FACT-Q265K的构建
组蛋白分子伴侣FACT在进化过程中高度保守（McCullough, L. L., B. Poe, Z.Connell, H. Xin and T. Formosa (2013). The FACT Histone Chaperone GuidesHistone H4 Into Its Nucleosomal Conformation in Saccharomyces cerevisiae.Genetics 195: 101 - 113）。本发明通过改变Ssrp1与组蛋白结合表面的氨基酸电荷属性，得到了影响FACT与组蛋白结合的突变位点Q265K，进而建立FACT突变体细胞系FACT-Q265K。
小鼠胚胎干细胞V6.5野生型及构建的突变细胞株均在 SL2i 培养液中进行正常培养。 SL2i（serum/LIF/2i）培养液成分包括 KnockOut DMEM 培养液中添加 15%胎牛血清、1x 青霉素/链霉素、1x GlutaMAX、1x非必需氨基酸（non-essential amino acids，NEAA）、1x 核苷、100 µM β-巯基乙醇、30 ng/mL LIF、1 µM PD0325901及3 µM CHIR99021培养条件为 37℃，5%二氧化碳。
使用 Lipo2000 转染试剂同时向细胞内转染表达特定 sgRNA 和 Cas9 的pX459质粒和携带同源重组模板的 pEASY-Blunt 质粒，两种质粒摩尔比为 1：3。细胞转染12小时后使用 1 µM 的 SCR7（DNA连接酶IV抑制剂）和 0.2 µg/mL的诺考达唑（nocodazole）处理细胞 12 hr。24 hr后使用2 µg/mL 的嘌呤霉素（puromycin）处理约48 hr，进行阳性细胞筛选。构建 FACT-Q265K的sgRNA 为 5’-CGAGTTTGGCCCTGCTTGAT-3’（如SEQ ID NO：1所示），同源重组模板选取基因组上突变位点上下游各 500 bp。（基因组为NCBI ReferenceSequence： NC_000068.8，在Ssrp1编码基因265K的对应碱基进行突变）。
在建立突变体细胞系的基础上，通过测序的方法进行了确认，结果如图2所示。
实施例2 FACT-Q265K对FACT复合物结合组蛋白能力的影响研究
为了进一步验证FACT-Q265K是否影响FACT复合物结合组蛋白的能力，在FACT亚基Supt16蛋白的C端加上eGFP标签，用亲和层析的方法提取FACT蛋白复合物，进而比较WT和FACT-Q265K突变体中FACT复合物中组蛋白的含量。
（1）用胰酶消化的方法收取细胞，磷酸盐缓冲液（PBS buffer）洗涤去除培养基，1000 g 离心5 min收集细胞。
（2）用裂解缓冲液（50 mM HEPES-KOH pH 7.4， 150 mM NaCl， 1 mM EDTA， 1%Triton X-100，0.1% SDS，蛋白酶抑制剂）在冰上裂解 30 min。
（3）超声破碎仪对裂解后的细胞样品进行超声破碎， 脱氧核糖核酸酶I（DNaseI）处理样品，在 4℃摇床转动30 min。离心收集上清，与GFP Trap磁珠在混合孵育2小时。
（4）孵育完成后用洗涤缓冲液洗涤磁珠3次，每次洗涤5 min。
（5）最后用针头吸干磁珠中残余的液体，向其中加入上样缓冲液，于100℃处理10min。
（6）离心后用上样枪头吸取不含磁珠的上清液置于新的1.5 mL离心管中，作为免疫共沉淀样品，利用蛋白质免疫印迹对样品进行分析。
结果如图3所示。实验中所用的针对H3和H2B的抗体都具有较好的特异性。图中no-Tag的细胞系为野生型，“Input”代表全细胞提取物，“IP”代表经过亲和纯化得到的蛋白复合物。
由图3中可以表明，在可溶性的全细胞裂解液中，组蛋白H3和H2B的表达量在野生型和突变型中基本一致，说明Q265K突变不影响组蛋白的表达；在FACT复合物相互作用的蛋白中，与野生型相比，Q265K突变使其结合的H3和H2B显著提高。实验结果表明，Q265K突变升高FACT结合组蛋白的能力。
实施例3 FACT-Q265K提升mSSRP1-MD蛋白结合组蛋白的水平
（1）向大肠杆菌BL21(DE3)中转化整合有目标蛋白序列的pGEX-4T-1（-GST）(GEHealthcare)。
（2）带有GST标签的重组蛋白的诱导条件为：0.1mM IPTG 18℃ 过夜，利用Glutathione agarose (GE Healthcare)进行纯化。
（3）相同质量的mSSRP1-MD野生型和FACT-Q265K突变的重组蛋白分别与GST beads结合，结合体系为A100 buffer （25 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA,and 0.01% TritonX-100），4℃充分结合2h后，分别将结合两种蛋白的GST beads均匀的分为多个等份。
（4）使均分后的GST beads在A100 buffer的体系中结合浓度梯度变化的另一种目标蛋白，4℃孵育过夜。
（5）用A100 buffer洗涤GST beads，随后除去上层液体。
（6）加入十二烷基磺酸钠溶液（SDS buffer），100℃处理3 min，使GST beads结合的蛋白质复合物充分变性。
（7）利用考蛋白免疫印迹的方法分析两种蛋白的结合情况。
通过在原核细胞中表达并纯化了带有GST标签的mSSRP1-MD蛋白（WT和Q265K突变型），通过体外下拉实验（pull-down assay）来观察SSRP1-MD结合组蛋白H3-H4的情况。结果如图4所示，GST-Ssrp1-M和组蛋白H3-H4由原核细胞表达并纯化，GST-Ssrp1-M结合组蛋白的浓度梯度如图中所示，Q265K使Ssrp1-M结构域结合组蛋白H3-H4的能力升高。可见SSRP1-MD可以直接结合组蛋白H3-H4，而FACT-Q265K突变体提升了两者的结合能力。
实施例4 Q265K突变型显著提高细胞对Curaxins的敏感度研究
（1）将前述构建的野生型和突变型细胞以相同起始量的细胞进行培养。
（2）正常培养24 hr后使用1 µΜ浓度的Curaxins处理细胞24 小时（Curaxins药物处理组）或不进行处理（无药物处理组）。
（3）对存活的细胞量进行计数：每个数据来自两个克隆的各三个技术重复合并后的结果。
FACT 在部分肿瘤细胞中的表达量较高，Curaxins是一种常见治疗癌症的药物，其工作原理是提高FACT与染色质的结合，药物与FACT协同作用，导致细胞基因表达发生改变，从而发挥抑癌作用。
本实验的结果如图5所示，从中可以观察到FACT-Q265K突变体显著提高细胞对Curaxins的敏感度，提示未来研究FACT突变可作为癌症治疗中的靶向位点。
综上所述，本发明基于对SSRP1 M domain结构的分析，筛选得到的FACT-Q265K突变体，在突变体细胞中，FACT与染色质的结合显著升高。该突变体提供了研究FACT功能机制的切入点。FACT-Q265K突变体与抗癌药物Curaxins的协同作用，FACT-Q265K突变显著提高了细胞对于Curaxins的敏感性，显示FACT的突变位点未来可能成为抗癌药物研究的重要靶点，同时帮助解决耐药性问题，提升癌症病人的预后生存率。基于FACT作用功能与突变位点机制的分析，可能为后续癌症病人诊断分流提供参考，助力相关疾病的科学治疗。
以上所述的实施例仅仅是对本发明的优选实施方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案作出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。
Claims (1)
1.一种抗癌药物敏感性增强的FACT突变体细胞系在筛选和制备抗癌靶向药物中的应用，其特征在于：所述抗癌药物敏感性增强的FACT突变体细胞系的构建方法为，通过改变Ssrp1与组蛋白结合表面的氨基酸电荷属性，得到影响FACT与组蛋白结合的突变位点Q265K，进而建立FACT突变体细胞系FACT-Q265K；所述FACT突变作为筛选和制备抗癌靶向药物中的靶向位点。
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