CN114958854B - Dcaf13抑制剂在制备治疗肺腺癌的药物中的应用 - Google Patents
Dcaf13抑制剂在制备治疗肺腺癌的药物中的应用 Download PDFInfo
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Abstract
本发明涉及一种靶向DCAF13基因的抑制剂及其应用，所述抑制剂为选自si13#1、si13#2或si13#3中的一种或多种的小干扰RNA。本发明发现DCAF13在肺腺癌的增殖和转移过程中发挥促进作用，通过敲低DCAF13的siRNA可以抑制肺腺癌的增殖和转移。本发明为研发靶向肺腺癌的靶向治疗方法提供了新的策略，具有广阔的临床应用前景和商业应用前景。
Description
技术领域
本发明涉及生物领域，具体涉及一种治疗肺腺癌的药物及其应用。
背景技术
肺癌作为全球最常见的恶性肿瘤，其在2020年全球癌症死亡率中排名第一，五年生存率低于20％。且该病发病率排名第二，仅次于女性乳腺癌，每年约有220万新发病例和180万死亡病例。在我国，肺癌患者存在确诊时间晚、预后差等问题，其防治形势十分严峻。
根据组织病理学，肺癌有两种主要类型：非小细胞肺癌(non-small cell lungcancer，NSCLC)和小细胞肺癌(small cell lung cancer，SCLC)。NSCLC约占所有病例的80-85％，包括腺癌、鳞状细胞癌和大细胞癌。肺腺癌(lung adenocarcinoma，LUAD)是最常见的NSCLC类型，约占所有恶性NSCLC的一半。
目前肺癌的治疗方案有手术、局部放射治疗和全身药物治疗，但均无法解决五年生存率低的问题，因此急需寻找新的、更好的治疗方案。目前临床一线治疗方案中已经应用了靶向肺癌最常见的突变基因表皮生长因子受体(epidermal growth factor receptor，EGFR)和ALK受体酪氨酸激酶(ALK receptor tyrosine kinase，ALK)的抑制剂，但是仅有不到25％的患者能从靶向治疗中受益，且在治疗期间常会产生耐药现象，致使其依然无法有效提高患者的五年生存率。因此，亟需确定参与肺腺癌进展的关键信号分子，发现潜在的治疗靶点，改善现有治疗方案。
泛素-蛋白酶体降解系统在人体内的多个基本生化过程中起关键作用，包括蛋白质的降解，细胞周期进程，细胞凋亡，DNA损伤修复和信号转导等。Cullin-ring连接酶(CRL)属于RINGE3泛素连接酶的超家族。CRL包含三个主要成分：一个cullin支架蛋白，一个锌指蛋白和一个底物识别单元(包括衔接蛋白DDB1和多种底物识别蛋白)。蛋白酶体泛素降解途径的异常常常会导致蛋白表达的异常，从而影响肿瘤的发生发展以及治疗。DCAF13(DDB1and CUL4 associated factor 13)是CRL中关键的一员底物识别蛋白，在E3泛素连接酶CRLs家族中发挥至关重要的的作用，但是对于他的癌症研究目前很少。根据研究报道，DCAF13在发育过程中发挥关键作用，例如CRL4-DCAF13介导SUV39H1histone lysinemethyltransferase(SUV39H1)多泛素化和降解是胚胎发育过程中的重要步骤。此外，CRL4B-DCAF13促进phosphatase and tensin homolog(PTEN)泛素化促进骨肉瘤细胞生长。上述结果提示DCAF13在癌症中可能发挥重要作用，但是目前尚未见关于DCAF13基因在肺腺癌中的功能与机制的任何报道。
发明内容
本发明的目的在于提供一种靶向DCAF13基因的治疗肺癌，特别是肺腺癌的药物及其应用。
针对上述发明目的，本发明提供以下技术方案：
本发明第一方面提供一种靶向DCAF13基因的抑制剂，所述抑制剂为选自si13#1、si13#2或si13#3中的一种或多种的小干扰RNA，其中si13#1的正义链和反义链序列分别如SEQ ID NO.1和SEQ ID NO.2所示，si13#2的正义链和反义链序列分别如SEQ ID NO.3和SEQID NO.4所示；si13#3的正义链和反义链序列分别如SEQ ID NO.5和SEQ ID NO.6所示。
所述SEQ ID NO.1的序列为CGAAUCUUUCCUGUAGACAdTdT。
所述SEQ ID NO.2的序列为UGUCUACAGGAAAGAUUCGdTdT。
所述SEQ ID NO.3的序列为CAGUGUAUACUGGGAUUGAdTdT。
所述SEQ ID NO.4的序列为UCAAUCCCAGUAUACACUGdTdT。
所述SEQ ID NO.5的序列为GUGUGAUGGAGAGGUUAGAdTdT。
所述SEQ ID NO.6的序列为UCUAACCUCUCCAUCACACdTdT。
本发明的第二方面提供一种包含上述小干扰RNA的载体。
优选的，所述载体为慢病毒或腺病毒。
本发明的第三方面提供上述小干扰RNA或包含上述小干扰RNA的载体在制备治疗肺癌的药物中的应用。
优选的，所述肺癌是非小细胞肺癌。
更优选的，所述非小细胞肺癌是腺癌、鳞状细胞癌和大细胞癌。
进一步优选的，所述肺癌是肺腺癌。
进一步优选的，所述肺腺癌是人肺腺癌细胞A549或NCI-H1299导致的癌症。
本发明的第四方面提供上述小干扰RNA在制备敲低肺腺癌细胞中DCAF13基因的药物的应用。
优选的，所述肺腺癌细胞为人肺腺癌细胞A549或NCI-H1299。
本发明的第五方面提供上述小干扰RNA在制备抑制肺腺癌细胞增殖的药物中的应用。
优选的，所述肺腺癌细胞为人肺腺癌细胞A549或NCI-H1299。
本发明的第六方面提供上述小干扰RNA在制备抑制肺腺癌细胞迁移的药物中的应用。
优选的，所述肺腺癌细胞为人肺腺癌细胞A549或NCI-H1299。
本发明的第七方面提供上述小干扰RNA在制备抑制肺腺癌细胞克隆形成能力和/或细胞活力的药物中的应用。
优选的，所述肺腺癌细胞为人肺腺癌细胞A549或NCI-H1299。
本发明具有积极有益的效果：
本申请的发明人意外发现，DCAF13在肺腺癌的增殖和转移过程中发挥促进作用，通过敲低DCAF13的siRNA(例如si13#1、si13#2、si13#3)可以抑制肺腺癌的增殖和转移。本发明的意外发现为研发靶向肺腺癌的靶向治疗方法提供了新的策略，具有广阔的临床应用前景和商业应用前景。
附图说明
图1是本发明小干扰RNA敲低DCAF13细胞的克隆形成实验结果；
图2是本发明小干扰RNA敲低DCAF13细胞的MTT实验结果；
图3是本发明小干扰RNA敲低DCAF13细胞的迁移能力实验结果；
图4是本发明小干扰RNA敲低DCAF13细胞的凋亡实验结果；
图5是本发明小干扰RNA敲低DCAF13细胞小鼠皮下成瘤实验结果。
具体实施方式
下面对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
如无特别说明，本申请试验例中使用的细胞为人肺腺癌A549和NCI-H1299细胞(购自中国科学院(上海)细胞库)。细胞在补充有10％胎牛血清和100U/ml青霉素-链霉素的F-12K或RPMI1640培养基(Gibco)中培养，并在5％CO2加湿培养箱中保持在37℃。试验例中使用的三个siRNA的序列分别为：
si13#1正义链(5'-3')：CGAAUCUUUCCUGUAGACAdTdT(SEQ ID NO.1)
si13#1反义链(5'-3')：UGUCUACAGGAAAGAUUCGdTdT(SEQ ID NO.2)
si13#2正义链(5'-3')：CAGUGUAUACUGGGAUUGAdTdT(SEQ ID NO.3)
si13#2反义链(5'-3')：UCAAUCCCAGUAUACACUGdTdT(SEQ ID NO.4)
si13#3正义链(5'-3')：GUGUGAUGGAGAGGUUAGAdTdT(SEQ ID NO.5)
si13#3反义链(5'-3')：UCUAACCUCUCCAUCACACdTdT(SEQ ID NO.6)
小干扰RNA转染严格按照GP-transfect-Mate转染试剂(GenePharma)的说明进行。
试验例1、细胞克隆实验
1、试验方法
为了检测DCAF13对细胞克隆形成能力的影响，使用了三种不同的小干扰RNA来抑制DCAF13的表达。siRNA转染48小时后，消化细胞并取1000个细胞均匀分布在新的3.5cm培养皿中。培养14天后，细胞用多聚甲醛固定15分钟，R250染色15分钟，观察拍照。
2、实验结果
为了评估DCAF13在细胞中的生物学功能，使用了三种不同的小干扰RNA来敲低DCAF13在人肺癌细胞中的蛋白表达。克隆形成实验结果表明，利用三种小干扰RNA敲低DCAF13与对照组相比，A549细胞的克隆点明显减少(图1A)，NCI-H1299中得到了同样的结果(图1B)，表明DCAF13敲低显著降低了肺腺癌细胞的克隆形成能力。
试验例2、MTT实验
1、试验方法
为了检测DCAF13对细胞增殖和活力的影响，siRNA转染48小时后，消化细胞并计数，分于96孔板中，每孔加入包含4000个细胞的200μl培养基，待细胞贴壁后，在第0，2，4，6天分别加入90μL新鲜培养液，再加入10μLMTT溶液，继续培养1小时后，在酶标仪490nm处测量各孔的吸光值。
2、试验结果
MTT试验结果显示，利用三种siRNA敲低DCAF13与对照组相比，A549细胞的活力明显削弱(图2)，表明DCAF13敲低显著抑制了A549细胞的生长。
试验例3、细胞迁移实验
1、试验方法
为了检测DCAF13对细胞迁移能力的影响，siRNA转染后48小时，将2×104个细胞铺板于Transwell的上室，上室使用100μL无血清或10％血清的培养基，下室使用含20％血清的600μL培养基。24小时后，观察细胞在板底的粘附情况。95％无水乙醇固定细胞15分钟，结晶紫染色细胞15分钟，观察拍照。
2、试验结果
Transwell实验结果表明，利用三种siRNA敲低DCAF13与对照组相比，发生迁移的A549(图3A)和NCI-H1299(图3B)细胞数量显著减少，表明DCAF13敲低显著抑制了肺腺癌细胞的迁移能力，预示着本申请的小干扰RNA可用于改善体内肺腺癌细胞的转移，进而控制肺腺癌的发展。
试验例4、细胞凋亡实验
1、试验方法
为了检测DCAF13对细胞凋亡能力的影响，siRNA转染48小时后，使用不含EDTA的胰酶消化细胞，终止消化后收集5*105个细胞，之后严格按照凋亡试剂盒(vazyme，A211-01)使用说明进行。洗涤细胞后用100μl1xBingBuffer重悬细胞，再加入5μl Annexin V-FITC和5μl PI Staining Solution对细胞进行染色，轻轻吹匀；避光、室温(20-25℃)孵育10min；加入400μl 1×Binding Buffer，轻轻混匀。染色后样品在1h内用流式细胞仪检测。
2、试验结果
细胞凋亡实验结果表明，利用三种siRNA敲低DCAF13与对照组相比，晚期凋亡的A549细胞数量显著增加(图4)，表明DCAF13敲低显著促进了A549细胞的晚期凋亡。
试验例5、小鼠皮下成瘤实验
1、试验方法
使用序列si13#1由吉凯基因(中国上海)制备慢病毒RNAi(载体为GV493)，转染A549细胞并用嘌呤霉素筛选。将1×106个shControl或sh13#1(即使用si13#1制备的慢病毒)细胞皮下分别注射到小鼠腋部的皮下。每周用电子卡尺测量肿瘤直径。肿瘤体积(mm3)计算为体积＝(短直径2×长直径)/2。四个星期后，处死小鼠并分离皮下肿瘤，称重。所有动物实验程序均按照动物伦理委员会批准的伦理规范进行。
2、试验结果
通过小鼠模型验证DCAF13对肺腺癌细胞体内功能的影响，小鼠皮下成瘤实验结果表明，DCAF13敲低组与对照组相比，肿瘤的大小、体积和重量均明显下降(图5)，表明DCAF13敲低可以在体内抑制肺腺癌细胞的生长。
上述实验结果表明，DCAF13在肺腺癌的增殖和转移过程中发挥促进作用，DCAF13可能成为肺腺癌新的治疗靶点，因此通过敲低DCAF13的siRNA、慢病毒或者DCAF13抑制剂有希望抑制肺腺癌的增殖和转移，进而为研发靶向肺腺癌的靶向治疗提供新的策略。
虽然已经对本发明的具体实施方案进行了描述，但是本领域技术人员应认识到，在不偏离本发明的范围或精神的前提下可以对本发明进行多种改变与修饰。因而，本发明意欲涵盖落在附属权利要求书及其同等物范围内的所有这些改变与修饰。
序列表
<110> 宁波大学附属人民医院
<120> DCAF13抑制剂在制备治疗肺腺癌的药物中的应用
<160> 6
<170> SIPOSequenceListing 1.0
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<213> 人工序列(Artificial Sequence)
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<213> 人工序列(Artificial Sequence)
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<213> 人工序列(Artificial Sequence)
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Claims (7)
1.一种靶向DCAF13基因的小干扰RNA在制备治疗肺癌的药物中的应用，其特征在于，所述小干扰RNA选自si13#1或si13#3的小干扰RNA，其中si13#1的正义链和反义链序列分别如SEQ ID NO.1和SEQ ID NO.2所示，si13#3的正义链和反义链序列分别如SEQ ID NO.5和SEQID NO.6所示；所述肺癌是非小细胞肺癌。
2.一种包含靶向DCAF13基因的小干扰RNA的载体在制备治疗肺癌的药物中的应用，其特征在于，所述小干扰RNA选自si13#1或si13#3的小干扰RNA，其中si13#1的正义链和反义链序列分别如SEQ ID NO.1和SEQ ID NO.2所示，si13#3的正义链和反义链序列分别如SEQID NO.5和SEQ ID NO.6所示；所述肺癌是非小细胞肺癌。
3.根据权利要求2所述的应用，其特征在于，所述载体为慢病毒或腺病毒。
4.根据权利要求1-3任一项所述的应用，其特征在于，所述非小细胞肺癌是腺癌、鳞状细胞癌和大细胞癌。
5.权利要求1中所述的小干扰RNA在制备敲低肺腺癌细胞中DCAF13基因的药物中的应用。
6.权利要求1中所述的小干扰RNA在制备抑制肺腺癌细胞增殖的药物中的应用。
7.根据权利要求6所述的应用，其特征在于，所述肺腺癌细胞为人肺腺癌细胞A549或NCI-H1299。
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