CN114712507B - 一种trim4在制备治疗卵巢癌药物的增敏剂或逆转耐药剂中的应用 - Google Patents

一种trim4在制备治疗卵巢癌药物的增敏剂或逆转耐药剂中的应用 Download PDF

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CN114712507B
CN114712507B CN202210329373.8A CN202210329373A CN114712507B CN 114712507 B CN114712507 B CN 114712507B CN 202210329373 A CN202210329373 A CN 202210329373A CN 114712507 B CN114712507 B CN 114712507B
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ovarian cancer
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CN114712507A (zh
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冯炜炜
车晓霞
建方方
王一唯
项怡宁
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Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
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Abstract

本发明涉及一种TRIM4在制备治疗卵巢癌药物的增敏剂或逆转耐药剂中的应用，TRIM4为靶标的产品用于改善PARP抑制剂耐药问题。

Description

一种TRIM4在制备治疗卵巢癌药物的增敏剂或逆转耐药剂中 的应用
技术领域
本发明属于增敏剂领域，特别涉及一种TRIM4在制备治疗卵巢癌药物的增敏剂或逆转耐药剂中的应用。
背景技术
卵巢癌是病死率最高的女性生殖系统恶性肿瘤，70％的卵巢癌患者就诊时已是临床晚期。卵巢癌首选治疗模式为手术联合以铂类为基础的化疗。虽然，大多数患者经过初始治疗可获得临床缓解，但仍有70％的患者在3年内复发，部分患者会经历多次复发，且复发间歇期越来越短，5年生存率只有40％左右。
发明内容
本发明所要解决的技术问题是提供一种TRIM4在制备治疗卵巢癌药物的增敏剂或逆转耐药剂中的应用。
本发明的一种TRIM4在制备治疗卵巢癌药物的增敏剂或逆转耐药剂中的应用。
本发明的一种TRIM4在制备治疗BRCA野生型卵巢癌药物的增敏剂或逆转耐药剂中的应用。
所述治疗卵巢癌药物为PARP抑制剂。
所述TRIM4为以TRIM4为靶标的制剂。
所述增敏剂或逆转耐药剂的制剂剂型包括口服液、胶囊剂、油滴剂、粉剂、片剂或注射剂。
有益效果
本发明中表明降调E3泛素连接酶(TRIM4)能显著增加卵巢癌PARP抑制剂的敏感性。相关研究成果将为BRCA野生型卵巢癌的维持治疗疗效的改善提供新的解决方案。
TRIM4为靶标的产品用于改善PARP抑制剂耐药问题。
附图说明
图1为CRISPR/cas9文库阴性筛选结果；其中A：RIGER分析图，排名靠前的基因，包括MRGBP、KPNA3、DLL4、USP47、POLD1、BRD2等；B：气泡图，差异大的基因已被标注出来；
图2为敲低TRIM4后，卵巢癌细胞中对尼拉帕尼的敏感性增加；其中A：7株卵巢癌细胞系中TRIM4蛋白的表达情况；B：7株卵巢癌细胞系中TRIM4 mRNA的表达情况；C和D为在高表达TRIM4的OVCA433及SKOV3细胞中，干扰TRIM4,CCK8法检测细胞生长及细胞对尼拉帕利的敏感性，可以看到敲低TRIM4后，尼拉帕利的IC50值降低；
图3为TRIM4参与卵巢癌细胞耐药的发生；A：成功构建SKOV3尼拉帕利耐药株(SKOV3-NR)，耐药指数为22.5；B：TRIM4在SKOV3-NR中mRNA表达较SKOV3亲本细胞高；C：TRIM4在SKOV3-NR中蛋白表达较亲本细胞高，**p<0.01；D：在SKOV3-NR中，敲低TRIM4，尼拉帕利IC50显著降低；
图4为TRIM4的互作蛋白hnRNPDL筛选；A：TRIM4的互作蛋白的火山图；B：GO分析TRIM4可能结合蛋白的功能富集，主要集中在mRNA剪切通路等；
图5为TRIM4和hnRNPDL相互作用的验证；A：相对于SKOV3，TRIM4在SKOV3-NR中mRNA表达上调，hnRNPDL表达不变，***p<0.001；B：在SKOV3-NR细胞中，TRIM4蛋白表达上调，hnRNPDL蛋白表达下调；C：在Hela细胞中，瞬转TRIM4，hnRNPDL过表达质粒，IP技术证明两者存在外源性结合；
图6为过表达hnRNPDL能改善耐药；A：在SKOV3-NR细胞中过表达hnRNPDL效率验证；B：在SKOV3-NR细胞中，过表达hnRNPDL，能显著降低尼拉帕利IC50。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1
卵巢癌PARP抑制剂耐药相关基因筛选的细胞模型构建方法：利用CRISPR/Cas9文库系统，从全基因组水平筛选卵巢癌细胞中影响PARP抑制剂敏感性的分子靶点。GeCKO v2文库包含123,411个sgRNA，靶向19,050个基因，平均每个基因6个位点。在低感染复数(MOI＝0.3)下转染SKOV3细胞并进行嘌呤霉素选择，构建稳定CRISPR/cas9敲除细胞系SKOV3-GeCKO。用临床试验中证实对全人群有效的PARP抑制剂尼拉帕利(Niraparib)4μM处理48h后撤药，继续扩增细胞至14天，以进行阴性筛选。分别收取Day7、Day14的细胞全基因组DNA，深度测序筛选出与对照组相比减少的sgRNA。
从CRISPR/Cas9基因敲除文库筛选中，鉴定了靶向1238个基因的sgRNA的子集，这些基因在尼拉帕利处理后的细胞中显著消耗(Z评分＞1.4)，表明这些基因可能与PARP抑制剂敏感性相关(如图1所示)。选取排名靠前的十余个基因，进行药物敏感性验证。利用siRNA敲低候选基因，CCK-8检测细胞生长，发现敲低TRIM4后，SKOV3细胞对尼拉帕利的IC50值显著降低。
另外，分析了7株卵巢癌细胞系(SKOV3，A2780，HEY，OVCA433，OVCAR3，OVCAR5，HO8910)中TRIM4的mRNA及蛋白水平，发现TRIM4在卵巢癌细胞系OVCA433和SKOV3中表达相对较高(如图2A,B所示)。进一步在OVCA433和SKOV3细胞中干扰TRIM4后，发现降调TRIM4可显著增强卵巢癌细胞对尼拉帕利的敏感性(如图2C，D所示)。
选取卵巢癌SKOV3细胞系，采用尼拉帕利低浓度逐步加量诱导法历时9个月成功构建SKOV3尼拉帕利耐药株(SKOV3-NR)，耐药指数为22.5(如图3A所示)。发现TRIM4在耐药株中mRNA和蛋白水平均升高(如图3B,C所示)，而敲低SKOV3-NR细胞中TRIM4后，尼拉帕利的IC50值显著降低(图3D)
为了进一步探讨TRIM4参与卵巢癌PARP抑制剂耐药的分子机制，在293T细胞中进行IP-MS以探索与TRIM4存在相互作用的蛋白，初步分析结果显示一系列mRNA剪切相关蛋白与之结合，其中以RNA结合蛋白hnRNPDL(heterogeneous nuclear ribonucleoprotein D-like protein)最为显著(如图4所示)。Co-IP实验也证实了TRIM4与hnRNPDL的结合(图5C)。RNA结合蛋白hnRNPDL是核不均一核糖核蛋白(hnRNPs)家族成员，主要通过结合外显子的3’-UTR段，参与RNA前体的剪接，从而影响mRNA的稳定性，参与多种肿瘤的增殖、迁移以及耐药等过程。发现SKOV3-NR细胞中hnRNPDL的蛋白表达明显下调，mRNA水平不变(图5A,B)；且在SKOV3-NR细胞中，过表达hnRNPDL后，显著增加了尼拉帕利的敏感性(图6B)。

Claims (3)

1.一种以TRIM4为靶标且敲低或干扰TRIM4的制剂在制备治疗卵巢癌药物尼拉帕利的增敏剂或逆转耐药剂中的应用。
2.一种以TRIM4为靶标且敲低或干扰TRIM4的制剂在制备治疗BRCA野生型卵巢癌药物尼拉帕利的增敏剂或逆转耐药剂中的应用。
3.根据权利要求1或2所述应用，其特征在于，所述增敏剂或逆转耐药剂的制剂剂型包括口服液、胶囊剂、油滴剂、粉剂、片剂或注射剂。
CN202210329373.8A 2022-03-30 2022-03-30 一种trim4在制备治疗卵巢癌药物的增敏剂或逆转耐药剂中的应用 Active CN114712507B (zh)

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CN111979290A (zh) * 2020-09-04 2020-11-24 大连医科大学附属第二医院 Spp1基因在制备增强卵巢癌患者对parp抑制剂敏感性的药物中的应用

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CN111979290A (zh) * 2020-09-04 2020-11-24 大连医科大学附属第二医院 Spp1基因在制备增强卵巢癌患者对parp抑制剂敏感性的药物中的应用

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