CN116077661A - Kdm1a抑制剂在制备治疗dnmt3a基因缺失癌症的药物中的用途 - Google Patents
Kdm1a抑制剂在制备治疗dnmt3a基因缺失癌症的药物中的用途 Download PDFInfo
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- CN116077661A CN116077661A CN202211079575.8A CN202211079575A CN116077661A CN 116077661 A CN116077661 A CN 116077661A CN 202211079575 A CN202211079575 A CN 202211079575A CN 116077661 A CN116077661 A CN 116077661A
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Abstract
本发明属于医药生物技术领域,主要涉及一种新的癌症治疗靶点赖氨酸特异性组蛋白去甲基化酶1(KDM1A)抑制剂在制备治疗DNMT3A基因缺失癌症的药物中的用途。本发明从小分子表观遗传学抑制剂库中筛选出一类KDM1A抑制剂,发现其能够选择性抑制DNMT3A缺失肿瘤细胞的增殖、迁移,诱导细胞凋亡。同时,在异位移植瘤模型小鼠中抑制肿瘤的发生发展并出现显著抑制肿瘤肝转移的现象,且对小鼠的毒性微弱。本发明首次揭示了第一种被发现的组蛋白去甲基化酶KDM1A在DNMT3A缺失癌症中扮演的重要角色,提示KDM1A可作为生物标志物用于DNMT3A基因缺失癌症患者的诊断、治疗或预后预测,同时提示靶向抑制KDM1A未来可能为临床上DNMT3A缺失型肿瘤患者提供一个良好的、可行的治疗手段。
Description
本发明要求中国专利申请CN202211004929.2的优先权,该优先权文件的说明书、说明书附图和权利要求书所记载的内容全文引入本发明的说明书并被作为本发明说明书原始记载的一部分。申请人进一步声明,申请人拥有基于该优先权文件修改本发明的说明书和权利要求书的权利。
技术领域
本发明属于医药生物技术领域,具体涉及一种新的癌症治疗靶点KDM1A抑制剂在制备治疗DNMT3A基因缺失癌症的药物中的用途。
背景技术
根据2020年最新全球癌症报告数据显示,肺癌新增224万例,发病率仅次于乳腺癌,约占11.4%,位于全球第二,但其死亡率仍占据全球首位,为18%,是癌症死亡的主要原因,其治疗手段括化疗、手术、分子靶向、免疫治疗等一直是临床上的研究热点。近年来,表观遗传学领域的研究异军突起,表观遗传学与癌症的发生发展密切相关,目前表观遗传学已成为指导新药研发、评价新药、开发新的治疗策略的重要工具,寻找新的表观遗传学治疗靶点将会为克服和治疗癌症提供新方法和新思路。DNA甲基化是最经典的表观遗传调控之一,是可以将甲基转移到胞嘧啶的5位的一种碱基共价修饰过程,参与基因表达的调控、转座子的沉默、基因组印记等体内多种重要的生理病理过程。根据其结构和功能的不同,DNMT甲基转移酶(DNA Methyltransferase, DNMT)家族主要包括三个具有功能性甲基化活性的主要成员,分别是在DNA复制期间维持甲基化的DNMT1,以及负责催化DNA的从头甲基化的DNMT3A和DNMT3B。近年来,多项临床研究表明DNA甲基化模式的改变与肿瘤的发生发展密切相关。DNMT3A催化结构域发生突变,使酶活性显著降低,导致DNMT3A突变为功能缺失性突变。研究表明这种功能缺失性突变占所有突变的90%,并导致癌症患者的不良预后。在肺腺癌和肺鳞癌中,DNMT3A在肿瘤组织中的表达量显著低于正常组织,且本发明研究前期数据表明DNMT3A基因缺失能够增加肺癌表型的恶性程度,以上均提示在肺癌中,DNMT3A可能作为抑癌基因而发挥作用,也提示了DNMT3A有作为协同致死基因的潜质。因此,寻找靶向DNMT3A基因缺失癌症的协同致死对,探索新型治疗手段至关重要。
发明内容
为了解决上述技术问题,本发明首次从表观遗传学的角度确证了KDM1A抑制剂可用于治疗DNMT3A基因缺失的癌症,并且在此基础上开发了KDM1A抑制剂在制备治疗DNMT3A基因缺失癌症的药物中的用途。
具体地,通过以下几个方面的技术方案实现本发明:
在第一个方面中,本发明提供了组蛋白赖氨酸去甲基酶1A(KDM1A)抑制剂在制备治疗DNMT3A基因缺失癌症的药物中的用途。
作为可选的方式,在上述用途中,所述癌症选自以下一种或多种:肺癌、白血病、胃癌、结肠癌、食管癌、胶质瘤、宫颈癌、子宫内膜癌、肝癌或肉瘤。
作为可选的方式,在上述用途中,所述KDM1A抑制剂是从转录或翻译水平上抑制KDM1A基因表达的物质。
优选地,所述KDM1A抑制剂是指小分子KDM1A抑制剂或者对KDM1A具有抑制作用的RNA药物。
作为可选的方式,在上述用途中,所述小分子KDM1A抑制剂选自以下一种或多种:ORY-1001/RG6016、ORY-2001、GSK-2879552、IMG-7289、OG-L002、INCB059872、CC-90011、CBB1007、GSK J2、GSK J4 HCl、GSK J5、GSK-LSD1 2HCl、GSK-LSD1-d4 2HCl、反式-2-(苯基-D5)环丙胺盐酸盐、RG6016、萘莫胺、DDP-38003、HE006、环丙胺(TCP)、反苯环丙胺(Parnate)、SP-2577、SP-2509、CC-90011、XHW-9e、TAK-418、Vafidemstat、Seclidemstat、Bomedemstat、RN-1 2HCl、反式苯环丙胺半硫酸盐、Iadademstat、LSD1/ER-IN-1、LSD1-IN-17、黄素腺嘌呤二核苷酸、2,4-吡啶酸、去甲乌药碱、山柑子碱、辣椒素、脱氢延胡索碱、葛根、黄芩、α-芒果苷、2,4-吡啶二羧酸或反式苯环丙胺。
作为可选的方式,在上述用途中,所述KDM1A抑制剂抑制DNMT3A基因缺失引起的癌细胞的恶性表型增加。
优选地,所述癌细胞的恶性表型增加包括癌细胞的增殖和迁移。
在第二个方面中,本发明提供了一种DNMT3A基因缺失癌症的生物标志物,所述生物标志物包含KDM1A基因或KDM1A蛋白,其中所述KDM1A基因或所述KDM1A蛋白在DNMT3A基因缺失癌症中上调,所述生物标志物用于DNMT3A基因缺失癌症患者的诊断、预后预测或用药指导。
作为可选的方式,在上述生物标志物中,所述癌症选自以下一种或多种:肺癌、白血病、胃癌、结肠癌、食管癌、胶质瘤、宫颈癌、子宫内膜癌、肝癌或肉瘤。
在第三个方面中,本发明提供了上述第二个方面所述的生物标志物在制备用于DNMT3A基因缺失癌症患者的诊断、预后预测或用药指导的试剂盒中的用途。
在第四个方面中,本发明提供了一种DNMT3A基因缺失细胞选择性杀伤药物的体外筛选方法,所述筛选方法包括以下步骤:
(1)构建DNMT3A基因缺失的癌细胞;
(2)在步骤(1)中构建DNMT3A基因缺失的癌细胞中加入候选药物,待所述候选药物分别与构建DNMT3A基因缺失的癌细胞以及配对野生型癌细胞孵育一段时间后,检测所述DNMT3A基因缺失的癌细胞和所述配对野生型癌细胞的细胞成活率;
(3)如果与所述配对野生型细胞相比,所述DNMT3A基因缺失的癌细胞对所述候选药物更敏感,则认为所述候选药物是所述DNMT3A基因缺失的癌细胞的选择性杀伤药物。
作为可选的方式,在上述体外筛选方法中,所述癌细胞选自以下一种或多种:肺癌、白血病、胃癌、结肠癌、食管癌、胶质瘤、宫颈癌、子宫内膜癌、肝癌或肉瘤。
本发明相对于现有技术,具有以下有益效果:
本发明首次发现,DNMT3A在某些癌症中可能具有抑癌基因的潜质,其缺失可促进某些癌症恶性表型增加。KDM1A抑制剂能够选择特异性的杀伤DNMT3A缺失的肿瘤细胞,未来为多种DNMT3A基因缺失的恶性肿瘤患者提供一种肿瘤精准治疗的策略。
除此之外,本发明首次发现,在肿瘤细胞中KDM1A与DNMT3A之间存在相互依赖关系,可协同调控肿瘤的发生、发展、转移。KDM1A在DNMT3A基因缺失的肿瘤细胞中表达量上调,其可以作为诊断、预后预测以及用药指导的肿瘤生物标志物,为后续患者的诊断和临床指导用药方面提供了新的作用靶点和理论基础,同时也提示该靶点还可以用于筛选制备治疗DNMT3A缺失癌症的药物,加速向临床应用转化。
附图说明
图1是CRISPR/Cas9构建DNMT3A敲除细胞株H460/H1299/PC9 DNMT3A KO并检测其DNMT3A/3B/1表达水平的检测结果。
图2是H460 WT/DNMT3A KO细胞对表观遗传小分子抑制剂库筛选流程及筛药结果。
图3是H460/H1299 WT及DNMT3A KO细胞对筛选出的KDM1A抑制剂的细胞存活率结果。
图4是H460/H1299 WT及H460/H1299 DNMT3A KO细胞在体外水平经KDM1A抑制剂处理后对DNMT3A敲除细胞生物学特性克隆形成能力的检测结果。
图5是H460/H1299 WT及H460/H1299 DNMT3A KO细胞在体外水平经KDM1A抑制剂对DNMT3A敲除细胞凋亡的检测结果。
图6是H460/H1299 WT及H460/H1299 DNMT3A KO细胞经KDM1A siRNA瞬时转染后KDM1A表达水平。
图7是H460/H1299 WT及H460/H1299 DNMT3A KO细胞经KDM1A siRNA瞬时转染后对于肺癌细胞克隆形成能力的的检测结果。
图8是H460/H1299 WT及H460/H1299 DNMT3A KO细胞经KDM1A siRNA瞬时转染后对于DNMT3A野生型及敲除细胞凋亡的检测结果。
图9是H460/H1299 WT及H460/H1299 DNMT3A KO细胞重输DNMT3A后DNMT3A的表达情况及经KDM1A抑制剂处理后细胞存活率结果。
图10是H460/H1299 WT及H460/H1299 DNMT3A KO细胞重输DNMT3A后并用KDM1A抑制剂处理后肺癌细胞克隆形成能力的检测结果。
图11是H460/H1299 WT及H460/H1299 DNMT3A KO细胞重输DNMT3A后并经KDM1A抑制剂处理后对于DNMT3A敲除和重输细胞凋亡的检测结果。
图12是H460/H1299 WT及H460/H1299 DNMT3A KO细胞荷瘤裸鼠经溶剂、KDM1A抑制剂处理后肿瘤体积、瘤重及抑瘤率的考察结果。
图13是H460/H1299 WT及H460/H1299 DNMT3A KO细胞荷瘤裸鼠经溶剂、KDM1A抑制剂处理后肿瘤体重及脏器指数的考察结果。
图14是H460 WT/DNMT3A KO细胞荷瘤裸鼠经溶剂、KDM1A抑制剂处理后肿瘤切片Ki67的检测结果。
图15是H460 WT/DNMT3A KO细胞荷瘤裸鼠经溶剂、KDM1A抑制剂处理后肿瘤切片TUNEL的检测结果。
图16是H460 WT/DNMT3A KO细胞荷瘤裸鼠经溶剂、KDM1A抑制剂处理后肿瘤肝转移结节的病理结果以及肿瘤切片HE的检测结果。
图17是H460 WT/DNMT3A KO细胞经KDM1A抑制剂处理后WT/KO细胞的迁移能力的检测结果。
图18是多种癌症细胞WT及其配对DNMT3A KO细胞经KDM1A抑制剂处理后WT/KO细胞的成活率的检测结果。
图19是DNMT3A敲除细胞KDM1A表达增高的检测结果。
具体实施方式
下面参照具体的实施例对本发明做进一步说明。应当理解,此处所描述的具体实施例仅用于解释本发明,并不用于限定本发明的范围。
实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件,或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可通过正规渠道购买得到的常规产品。
下面实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为市售产品。
实验例1:对DNMT3A缺失的基因稳定敲除细胞株的验证以及靶向DNMT3A缺失的基因的筛选和初步鉴定
1. 材料
细胞株:人非小细胞肺癌细胞NCI-H460,NCI-H1299购自ATCC;发明人实验室构建的NCI-H460 DNMT3A KO,NCI-H1299 DNMT3A KO。
2. 方法
1)对DNMT3A缺失的基因稳定敲除细胞株的验证
蛋白质免疫印迹实验:分别采用分子生物学领域的常规方法进行蛋白提取、蛋白浓度测定(BCA法)和SDS聚丙烯酰胺凝胶电泳。
2)靶向DNMT3A缺失的基因的初步筛选
CCK-8实验
(1)细胞铺板:取对数生长期细胞,弃掉培养基,PBS洗一遍,经0.25%的胰蛋白酶消化处理后,用含10%胎牛血清的细胞培养液将细胞吹打均匀,分散成单细胞悬液,经计数后调整细胞悬液浓度为2~3×104/mL,以每孔100 μL细胞悬液(即3×103个)细胞均匀铺入96孔板中,置于37 ℃,5%CO2饱和湿度的培养箱中培养。
(2)加药:铺板12-24 h后待细胞长至合适密度,根据实验需求设计不同的加药浓度梯度加药,每个浓度设置3个复孔,采用梯度稀释法稀释,将药物溶解于含10%血清的培养基中,96孔板每孔加入100 μL,加药后继续放置于37 ℃,5%CO2饱和湿度培养箱中培养48h。
(3)呈色:药物作用48h后,弃去原培养基,每孔加入100 μL含10% CCK8溶液(2.5mg/mL)的新鲜细胞培养液,放于37 ℃,5% CO2饱和湿度培养箱中继续孵育3-4 h。
(4)比色检测:将96孔板细胞板置于酶标仪中,在450 nm波长处检测各孔测OD值。使用SPSS 20.0软件计算IC50值。细胞抑制率=1-100% × 实验组OD值/对照组OD值。实验结果以平均值±标准误(mean ± SEM)表示。
3)KDM1A抑制剂对DNMT3A敲除肺癌细胞活力的影响
CCK-8实验:操作步骤同实验例1的CCK-8实验。
3. 实验结果
如图1A-图1B所示,申请人前期利用CRISPR/Cas9技术成功构建H460/H1299/PC9WT与H460/H1299/PC9 DNMT3A KO敲除细胞,并通过蛋白免疫印迹实验验证敲除效率,结果表明,敲除细胞株的DNMT3A蛋白表达量相比于对照组几乎不表达。且前期数据表明,DNMT3A基因敲除后细胞的恶性表型及耐药性增强,说明DNMT3A可能是一种抑癌基因。如图2A-图2B所示,对448种表观遗传学抑制剂化合物库进行筛选,发现两种KDM1A特异性抑制剂SP-2509、Seclidemstat排名前三位,选择性最强。图3A显示,H460/H1299/PC9WT/DNMT3A KO细胞对筛选出的KDM1A抑制剂的进行细胞存活率检测,发现SP-2509、Seclidemstat对H460/H1299/PC9 DNMT3A KO细胞具有选择性抑制作用。
4. 结论
蛋白水平确证前期成功建立了稳定敲除DNMT3A基因的人非小细胞肺癌H460/H1299/PC9 DNMT3A KO细胞系。并通过表观遗传抑制剂化合物库初步筛选出靶向KDM1A可能为抑制DNMT3A缺失细胞增殖的分子靶标,发现KDM1A抑制剂SP-2509、Seclidemstat对H460/H1299 /PC9 DNMT3A KO细胞具有选择性抑制作用。
实验例2:体外水平研究KDM1A抑制剂对DNMT3A敲除细胞生物学特性以及对DNMT3A敲除肺癌细胞凋亡的影响。
1. 材料
细胞株:人源非小细胞肺癌细胞NCI-H460,NCI-H1299购自ATCC;发明人实验室构建的NCI-H460 DNMT3A KO,NCI-H1299 DNMT3A KO。
2. 方法
1)在体外细胞水平,化学干预KDM1A表达对DNMT3A敲除肺癌细胞克隆形成能力的影响。
平皿克隆实验
(1)细胞接种
细胞接种:取3-5代状态良好的细胞,待细胞生长到合适密度时,弃掉培养液,胰蛋白酶消化后用加入培养液,收集到4 mL离心管中,分散成单个细胞悬液,分别计数3次,取均值,调整细胞悬液至适当细胞密度。将细胞以300个/孔的密度接种于每孔含2 mL培养液的六孔板(35 mm3)中,摇匀后放入饱和湿度,37 ℃,5% CO2的培养箱中培养。
(2)克隆培养:分别在接种后第2、4、6、8天更换新鲜的完全细胞培养液后加药,在饱和湿度,37 ℃,5% CO2的培养箱中培养7-10天后,显微镜下可观察到各组集落形成,且每个集落至少由50个细胞组成即可终止培养。
(3)染色:待集落长成后,终止细胞培养。吸弃培养液,用1 mL PBS/孔清洗3次,然后加入1 mL甲醇/孔室温固定15 min。吸弃固定液,1 mL PBS/孔清洗3次,加入1 mL 0.1%结晶紫染色液/孔,室温染色30 min。染色后,用流水轻轻冲掉染色液直至空白处干净透明,通风处倒置晾干,相机拍照,计算克隆数(每个克隆中细胞数超过50个)。
(4)统计克隆数:克隆形成率(%)=(克隆数/细胞数)×100%。
2)在体外细胞水平,化学干预KDM1A表达对DNMT3A敲除肺癌细胞凋亡的影响。
Annexin V-FITC/PI双染实验
(1)收集细胞:细胞贴壁16-20 h后,使用培养液将药物稀释至所需浓度,置于培养箱中继续培养48 h取加药处理后的细胞,弃掉原培养液,PBS清洗一遍,用胰酶消化处理后,加入2 mL细胞培养液终止消化,转移至4 mL离心管,放入4 ℃离心机中,调整转速300 g,离心5 min。用预冷的PBS清洗收集的细胞两遍。
(2)孵育缓冲液:将收集的细胞用100 μL 1 × Binding Buffer重悬,随后加入5μL Annexin V-FITC与5 μL PI混匀,避光于室温孵育15 min,上机前5 min补加400 μL 1× Binding Buffer。
(3)检测:过筛网,用流式细胞仪检测。
3. 实验结果
如图4A-图4B所示,与野生型相比,KDM1A抑制剂SP-2509在0.5、1、2 μM各浓度可以显著的抑制DNMT3A缺失型肺癌细胞的克隆形成能力,且具有一定的浓度依赖性,作用明显强于对应的三株肺癌野生型细胞。KDM1A另一种小分子抑制剂Seclidemstat对DNMT3A缺失型肺癌细胞也具有显著抑制作用。上述研究表明KDM1A抑制剂显著降低DNMT3A敲除细胞增殖能力。如图5A-图5D所示,Annexin V-FITC/PI双染法检测分析了KDM1A小分子抑制剂SP-2509对肺癌细胞凋亡的影响。与野生型相比,SP-2509在5和10 μM浓度可以显著诱导H460DNMT3A缺失细胞发生凋亡,凋亡率分别为23.1% ± %1.8和25.05% ± 4.5%,而同等浓度对野生型细胞无明显影响。在H1299配对细胞也出现相似现象。这提示KDM1A抑制剂SP-2509可以选择性的诱导DNMT3A缺失肺癌细胞发生凋亡;另一种KDM1A抑制剂Seclidemstat在5 μm和10 μm浓度均可以显著的诱导DNMT3A敲除细胞的凋亡水平。KDM1A抑制剂可以选择性诱导DNMT3A敲除细胞的凋亡。
4. 结论
与野生型相比,KDM1A抑制剂SP-2509和Seclidemstat可以显著的抑制DNMT3A缺失型肺癌细胞的克隆形成能力,呈一定的浓度依赖性。并且可以选择性诱导DNMT3A敲除细胞的凋亡。
实验例3:基因干预KDM1A对DNMT3A敲除肺癌细胞克隆形成能力的影响以及对凋亡的影响。
1)考察KDM1A瞬转敲低后的沉默效率以及基因干预KDM1A对DNMT3A敲除肺癌细胞克隆形成能力的影响
RNA干扰实验
(1)细胞铺板:转染前一天将细胞接种于6孔板中,以RPMI-1640培养液作为基础培养液,加入10%胎牛血清,在饱和湿度,37 ℃,5% CO2培养箱中培养,保证转染时细胞密度达到70%-90%,转染前弃去原培养液,PBS冲洗一次,更换含有10%胎牛血清的新鲜RPMI-1640培养液放入培养箱中待用。
(2)转染:待细胞密度生长至70-90%时开始转染,取2个1.5ml EP管,首先取7.5 μL的Lipofectamine® 3000转染试剂于125 μL的Opti-MEM®培养基中,放入第一个EP管中,充分混匀。接着取2.5 μg的siRNA加入到125 μL Opti-MEM®培养基中稀释,放入第二个EP管中,充分混匀待用。将上述后者的混合液加入前者中,1:1混合,室温下静置10-15 min形成RNA- Lipofectamine® 3000脂质体复合物。
(3)将脂质体复合物以画同心圆的方式逐滴滴加到适量体积细胞培养液的孔板中,轻轻震荡摇匀,放入培养箱中,转染6-8 h后,更换新鲜培养液,在饱和湿度,37 ℃,5%CO2培养箱中培养48-72 h后终止培养。
蛋白质免疫印迹实验:操作步骤同实验例1的蛋白质免疫印迹。
平皿克隆实验:操作步骤同实验例2的平皿克隆实验。
2)基因干预KDM1A表达对DNMT3A敲除肺癌细胞凋亡的影响。
Annexin V-FITC/PI双染实验:操作步骤同实验例2的AnnexinV-FITC/PI双染实验。
3. 实验结果
通过RNA干扰技术,将两条不同序列的小干扰RNA以100 nM的浓度通过脂质体转染进入细胞内,瞬时沉默KDM1A的表达后,通过蛋白免疫印迹实验对沉默效率进行了验证。如图6A-图6B所示,结果表明H460/H1299 KDM1A瞬时转染后si#1和si#2在蛋白水平均有明显敲低效果,且敲低效率均大于70%。如图7A-图7D所示,采用两条不同序列的KDM1A-siRNA,用不同的浓度梯度进行转染,考察对于肺癌细胞克隆形成能力的影响,KDM1A-siRNA两条链在40和80 nm时,可以显著的选择性抑制H460/H1299 DNMT3A-KO细胞的克隆形成能力。如图8A-图8B所示,利用Annexin V-FITC/PI双染法检测分析了KDM1A两条不同序列的siRNA对肺癌细胞凋亡的影响。结果显示,在采用100 nM的si#1和150 nM的si#2对KDM1A进行基因干扰后,可以选择性诱导H460/H1299 DNMT3A-KO细胞的凋亡。
4. 结论
通过表观遗传抑制剂化合物库初步筛选出靶向KDM1A可能为抑制DNMT3A缺失细胞增殖的分子靶标。化学抑制和基因干预KDM1A后,可显著抑制DNMT3A敲除肺癌细胞的生长;恢复DNMT3A表达后,与受到KDM1A抑制剂的杀伤效果减弱,进一步证实肺癌中靶向KDM1A可能为抑制DNMT3A缺失细胞增殖的分子靶标。
实验例4:DNMT3A重表达后蛋白水平的验证以及KDM1A抑制剂对DNMT3A重表达后细胞存活率和克隆形成能力的影响
1. 材料
细胞株:人源非小细胞肺癌细胞NCI-H460,NCI-H1299购自ATCC;发明人实验室构建的NCI-H460 DNMT3A KO,NCI-H1299 DNMT3A KO。
2. 方法
1)DNMT3A重表达后蛋白水平的验证。
蛋白质免疫印迹:操作步骤同实验例1的蛋白质免疫印迹。
2)在体外细胞水平,KDM1A抑制剂对DNMT3A重表达后细胞存活率和克隆形成能力的影响
CCK-8实验:操作步骤同实验例1的CCK-8实验。
平皿克隆实验:操作步骤同实验例2的平皿克隆实验。
Annexin V-FITC/PI双染实验:操作步骤同实验例2的AnnexinV-FITC/PI双染实验。
3. 实验结果
采用再营救(Rescue)实验,在DNMT3A敲除细胞中重新过表达DNMT3A,如图9A-9B显示,通过蛋白免疫印迹实验检测过表达成功后,用CCK-8实验考察DNMT3A表达水平对细胞存活率的影响,在H460/H1299 DNMT3A-KO细胞中重输DNMT3A后,减弱了KDM1A抑制剂对DNMT3A敲除肺癌细胞活力的抑制作用。如图10A-图10B显示,在H460 DNMT3A-KO细胞中重表达DNMT3A后,减弱了KDM1A抑制剂对DNMT3A敲除肺癌细胞克隆形成能力的抑制作用。如图11A-图11B显示,为了探究KDM1A抑制剂在DNMT3A敲除肺癌细胞中重新表达DNMT3A后对细胞凋亡的影响,采用Annexin V-FITC/PI双染法进行了检测,在H460/H1299 DNMT3A-KO细胞中重输DNMT3A后,减弱了KDM1A抑制剂SP-2509和Seclidemstat对DNMT3A-KO细胞的诱导凋亡作用。
4. 结论
在DNMT3A敲除细胞中重新过表达DNMT3A后,减弱了KDM1A抑制剂对DNMT3A敲除的肺癌细胞活力的抑制作用及克隆形成能力的抑制作用。同时减弱了KDM1A抑制剂对DNMT3A敲除的肺癌细胞的诱导凋亡作用。
实验例5:体内水平研究KDM1A抑制剂对DNMT3A敲除细胞生物学特性的影响
1. 材料
实验动物:BALB/c Nude小鼠80只,雄性,6-8周龄,体重18-22 g,SPF 级。实验动物质量合格证号:NO.202136867。饲料为块状消毒标准饲料。
2.方法
1)KDM1A抑制剂对DNMT3A敲除肺癌异位移植瘤肿瘤生长的影响
裸鼠皮下异位移植瘤模型的建立:选择生长状态良好的置于5% CO2、37 °C恒温细胞培养箱中培养的4-5代人肺癌细胞H460 /H1299 WT、H460/H1299 DNMT3A-KO细胞,弃掉原含10%FBS的RMPI 1640培养液,PBS清洗一遍,去除残留培养液,用0.25%的胰酶消化,加入有血清培养液终止,轻柔吹打,制备成单细胞悬液,300 g离心5 min,弃掉上清液,用无血清RMPI-1640培养液重悬调整细胞密度至1×107个/ml,即0.2 mL中含有2×106个细胞。在SPF级动物实验室内,用酒精棉球给BALB/c Nude鼠腋皮下消毒后,皮下接种0.2 mL细胞悬液,建立裸鼠皮下异位移植瘤模型。
动物分组:当小鼠荷瘤部位肿瘤生长到80-150 mm3左右时,根据移植瘤体积大小,按照组间一致原则,将荷瘤小鼠随机分为H460/H1299 WT荷瘤模型:溶剂对照组(Control组)、SP-2509组(25 mg/kg);H460 /H1299 DNMT3A-KO组:溶剂对照组(Control组)、SP-2509组(25 mg/kg)共8组,每组10只。
药物处理:拟临床给药途径与方法,及查阅文献对上述各组荷瘤裸鼠分别进行以下方式处理:
溶剂对照组:腹腔注射对照溶剂,给予容积为0.1 mL/10 g,每两天一次,连续给药3周;
SP-2509组:腹腔注射SP-2509注射液,给药浓度为25 mg/kg,给药容积为0.1 ml/10 g,两天一次,连续给药3周
药效学指标考察
体重:给药前一天测量体重,给药期间,每隔一天测量动物体重
肿瘤体积:每两天测量一次,小鼠肿瘤长径a(mm)、短径b(mm)记录数据。计算肿瘤体积(Tumor volume, TV)及相对肿瘤体积(Relative tumor volume, RTV)。
肿瘤体积(TV)=1/2×a×b2(a:长径,b:短径)
RTV=Vt/V0(V0为给药前测量所得肿瘤体积,Vt为每一次测量时的肿瘤体积)
肿瘤重量:实验结束后处死小鼠,完整剥离肿瘤组织,用万分之一电子天平称量瘤重。
抑瘤率(TIR %)=(1-给药组平均瘤重/对照组平均瘤重)×100%
2)KDM1A抑制剂对DNMT3A敲除肺癌细胞荷瘤裸鼠体重和脏器指数的影响
操作步骤同实验例5的皮下荷瘤及动物分组
安全性考察
给药前一天测量体重,给药期间,每隔一天测量动物体重,测量肿瘤长径a(mm)、短径b(mm)并记录数据。给药周期结束后,剥取肿瘤组织和重要脏器进行称重,并对瘤组织进行拍照。
脏器指数:实验结束后脱颈处死各组荷瘤小鼠,完整剥离心肝脾肺肾脏器,对心、肝、脾、肺、肾5种脏器用万分之一电子天平称量各脏器进行称重,计算各组的脏器指数。
脏器指数=各脏器重量/体重×100%
3)KDM1A抑制剂对DNMT3A敲除肺癌异位移植瘤中细胞增殖的影响
免疫组织化学(Ki67)
(1)石蜡切片的制备:将肿瘤组织从4%多聚甲醛中取出,切成6×6×3的小块放入包埋盒中,用梯度乙醇进行脱水(乙醇浓度从低到高)、浸入二甲苯中透明后浸入石蜡,浸蜡完成后进行包埋。提前打开包埋机,左右蜡嘴均为65 ℃,平台温度为42 ℃,将包好的蜡块于室温或冰箱中冷却过夜,第二天取出石蜡块,于切片机上进行切片,厚度为5μm。将切好的薄片贴于玻片上;
(2)脱蜡与水化:将石蜡切片置于65 ℃烘箱中烘片12h,利用二甲苯脱蜡与梯度乙醇(浓度从高到底)进行水化,PBS冲洗3次,每次5 min;
(3)抗原修复:将组织切片浸泡于1×的柠檬酸盐溶液中,于微波炉中加热10 min后取出,自然冷却。PBS洗3次,每次5 min;
(4)消除内源性过氧化物酶:用组化笔将组织画圈,过氧化物酶封闭液(3% H2O2)室温孵育30 min,PBS冲洗3次,每次5 min;
(5)封闭非特异性抗原:滴加约200 μL山羊血清封闭液至组织片上,放入湿盒中室温孵育1 h;
(6)抗体孵育:按1:1000的比例配制一抗,甩掉封闭液,滴加一抗至组织片上,于湿盒中4 ℃孵育过夜。第二天PBS洗3次,每次5 min。然后加入带生物素标记山羊抗小鼠/兔IgG,室温孵育1 h,PBS洗3次,每次5 min。
(7)生物素标记滴加HRP生物素,室温孵育0.5 h,PBS冲洗3次,每次5 min;
(8)显色:滴加DAB显色液, DAB显色液现用现配,将20×DAB显色液稀释到1×,每个组织滴加30 μL。染色1-2 min,在显微镜下判断是否终止显色。将切片置于纯净水中终止显色,纯净水洗3次,每次5 min。
(9)复染:20%苏木素染液染色2-3 min,PBS 冲洗3次,每次5 min;
(10)封片:将切片按照以下流程脱水:75%、85%、95%和无水乙醇各5min,二甲苯I、二甲苯II各3 min。最后用1:1的二甲苯/中性树脂封片,在正置显微镜下拍照观察。
4)KDM1A抑制剂对DNMT3A敲除肺癌异位移植瘤中细胞凋亡的影响
(1)TUNEL实验
A.石蜡组织脱蜡,再水化,步骤同前;
B.通透:将切片放入200 mL的0.1 M的柠檬酸盐溶液中,PH为6.0,在微波炉中750W激发1 min,然后加入80 mL的双蒸水(20-25 ℃)迅速冷却,然后把切片转移到PBS溶液中,注意不要使用蛋白酶K孵育;
C.封闭:将切片浸没于0.1 M,PH为7.5的Tris-HCl中,其中包含3% BSA和20%正常牛血清,孵育30 min。然后用PBS洗两次,每次5 min;
D.荧光标记:配制TUNEL反应混合液,现用现配,每个样本滴加50 μL,放入湿盒中于37 ℃烘箱中孵育1 h。PBS洗三次,每次5 min;
E.检测:每个组织样本滴加一滴PBS,盖上盖玻片,于荧光显微镜下观察,激发波长为450-500 nm,发射波长为515-565 nm(绿色),随机选择5个视野,在显微镜下采集图像,计算每个切片的TUNEL阳性染色率。
(2)苏木素-伊红染色法
A. 苏木素染色
将洗好的组织切片放入苏木素中染3-8 min,自来水洗,1%盐酸酒精分化数秒,自来水冲洗,0.6%氨水返蓝,流水冲洗。
B. 伊红染色
0.5%伊红染色液染3-5 min,置于蒸馏水中涮一下。
C. 脱水封片
将切片按照以下流程脱水和透明:75%、85%、95%和无水乙醇各5 min,二甲苯I、二甲苯II各3 min。最后用1:1的二甲苯/中性树脂封片,在正置显微镜下拍照观察,采集图像。
蛋白质免疫印迹:操作步骤同实验例1的蛋白质免疫印迹。
3.实验结果
如图11A-图11B所示,为与对照组相比,在H460WT/H1299 WT荷瘤模型中,SP-2509(25mg/kg)对瘤重和瘤体积无显著影响;而在H460/H1299 WT DNMT3A-KO荷瘤模型中,相比于对照组,SP-2509(25mg/kg)可以显著降低瘤重和瘤体积,抑瘤率分别达到了67.62%,50.13%。综上所述,SP-2509可选择性抑制DNMT3A敲除肺癌异位移植瘤的生长。
图12A-图12D显示,与溶剂对照组相比,给予SP-2509治疗后,裸鼠体重略有下降,随后体重稳定在一定范围,但各组裸鼠体重比较均无统计学意义。表明SP-2509对小鼠体重无显著影响。同时,与溶剂对照组相比,脏器指数均无统计学意义(P>0.05),说明KDM1A抑制剂SP-2509对心、肝、脾、肺、肾均无明显的毒副作用。综上所述,KDM1A抑制剂SP-2509无明显毒副作用,安全性较好。
图13A显示,KDM1A抑制剂对裸鼠异位移植瘤增殖能力的影响,对肿瘤组织内增殖标记物Ki-67进行检测,其中棕色点代表Ki-67阳性表达。相比于H460 WT对照组,SP-2509(25mg/kg,)并未显著影响Ki-67阳性细胞数量;而相比于H460 DNMT3A-KO对照组,SP-2509(25mg/kg)可以使其Ki-67阳性细胞数显著减少。因此,SP-2509可以显著的选择性抑制H460DNMT3A-KO异位移植瘤模型的增殖,从而抑制裸鼠皮下移植瘤的生长。进一步证实KDM1A抑制剂对DNMT3A缺失肿瘤的选择性抗肿瘤活性与抑制细胞增殖相关。
如图14A所示,使用TUNEL原位凋亡检测试剂盒对瘤组织的凋亡水平进行检测,其中绿色荧光代表凋亡细胞,蓝色荧光代表细胞核。相比于H460 WT溶剂对照组,SP-2509(25mg/kg)组凋亡细胞数并未显著增加;而相比于H460 DNMT3A-KO溶剂对照组,SP-2509组凋亡细胞数显著增加。以上结果提示,SP-2509可以显著的选择性诱导H460 DNMT3A-KO异位移植瘤模型的凋亡,从而抑制裸鼠皮下移植瘤的生长。综上表明,KDM1A抑制剂可显著诱导DNMT3A缺失肿瘤的凋亡,与体外Annexin V-FITC/PI双染实验结果一致。
如图15A所示,在裸鼠异位移植瘤模型中,出现肝脏转移现象,对肝转移结节数量进行统计,并对肝转移结节进行H&E染色。结果显示相比于H460野生型细胞荷瘤模型,H460DNMT3A敲除细胞荷瘤模型中肝转移结节数量显著增加,且在给予SP-2509之后显著抑制了肝转移现象。H&E染色观察到在对照组可以观察到肝转移结节细胞致密分布在肝脏中,而在给予SP-2509后消失。综上表明,SP-2509可以显著抑制H460 DNMT3A-KO细胞裸鼠异位移植瘤模型的肝脏转移现象。
4. 结论
与野生型相比,KDMIA抑制剂SP-2509显著抑制了NCI-H460/H1299 DNMT3A敲除肺癌异位移植瘤的生长,且对荷瘤裸鼠的体重和脏器指数无显著影响。SP-2509可显著降低NCI-H460 DNMT3A敲除肿瘤中细胞增殖标记物Ki67的表达,并诱导DNMT3A敲除肿瘤细胞的凋亡,同时显著抑制H460 DNMT3A-KO细胞裸鼠异位移植瘤模型的肝脏转移现象。
实验例6:体外水平研究KDM1A抑制剂对DNMT3A敲除细胞迁移的影响
1. 材料
细胞株:人非小细胞肺癌细胞NCI-H460,NCI-H1299购自ATCC;发明人实验室构建的NCI-H460 DNMT3A KO,NCI-H1299 DNMT3A KO。
2. 方法
KDM1A抑制剂对DNMT3A敲除细胞迁移的影响
Transwell实验;
(1)包被Transwell板:基质胶用无血清培养液按1:8比例稀释后,用移液枪取50 μL混合好的基质胶,加入小室上层使其均匀覆盖整个小室,稍停留吸出小室内剩余基质胶,重复一次。放置于37℃条件下,凝固30 min使用。
(2)加药:下室加入含血清的培养液(500 μL),并加入待测药物SP-2509使药物终浓度为SP-2509 (5 μM)。上室加入无血清培养液配置的药物SP-2509使药物终浓度为SP-2509(5 μM)。
(3)将提前饥饿的细胞用胰酶消化,用无血清培养液制备细胞悬液,H460/H1299WT/DNMT3A-KO细胞个数约为4×105/mL,每孔加入100 μL细胞悬液使药物终浓度为SP-2509(5 μM),超净台静置30 min后,将Transwell板放入37℃培养箱里。
(4)检测:培养箱中培养36h后用棉棒擦去上室表面细胞,用37℃水浴锅预热的PBS清洗小室膜的底部,钙黄绿素染(钙黄绿素:PBS为1:1000)放入培养箱染色30 min后将小室用荧光倒置显微镜进行拍摄,并计数。
3. 实验结果
如图16和图17所示,KDM1A抑制剂SP-2509选择性抑制DNMT3A-KO细胞的迁移。
4. 结论
KDM1A抑制剂SP-2509选择性抑制DNMT3A-KO细胞的迁移。
实验例7:选取KDM1A抑制剂考察其对多种恶性肿瘤DNMT3A缺失细胞生存率的影响
1. 材料
细胞株:人白血病细胞NB-4购自ATCC;人子宫内膜癌细胞ECC-1购自ATCC;人黑色素瘤细胞A-375购自ATCC;人胃癌细胞AGS(购自ATCC);人胶质瘤细胞U251(购自ATCC)人宫颈癌细胞KB(购自ATCC);人结肠癌细胞Colo-205(购自ATCC);人食管癌细胞KYSE-150(购自ATCC);人肝癌细胞Hep-G2(购自ATCC);人横纹肌肉瘤细胞RD(购自ATCC)。
2. 方法
1)细胞转染
(1)细胞培养:接种汇合度为50%的细胞于60 mm培养皿中,以RPMI-1640培养液作为基础培养液,常规加入10%胎牛血清,在饱和湿度,37℃,5% CO2培养箱中培养至细胞汇合度达到70%-90%,转染前弃去原培养液,PBS清洗两次,加入适量Opti-MEM培养基放入培养箱待用。
(2)Opti-MEM培养基稀释lipofectamine 3000,根据倍增系数。取Opti-MEM培养基250 μL,加入lipofectamine 3000脂质体5 μL并充分混匀。另取Opti-MEM培养基250 μL,加入10 μg质粒,充分混匀。将上述两支离心管内液体混合,轻轻吹打,混匀后室温孵育10min,使DNA-脂质体复合物形成。
(3)转染:从培养箱中取出细胞,将上步制备的DNA-脂质体复合物逐滴加入培养皿中,轻轻混匀放入培养箱。转染6 h之后弃去培养液,换成含血清的培养基,在饱和湿度,37℃,5% CO2培养箱中继续培养48 h,培养48 h期间可更换培养基一次。
(4)按上述步骤分别转染Sramble siRNA和DNMT3A siRNA。
2)MTT检测细胞存活率
(1)接种细胞:取对数生长期的细胞,分别用胰酶消化,将细胞重悬于培养液,计数3次,取均值,调整细胞悬液至等密度。用含10%血清培养液制成单个细胞悬液,分别以3000-5000个/孔的密度接种于96孔板,100 μL/孔。分别于饱和湿度,37℃,5%CO2培养箱中培养72h。
(2)呈色:到达时间点后,每孔加入MTT溶液10 μL,继续孵育3~5 h,终止培养,弃去上清,每孔加入二甲基亚砜溶液100 μL,震荡溶解结晶物;
(3)比色:将96孔板置于酶标仪中,490 nm波长处检测各孔OD值,与Scramble细胞相比计算各组细胞增殖率。
细胞存活率% = 药物处理后DNMT3A siRNA 细胞 OD值平均值/药物处理后Scramble细胞组OD值平均值×100%
3. 实验结果
KDM1A抑制剂SP-2509对多种恶性肿瘤DNMT3A敲低细胞(白血病、子宫内膜癌及黑色素瘤细胞)存活率的影响如图18所示,MTT结果显示,与对照组细胞相比,KDM1A抑制剂SP-2509对DNMT3A敲低细胞的生长抑制更强,说明KDM1A抑制剂在其他类型的DNMT3A敲低癌症中也具有选择抑制作用。
4. 结论
KDM1A抑制剂在其他类型的DNMT3A敲低癌症中也具有选择抑制作用。
实验例8:DNMT3A敲除细胞KDM1A表达的检测
1. 材料
细胞株:人非小细胞肺癌细胞NCI-H460,NCI-H1299购自ATCC;发明人实验室构建的NCI-H460 DNMT3A KO,NCI-H1299 DNMT3A KO。
2. 方法
蛋白质免疫印迹:操作步骤同实验例1。
Realtime-PCR:操作步骤同实验例1。
3. 实验结果
如图19A和图19B所示,与野生型细胞相比,DNMT-3A敲除的H460和H1299细胞中,KDM1A在mRNA和蛋白水平均上调,提示两者具有负向调控作用。
4. 结论
DNMT3A负向调控KDM1A表达。
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。
Claims (10)
1.组蛋白赖氨酸去甲基酶1A(KDM1A)抑制剂在制备治疗DNMT3A基因缺失癌症的药物中的用途。
2.根据权利要求1所述的用途,其特征在于:所述癌症选自以下一种或多种:肺癌、白血病、胃癌、结肠癌、食管癌、胶质瘤、宫颈癌、子宫内膜癌、肝癌或肉瘤。
3.根据权利要求1所述的用途,其特征在于:所述KDM1A抑制剂是从转录或翻译水平上抑制KDM1A基因表达的物质或底物活性,优选地,所述KDM1A抑制剂是指小分子KDM1A抑制剂或者对KDM1A具有抑制作用的RNA药物。
4.根据权利要求3所述的用途,其特征在于:所述小分子KDM1A抑制剂选自以下一种或多种:ORY-1001/RG6016、ORY-2001、GSK-2879552、IMG-7289、OG-L002、INCB059872、CC-90011、CBB1007、GSK J2、GSK J4 HCl、GSK J5、GSK-LSD1 2HCl、GSK-LSD1-d4 2HCl、反式-2-(苯基-D5)环丙胺盐酸盐、RG6016、萘莫胺、DDP-38003、HE006、环丙胺(TCP)、反苯环丙胺(Parnate)、SP-2577、SP-2509、CC-90011、XHW-9e、TAK-418、Vafidemstat、Seclidemstat、Bomedemstat、RN-1 2HCl、反式苯环丙胺半硫酸盐、Iadademstat、LSD1/ER-IN-1、LSD1-IN-17、黄素腺嘌呤二核苷酸、2,4-吡啶酸、去甲乌药碱、山柑子碱、辣椒素、脱氢延胡索碱、葛根、黄芩、α-芒果苷、2,4-吡啶二羧酸或反式苯环丙胺。
5.根据权利要求1至4中任一项所述的用途,其特征在于:所述KDM1A抑制剂抑制DNMT3A基因缺失引起的癌细胞的恶性表型增加,优选地,所述癌细胞的恶性表型增加包括癌细胞的增殖和迁移。
6.一种DNMT3A基因缺失癌症的生物标志物,其特征在于:所述生物标志物包含KDM1A基因或KDM1A蛋白,其中所述KDM1A基因或所述KDM1A蛋白在DNMT3A基因缺失癌症中上调,所述生物标志物用于DNMT3A基因缺失癌症患者的诊断、预后预测或用药指导。
7.根据权利要求6所述的生物标志物,其特征在于:所述癌症选自以下一种或多种:肺癌、白血病、胃癌、结肠癌、食管癌、胶质瘤、宫颈癌、子宫内膜癌、肝癌或肉瘤。
8.权利要求6或权利要求7所述的生物标志物在制备用于DNMT3A基因缺失癌症患者的诊断、预后预测或用药指导的试剂盒中的用途。
9.一种DNMT3A基因缺失细胞选择性杀伤药物的体外筛选方法,其特征在于:所述筛选方法包括以下步骤:
(1)构建DNMT3A基因缺失的癌细胞;
(2)在步骤(1)中构建DNMT3A基因缺失的癌细胞中加入候选药物,待所述候选药物分别与构建DNMT3A基因缺失的癌细胞以及配对野生型癌细胞孵育一段时间后,检测所述DNMT3A基因缺失的癌细胞和所述配对野生型癌细胞的细胞成活率;
(3)如果与所述配对野生型细胞相比,所述DNMT3A基因缺失的癌细胞对所述候选药物更敏感,则认为所述候选药物是所述DNMT3A基因缺失的癌细胞的选择性杀伤药物。
10.根据权利要求9所述的体外筛选方法,其特征在于:所述癌细胞选自以下一种或多种:肺癌、白血病、胃癌、结肠癌、食管癌、胶质瘤、宫颈癌、子宫内膜癌、肝癌或肉瘤。
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