CN115192714A - Hdac6抑制剂在制备治疗dnmt3a基因缺失癌症的药物中的用途 - Google Patents
Hdac6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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Abstract
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组蛋白去乙酰化酶6(HDAC6)抑制剂在制备治疗DNMT3A基因缺失癌症的药物中的用途。本发明基于DNMT3A缺失癌症的恶性程度较高,从表观遗传学抑制剂库中筛选出一类HDAC6抑制剂,所述抑制剂能够选择性阻止DNMT3A缺失癌症细胞的增殖、诱导细胞凋亡并抑制异位移植瘤模型小鼠肿瘤的发生发展,同时对小鼠的毒性很小。本发明首次揭示了HDAC6在DNMT3A缺失癌症中的关键作用,并突出了靶向抑制HDAC6可能是DNMT3A缺失癌症的可行治疗方法。
Description
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HDAC6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
根据全球最新数据显示,肺癌的发病率仅次于乳腺癌,排名第二,但其死亡率仍占据榜首,是癌症相关死亡的主要原因,对其的治疗手段是人们一直研究的重点。近年来,随着全基因组染色质图谱等新技术的发展,推动了表观遗传研究的浪潮,从表观遗传角度开发新的治疗策略将成为现代生物学和医学最具创新性的研究领域之一。DNA甲基化是真核生物基因组中常见的一种碱基共价修饰过程,亦是最早被发现的表观遗传改变,其主要通过调节基因表达,基因印记,维持染色体完整性等参与体内多种重要的生理过程。依据结构和功能的不同,哺乳动物细胞中的DNA甲基化模式主要是由两大类DNA甲基转移酶(DNAMethyltransferase,DNMT),即维持性DNA甲基转移酶(DNMT1)和从头甲基化DNA酶(DNMT3A和DNMT3B)之间复杂的相互作用而建立和维持的。
近期,DNA甲基化模式的改变被发现与肿瘤的发生发展紧密相关。大量研究证明,DNMT3A突变,尤其是催化结构域突变,会显著降低酶活性,表现出局部DNA低甲基化,导致DNMT3A突变为功能缺失性突变,而这种功能缺失性突变占所有突变的90%,并且与癌症患者的预后不良相关。本发明研究前期表明DNMT3A基因缺失可促进肺癌恶性表型,提示DNMT3A在肺癌中可能充当抑癌基因角色。然而,针对功能缺失性突变缺少分子靶向的方法,因此,寻找靶向DNMT3A缺失癌症的治疗方法成为了亟待解决的问题。
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角度证明了HDAC6抑制剂能够用于治疗DNMT3A基因缺失的癌症,并且在此基础上开发了HDAC6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了本发明:
在第一个方面中,本发明提供了组蛋白去乙酰化酶6(HDAC6)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、子宫内膜癌、黑色素瘤、胃癌、胶质瘤、宫颈癌、结肠癌、食管癌、肝癌或肉瘤。
作为可选的方式,在上述用途中,所述HDAC6抑制剂是从转录或翻译水平上抑制HDAC6基因表达的物质。
优选地,所述HDAC6抑制剂是小分子HDAC6抑制剂或者对HDAC6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HDAC6抑制剂选自以下一种或多种:WT-161、tubastatin A、tubacin、M344、ACY-63、ACY-216、ACY-241(citarinostat)、ACY-251、ACY-257、ACY-738、ACY-775、ACY-1215(ricolinostat)、ISOX、ST-3-06、ST-2-92、nexturastat A、nexturastat B、HPOB、CAY10603、BRD9757、TCS HDAC6 20b、伏立诺他(vorinostat)、HPB、mTOR/HDAC6-IN-1、HDAC6-IN-3、CG347B、QTX125、QTX125 TFA、AES-135、ACY-1083、SW-100、J22352、丁苯羟酸(bufexamac)、BRD73954、SS-208、MPT0G211、MPT0G211甲磺酸盐、KA2507、KA2507盐酸盐、RTS-V5、CRA-026440、NN-390、droxinostat(NS41080)、SKLB-23bb、MPI_5a、resminostat、SR-4370、PTACH(NCH-51)、LMK-235、米罗地辛(romidepsin)(FK 228)、XP5、AES-350、依布硒林氧化物、MPT0E028、MC1742、FNDR-20123或FNDR-20123游离碱。
优选地,所述小分子HDAC6抑制剂是WT-161。
作为可选的方式,在上述用途中,所述HDAC6抑制剂抑制DNMT3A基因缺失引起的癌细胞的恶性表型。
优选地,所述癌细胞的恶性表型包括癌细胞的增殖。
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HDAC6基因或HDAC6蛋白,其中所述HDAC6基因或所述HDAC6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、子宫内膜癌、黑色素瘤、胃癌、胶质瘤、宫颈癌、结肠癌、食管癌、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,其特征在于所述筛选方法包括以下步骤:
(1)制备DNMT3A基因缺失的癌细胞;
(2)向步骤(1)中制备得到的癌细胞中加入候选药物,待所述候选药物与所述癌细胞孵育一段时间后,检测所述癌细胞中DNMT3A基因缺失细胞与配对野生型细胞的细胞成活率;和
(3)如果所述DNMT3A基因缺失细胞对药物更敏感,则认为所述候选药物是所述DNMT3A基因缺失细胞选择性杀伤药物。
作为可选的方式,在上述体外药物筛选方法中,所述癌细胞选自以下一种或多种:肺癌、白血病、子宫内膜癌、黑色素瘤、胃癌、胶质瘤、宫颈癌、结肠癌、食管癌、肝癌或肉瘤。
作为可选的方式,在上述体外药物筛选方法中,所述DNMT3A基因缺失细胞选择性杀伤药物是HDAC6抑制剂。
作为可选的方式,在上述体外药物筛选方法中,所述筛选方法包括以下步骤:
(1)制备DNMT3A基因缺失的癌细胞;
(2)向步骤(1)中制备得到的癌细胞中加入候选药物,待所述候选药物与所述癌细胞孵育一段时间后,检测所述癌细胞中HDAC6基因或所述HDAC6蛋白的水平及其底物活性;
(3)如果所述癌细胞中所述HDAC6基因或所述HDAC6蛋白及底物活性水平降低,则认为所述候选药物是所述HDAC6抑制剂。
作为可选的方式,在上述体外药物筛选方法中,制备DNMT3A基因缺失的癌细胞的方法,以及检测方法均是分子生物学领域的常规实验方法。
在第五个方面中,本发明提供了一种癌症治疗的靶点,所述癌症是DNMT3A基因缺失癌症,所述靶点是HDAC6基因或HDAC6蛋白。
优选地,所述癌症选自以下一种或多种:肺癌、白血病、子宫内膜癌、黑色素瘤、胃癌、胶质瘤、宫颈癌、结肠癌、食管癌、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特性,DNMT3A缺失可促进某些癌症恶性表型。HDAC6抑制剂能够选择性的杀伤DNMT3A缺失癌细胞,这能够作为多种DNMT3A缺失恶性肿瘤的一个治疗选择。
此外,本发明还首次发现HDAC6在DNMT3A缺失癌症中上调,其可以作为诊断、预后和用药判断的肿瘤生物标志物,为后续患者的诊断和临床指导用药方面提供了理论基础,同时该靶点还可以用于筛选制备治疗DNMT3A缺失癌症的药物。
附图说明
图1是LentiCRISPRv2表达载体图谱及LentiCRISPRv2线性载体琼脂糖凝胶电泳结果。
图2是LentiCRISPRv2与DNMT3A sgRNA连接的重组质粒测序结果。
图3是对DNMT3A敲除细胞株H460/H1299 DNMT3A KO中DNMT3A表达水平的检测结果。
图4是DNMT3A敲除细胞株H460 DNMT3A KO瞬时过表达DNMT3A后对DNMT3A表达水平及对DNMT3A敲除和过表达细胞克隆形成能力的检测结果。
图5是对DNMT3A敲除和过表达细胞肿瘤球形成能力的检测结果。
图6是对DNMT3A敲除和过表达细胞迁移能力的检测结果。
图7是考察DNMT3A敲除和过表达细胞对顺铂、依托泊苷、紫杉醇及长春新碱的药物敏感性变化。
图8是H460 WT/DNMT3A KO细胞对表观遗传小分子抑制剂库筛选流程及H460/H1299 WT/DNMT3A KO细胞对筛选出的HDAC6抑制剂的细胞存活率结果。
图9是H460/H1299 WT及H460/H1299 DNMT3A KO细胞经HDAC6 siRNA处理后HDAC6表达水平及细胞存活率结果。
图10是H460/H1299 WT及H460/H1299 DNMT3A KO细胞重输DNMT3A后DNMT3A的表达情况及经HDAC6抑制剂处理后细胞存活率结果。
图11是H460/H1299 WT及H460/H1299 DNMT1/3B KO细胞DNMT1/3B的表达情况及经HDAC6抑制剂处理后细胞存活率结果。
图12是H460/H1299 WT及H460/H1299 DNMT3A KO细胞经HDAC6抑制剂及siRNA处理后克隆形成结果。
图13是H460/H1299 WT及H460/H1299 DNMT3A KO细胞经HDAC6抑制剂及siRNA处理后细胞凋亡结果。
图14是HDAC6抑制剂对多种DNMT3A缺失恶性肿瘤细胞的生存率结果。
图15是H460 WT/DNMT3A KO细胞荷瘤裸鼠经溶剂、HDAC6抑制剂处理后肿瘤体积、瘤重及抑瘤率的考察结果。
图16是H460 WT/DNMT3A KO细胞荷瘤裸鼠经溶剂、HDAC6抑制剂处理后肿瘤体重及脏器指数的考察结果。
图17是H460 WT/DNMT3A KO细胞荷瘤裸鼠经溶剂、HDAC6抑制剂处理后肿瘤切片Ki67的检测结果。
图18是H460 WT/DNMT3A KO细胞荷瘤裸鼠经溶剂、HDAC6抑制剂处理后肿瘤组织中DNMT3A、PARP、cleaved-PARP蛋白的检测结果。
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:DNMT3A缺失细胞的构建及对肺癌恶性表型的影响
1.材料:细胞株:人非小细胞肺癌细胞NCI-H460,NCI-H1299购自ATCC。
2.方法
1)DNMT3A敲除质粒的构建
DNMT3A small guide RNA的合成
通过在线工具(Addgene),选择sgRNA,序列如下:
表1:DNMT3A基因的small guide RNA
该序列委托上海生工进行合成。
sgRNA退火
将上述sgRNA序列用ddH2O稀释成5μM进行sgRNA序列退火反应。将上述混合物置于PCR仪中运行以下程序:95℃5min,自然冷却到室温,退火后的双链sgRNA序列可立刻使用或保存于-20℃。
质粒扩增
(1)转化
从-80℃冰箱中取出感受态细胞,使其在冰上慢慢融化。在感受态细胞中加入质粒,冰浴静置30min。42℃水浴中热激45sec,然后快速冰浴2min。加LB培养基于管中,置于37℃,200rpm的摇床上,培养1h,复苏菌体。取已转化的细胞加至LB琼脂培养基上(含100μg/mL氨苄青霉素),将平板正置于37℃环境中,待液体被吸收后倒置平板,培养过夜。
(2)质粒提取
观察平板上菌落的生长状况,用枪头挑起生长良好的菌落,在液体培养基(含100μg/mL Amp)中继续培养,37℃,200rpm,培养12-16h,一部分菌液进行保菌,-20℃保存;剩余菌液用于质粒提取,具体步骤如下:把500μL的平衡液BL加入吸附柱,离心1min(12000rpm/min),倒掉废液。将菌液加至离心管中,离心1min(12000rpm/min),弃去上清。向细菌沉淀中加入500μL溶液P1后彻底打散沉淀。加500μL溶液P2后,温柔翻转使菌体裂解。加500μL溶液P4后,立即温柔翻转,摇匀,室温放置10min,离心10min(12000rpm/min)。将上清液加到过滤柱CS中,离心2min(12000rpm/min),收集滤液。把大约0.3倍滤液体积的异丙醇加入至滤液中,混匀后转移到吸附柱CP4中。室温离心1min(12000rpm/min),倒掉废液。取500μL去蛋白液PD加入至吸附柱CP4中,离心1min(12000rpm/min),倒掉废液。取600μL漂洗液PW加至柱CP4中,室温静置2min,离心1min(12000rpm/min),弃去废液;再漂洗一次将吸附柱CP4重新放回,离心2min(12000rpm/min),去除漂洗液。将吸附柱CP4放于一个干净的离心管中,滴加100μL洗脱缓冲液TB于吸附膜的中央,室温放置2min,离心1min(12000rpm/min)后,质粒溶液可直接使用或保存于-20℃。
线性化载体的回收
(1)LentiCRISPRv2质粒线性化
根据LentiCRISPRv2质粒图谱选择BsmBI限制性内切酶对质粒进行线性化,将反应混合物置于PCR仪中运行以下程序:37℃40min,线性化质粒可立刻使用或保存于-20℃。
(2)琼脂糖凝胶回收
连接反应及连接产物的鉴定与扩增
(1)连接反应
将退火得到的双链sgRNA序列用ddH2O稀释4倍后与回收的线性化载体进行连接,将上述混合物置于PCR仪中运行以下程序:16℃连接过夜,连接产物可立刻使用或者储存于-20℃。
(2)连接产物的扩增与鉴定
连接产物转化、扩增、提取步骤如前所示,重组质粒委托上海生工测序,测序结果采用ContingExpress软件进行序列比对。
2)DNMT3A稳定敲除细胞的构建
细胞培养
人非小细胞肺癌NCI-H460细胞株和人非小细胞肺癌NCI-H1299细胞株培养及传代:以含10%胎牛血清的RPMI-1640为培养液,在饱和湿度,37℃,5%CO2细胞培养箱中培养。每天观察细胞状态,待细胞长到80-90%时,弃去原培养液,PBS轻柔冲洗一次,用0.25%的胰蛋白酶(含EDTA)消化细胞。每天根据实验目的不同,以1:2或者1:3的比例进行传代培养,3代之后取生长状态良好,形态正常且处于对数生长期的细胞进行实验。
人肾上皮293T细胞株培养及传代:以含10%胎牛血清的高糖型DMEM为培养液,在饱和湿度,37℃,5%CO2细胞培养箱中培养。每天观察细胞状态,待细胞长到80-90%时,弃去原培养液,PBS轻柔冲洗一次,用0.25%的胰蛋白酶(含EDTA)消化细胞。每天根据实验目的不同,以1:2或者1:3的比例进行传代培养,3代之后取生长状态良好,形态正常且处于对数生长期的细胞进行实验。
慢病毒的包装及感染
(1)慢病毒的包装
准备细胞:转染前一天将293T细胞接种于100mm培养皿中,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,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。提前将转染试剂从4℃取出备用,将40μL Lipofectamine 3000转染试剂加入到500μL Opti-MEM培养基中稀释,充分混匀待用。将20μg重组质粒加入到500μLOpti-MEM培养基中稀释,混合均匀后,再添加40μL P3000助转染试剂,充分混匀待用。在已稀释的Lipofectamine 3000转染试剂中加入已稀释的重组质粒,1:1充分混合,室温下孵育10-15min形成DNA-Lipofectamine 3000脂质体复合物。取出准备好的细胞,将脂质体复合物以画同心圆的方式逐滴滴加到孔板中,轻轻震荡摇匀,放入培养箱中,转染6-8h后,弃去原有培养液,更换新鲜完全培养液,在饱和湿度,37℃,5%CO2培养箱中产毒48-72h后终止培养进行收取慢病毒。
(2)慢病毒感染
将NCI-H460和NCI-H1299提前一天铺板至六孔板,以保证感染时的密度为30-40%,同时准备一个对照组细胞不感染病毒。提前取出病毒在冰上溶解,去除待转染细胞的培养液,每个孔中加入5mL病毒上清,同时加入破膜剂Polybrene,使其最终的浓度为5μg/mL(也可以根据自己的具体情况加入一定浓度的破膜剂)。24h后,弃去病毒上清液,再向每个孔中加入5mL病毒上清,同时加入破膜剂Polybrene,使其最终的浓度为5μg/mL。24h后,弃去病毒上清液,向每个孔中加入5mL完全培养基继续培养24h,目的是让细胞适应一段时间,后续加入相应浓度的抗生素进行筛选。
DNMT3A敲除阳性细胞的筛选
(1)最低致死浓度筛选
将NCI-H460和NCI-H1299以适当密度接种于24孔板中,以RPMI-1640培养液作为基础培养液,加入相应浓度的胎牛血清,在饱和湿度以及37℃,5%CO2培养箱中培养,直至细胞密度达到50%左右。弃去24孔板中的培养液,用PBS清洗一次,更换为嘌呤霉素终浓度分别为0、0.2、0.4、0.6、0.8、1、1.2、1.4、1.6、1.8、2μg/mL的完全培养液,做好标记,在饱和湿度以及37℃,5%CO2培养箱中培养。加药细胞每隔一天用PBS清洗一次,并更换含有相应嘌呤霉素的完全培养液,在饱和湿度以及37℃,5%CO2培养箱中连续培养3天。3天之后,观察24孔板中各个孔细胞的生长状态,选择3天能杀死孔内全部细胞的最低嘌呤霉素浓度为其对NCI-H460和NCI-H1299的最低致死浓度。
(2)将慢病毒感染完成的NCI-H460和NCI-H1299用PBS清洗一次,并更换含有嘌呤霉素最低致死浓度的完全培养液,在饱和湿度以及37℃,5%CO2培养箱中连续培养3天,存活下来的细胞进行进一步培养将得到DNMT3A敲除阳性细胞。
蛋白质免疫印迹
分别进行蛋白提取、蛋白浓度测定(BCA法)和SDS聚丙烯酰胺凝胶电泳。
3)平皿克隆实验
(1)细胞接种:取生长状态良好的细胞,胰蛋白酶消化后用完全培养液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。将细胞以每孔300个的密度接种于每孔含2mL完全培养液的六孔板中培养,接种后十字方向晃动培养皿,使细胞尽量均匀分布。在饱和湿度,37℃,5%CO2的培养箱中培养。
(2)克隆培养:细胞在饱和湿度,37℃,5%CO2的培养箱中培养7-10天,每2天更换一次培养液,直至显微镜下观察到各组集落形成,且每个集落至少由50个细胞组成。
(3)染色:待集落长成后,终止细胞培养。弃去培养基,用PBS轻轻清洗六孔板3次,然后用甲醛室温固定15min。弃去固定液后,PBS清洗六孔板3次,然后每个皿加入1mL 0.1%结晶紫染色液,室温染色30min。染色后,用流水轻轻洗掉染色液(以细胞被染色,空白处干净透明为标准),放置于通风处自然晾干,相机照相。
4)肿瘤球形成实验
(1)配制特殊培养液:在新鲜的无血清的Ham’s F-12/DM培养液中加入生长因子,均匀混合,使B27终浓度达到1×、bFGF终浓度达到20ng/mL、EGF终浓度达到20ng/mL,现配现用。
(2)细胞接种:取生长状态良好的细胞,胰蛋白酶消化后用完全培养液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。将细胞以每孔3000个的密度接种于每孔含2mL完全培养液的低粘附6孔板中培养,接种后十字方向晃动培养板,使细胞尽量均匀分布。在饱和湿度,37℃,5%CO2的培养箱中培养。
(3)计数:细胞在饱和湿度,37℃,5%CO2的培养箱中培养10-14天,每2天补加适量培养液,待肿瘤球直径大于2mm,显微镜下计数并拍照。
5)划痕实验
(1)细胞接种:取生长状态良好的细胞,胰蛋白酶消化后用完全培养液分散成单个细胞悬液,调整细胞悬液至适当细胞密度。将细胞以每孔25万个的密度接种于每孔含500μL完全培养液的24孔板中培养,接种后十字方向晃动培养板,使细胞尽量均匀分布。在饱和湿度,37℃,5%CO2的培养箱中培养。
(2)划痕:用10μL枪头进行划痕,十字交叉,尽可能划直线(切勿划伤板底,否则无法统计),然后用恢复至室温的PBS缓冲液清洗细胞2次,洗掉细胞碎片。
(3)拍照:置于倒置显微镜进行拍照,记为0h划痕照片。换无血清的培养液(排除细胞增殖的影响),将细胞置于饱和湿度,37℃,5%CO2的培养箱中培养24h后,置于倒置显微镜进行拍照,记为24h划痕照片。
6)细胞存活率实验
(1)细胞接种:取生长状态良好的细胞,胰蛋白酶消化后用完全培养液分散成单个细胞悬液,经计数后以每孔100μL的体积、适当细胞密度接种于96孔板,于饱和湿度,37℃,5%CO2培养箱中培养过夜。
(2)加药:根据不同实验需要,加入不同浓度梯度的药物,将加药细胞继续培养48h,倒置显微镜下观察细胞状态。
(3)呈色:待药物达到作用时间点后,每孔加入10μL浓度为2.5mg/mL的MTT溶液,继续37℃培养箱中孵育4h。弃去上清,每孔加入二甲基亚砜溶液100μL,震荡仪上适当速度震荡5min,使紫色结晶溶解。如药物能与MTT产生反应,需先丢弃培养液,小心用PBS缓冲液冲洗2-3次,再加入含有相应量MTT的培养液孵育培养4h。
(4)比色:将96孔板置于酶标仪中,检测490nm波长处各孔OD值,计算药物对细胞增值的影响。
3.实验结果
如图1A-图1B所示,为本实验所用LentiCRISPRv2质粒图谱及琼脂糖凝胶电泳结果显示,经BsmBI限制性内切酶作用后LentiCRISPRv2质粒的条带位置与预计的长度基本一致,约在12000bp左右。图2显示,DNMT3A sgRNA1,DNMT3A sgRNA2,DNMT3A sgRNA3已成功连入LentiCRISPRv2质粒,且均未发生基因突变,形成重组质粒。图3显示,成功制备出病毒液并通过慢病毒感染至肺癌细胞并筛选DNMT3A敲除细胞,利用Western Blot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。如图4A-图4B、图5A-图5B、图6A-图6B和图7A-图7D所示,DNMT3A敲除细胞相较于对照细胞,其增殖能力,自我更新能力,迁移能力及耐药性均有所增强。
4.结论
成功建立了稳定敲除DNMT3A基因的人非小细胞肺癌H460 DNMT3A KO及NCI-H1299DNMT3A KO细胞系。细胞水平证明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。
实验例2: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;已构建的NCI-H460 DNMT3A KO,NCI-H1299 DNMT3A KO。
2.方法
1)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,用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吹匀细胞,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100μL细胞悬液均匀铺入96孔板中,放置在37℃,5%CO2饱和湿度培养箱中培养。
(2)加药:12-24h后待细胞长至合适密度,根据实验需要,设置3个复孔,加入不同浓度梯度的药物,并将加药后细胞板继续放置于37℃,5%CO2饱和湿度培养箱中培养72h。
(3)呈色:弃去原培养液,每孔加入100μL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4h。
(4)比色:将细胞板置于酶标仪中,在450nm波长处检测各孔吸光度值。2)HDAC6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2的CCK-8实验。
3)基因干预HDAC6对DNMT3A敲除肺癌细胞活力的影响
RNA干扰实验
(1)细胞铺板:按实验所需将细胞均匀铺入适当孔径的细胞皿中。
(2)转染液制备:待细胞密度生长至70-90%时开始转染。用适当体积Opti-MEM培养液分别稀释一定浓度小干扰RNA(Small interfering RNA,siRNA)与转染试剂Lipofectamine 3000,将稀释好的siRNA加入到稀释好的Lipofectamine3000中轻轻混匀,室温静置10-15min。
(3)以同心圆方式滴加至适量体积细胞培养液的细胞板中,于37℃、5%CO2饱和湿度培养箱中继续培养48-72h。
蛋白质免疫印迹:操作步骤同实验例1的蛋白质免疫印迹。
CCK-8实验:操作步骤同实验例2的CCK-8实验。
4)改变细胞中DNMT3A表达水平对HDAC6抑制剂的作用
构建DNMT3A稳定重输肺癌细胞
(1)质粒构建
通过NCBI数据库查询DNMT3A基因序列(Gene ID:1788),提供NM_022552.5→NP_072046.2转录本信息交由南京擎科生物合成。
(2)质粒的转化与提取:操作步骤同实验例1的转化与质粒提取。
(3)慢病毒包装与感染:操作步骤同实验例1的慢病毒包装与感染。
(4)DNMT3A稳定重输阳性细胞的筛选:操作步骤同实验例1的阳性细胞筛选。
蛋白质免疫印迹:操作步骤同实验例1的蛋白质免疫印迹。
CCK-8实验:操作步骤同实验例2的CCK-8实验。
5)DNMT3A缺失细胞对HDAC其他亚型抑制剂的作用
CCK-8实验:操作步骤同实验例2的CCK-8实验。
6)HDAC6抑制剂与DNMT其他亚型(DNMT1和DNMT3B)缺失细胞生存率的作用
构建DNMT1、DNMT3B稳定敲除肺癌细胞株:操作步骤同实验例1中DNMT3A稳定敲除细胞的构建。
蛋白质免疫印迹:操作步骤同实验例1的蛋白质免疫印迹。
CCK-8实验:操作步骤同实验例2的CCK-8实验。
3.实验结果
如图8A-图8G所示,对448种表观遗传学抑制剂化合物库进行筛选,发现HDAC6特异性抑制剂WT-161排名最高,选择性最强。图9A-图9D显示,瞬时转染HDAC6小干扰RNA后,发现HDAC6蛋白的表达水平降低,与野生型细胞相比,沉默HDAC6后,显著抑制了DNMT3A敲除细胞的生长。如图10A-图10B所示,在DNMT3A敲除细胞中重表达DNMT3A后,减弱了HDAC6抑制剂对DNMT3A敲除肺癌细胞活力的抑制作用。图11A-图11D显示,与野生型相比,在NCI-H460/NCI-H1299细胞敲除DNMT3B后,对HDAC6抑制剂的敏感性无显著差异,而在NCI-H460/NCI-H1299细胞敲除DNMT1后,对HDAC6抑制剂的敏感性则略高于WT细胞,IC50差异倍数低于1倍,同类细胞对比明显弱于对DNMT3A敲除细胞的杀伤作用。结果表明,靶向HDAC6选择性杀伤DNMT3A为特异性的。
4.结论
通过表观遗传抑制剂化合物库初步筛选出靶向HDAC6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HDAC6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HDAC6抑制剂的杀伤效果减弱,进一步证实肺癌中靶向HDAC6可能为抑制DNMT3A缺失细胞增殖的分子靶标。
实验例3:体外水平研究HDAC6抑制剂对DNMT3A敲除细胞生物学特性的影响
1.材料
细胞株:人非小细胞肺癌细胞NCI-H460,NCI-H1299购自ATCC;已构建的NCI-H460DNMT3A KO,NCI-H1299 DNMT3A KO。
2.方法
1)化学和基因干预HDAC6表达对DNMT3A敲除肺癌细胞克隆形成能力的影响平皿克隆:操作步骤同实验例1的平皿克隆。
2)化学和基因干预HDAC6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:取加药处理的细胞,弃掉原培养液,PBS清洗一遍,用0.25%的胰蛋白酶消化处理后,加入2mL完全细胞培养液终止消化,并转移至4mL离心管,放入4℃离心机中,调整转速300g,离心5min。用冷的PBS清洗收集的细胞柄两遍。
(2)孵育缓冲液:用300μL 1×Binding Buffer重悬细胞,加入5μL Annexin V-FITC混匀,室温避光孵育15min;上机前5min加入5μL PI,并补加200μL 1×BindingBuffer。
(3)检测:过筛网,用流式细胞仪检测。
3.实验结果
如图12A-图12D所示,与野生型相比,HDAC6抑制剂WT-161显著抑制DNMT3A敲除肺癌细胞的克隆形成。同样,与野生型细胞相比,使用小干扰RNA瞬时沉默HDAC6表达后,也显著抑制DNMT3A敲除肺癌细胞的克隆形成。上述研究表明HDAC6抑制或沉默可显著降低DNMT3A敲除细胞增殖能力。图13A-图13D显示,与野生型相比,HDAC6抑制剂显著诱导了NCI-H460/H1299DNMT3A敲除细胞的凋亡。而使用HDAC6 siRNA在NCI-H1299 WT/DNMT3A KO细胞中沉默HDAC6后,同样,与野生型相比,沉默HDAC6后显著诱导了DNMT3A敲除细胞的凋亡。
4.结论
化学和基因干预HDAC6表达后可显著降低DNMT3A敲除肺癌细胞的增殖能力,并诱导其凋亡。在DNMT3A敲除肺癌细胞中恢复DNMT3A表达后,HDAC6抑制剂对其促凋亡作用减弱。
实验例4:选取HDAC6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60mm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250μL,加入lipofectamine 3000脂质体5μL并充分混匀。另取Opti-MEM培养基250μL,加入10μg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6h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48h,培养48h期间可更换培养基一次。
(4)按上述步骤分别转染LentiCRISPRv2和LentiCRISPRv2-DNMT3A KO表达载体。
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,100μL/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10μL,继续孵育3~5h,终止培养,弃去上清,每孔加入二甲基亚砜溶液100μL,震荡溶解结晶物;
(3)比色:将96孔板置于酶标仪中,490nm波长处检测各孔OD值,与LentiCRISPRv2空白表达载体细胞相比计算各组细胞增殖率。
细胞存活率%=药物处理后LentiCRISPRv2-DNMT3A KO OD值平均值/药物处理后LentiCRISPRv2空白表达载体细胞组OD值平均值×100%
3.实验结果
HDAC6抑制剂对多种恶性肿瘤DNMT3A缺失细胞(白血病、子宫内膜癌及黑色素瘤细胞)存活率的影响如图14所示,MTT结果显示,与对照组细胞相比,HDAC6抑制剂对DNMT3A缺失细胞的生长抑制更强,说明HDAC6抑制剂在其他类型的DNMT3A缺失癌症中也具有选择抑制作用。
4.结论
HDAC6抑制剂在其他类型的DNMT3A缺失癌症中也具有选择抑制作用。实验例5:体内水平研究HDAC6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100只,雄性,5-6周龄,体重18-22g,SPF级。购自江苏集萃药康生物科技有限公司。实验动物质量合格证号:NO.202136867。
2.方法
1)HDAC6抑制剂对DNMT3A敲除肺癌异位移植瘤肿瘤生长的影响
皮下荷瘤
生长状态良好的NCI-H460/H1299 WT和DNMT3A KO细胞,弃掉原培养液,PBS清洗一遍,去除残留培养液,用0.25%的胰蛋白酶消化处理后,加入有血清培养液终止,制备成单细胞悬液,300g离心5min,弃掉上清液,加无血清培养液重悬,调整细胞密度至1×107个/mL,即0.2mL中含有2×106个细胞。在SPF级动物房内,用酒精棉给BALB/c Nude鼠腋皮下消毒后,皮下接种0.2mL细胞悬液。
动物分组
当荷瘤部位肿瘤生长到50-80mm3左右时,测量瘤体积大小并做好标记,按照组间一致原则,将荷瘤小鼠随机分组,共分为6组,每组设10只小鼠,即:
NCI-H460 DNMT3A KO细胞:溶剂对照组(Control)、低剂量组(WT-16150mg/kg)、高剂量组(WT-161 75mg/kg);NCI-H460 WT:溶剂对照组(Control)、低剂量组(WT-161 50mg/kg)、高剂量组(WT-161 75mg/kg);NCI-H1299 DNMT3A KO细胞:溶剂对照组(Control)、给药组(WT-161 50mg/kg);NCI-H1299 DNMT3A KO细胞:溶剂对照组(Control)、给药组(WT-16150mg/kg)。
药效学考察
从给药当天起计算为实验第一天,每两天称量一次裸鼠体重,测量肿瘤长径a(mm)、短径b(mm)并记录数据。
肿瘤体积(TV)=1/2×a×b2(a:长径,b:短径);
抑瘤率(TIR%)=(1-给药组平均瘤重/对照组平均瘤重)×100%。
2)HDAC6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4的皮下荷瘤及动物分组
安全性考察
从给药当天起计算为实验第一天,每两天称量一次裸鼠体重,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。
体重(g);
脏器指数:实验结束后对心、肝、脾、肺、肾5种脏器进行称重。
脏器指数=脏器重/体重×100
3)HDAC6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)制备石蜡切片:取4%多聚甲醛固定后的肿瘤组织,剪成长1-3cm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,利用包埋机石蜡包埋,盖上包埋盒,待冷却凝固后开始切片,将切好的薄片贴于玻片上;
(2)脱蜡与水化:将石蜡切片放于60℃烘箱中烘片1h,利用二甲苯脱蜡与梯度乙醇(浓度从高到底)进行水合,PBS冲洗3次,每次5min(组化笔在组织周围画圈标记);
(3)消除内源性过氧化物酶:过氧化物酶封闭液(3%H2O2)孵育30min,PBS冲洗3次,每次5min;
(4)抗原修复:切片置于柠檬酸钠缓冲液缓冲液中,350W加热10min,室温冷却后,PBS洗3次,每次5min;
(5)封闭非特异性抗原:滴加约200μL山羊血清封闭液至组织片上,室温孵育1h;
(6)抗体孵育:滴加约200μL适当比例稀释的目的一抗至组织片上,4℃过夜,PBS冲洗3次;滴加带生物素标记的二抗,室温孵育1h,PBS冲洗3次;滴加HRP生物素,室温孵育1h,PBS冲洗3次;
(7)显色:滴加DAB显色液,镜下观察显色后,流水冲洗3次,每次5min;
(8)复染:20%苏木素染液染色约5min,PBS冲洗3次,每次5min;
(9)封片:乙醇梯度脱水,二甲苯中透明,采用中性树胶封片。
4)HDAC6抑制剂对DNMT3A敲除肺癌异位移植瘤中细胞凋亡的影响
TUNEL实验
(1)石蜡组织脱蜡,再水化,步骤同前;
(2)通透:Proteinase K工作液(20μg/mL)中室温孵育15-30min。PBS冲洗2次;
(3)组织周围变干后,滴加50μL TUNEL反应混合液(现用现配),盖上盖玻片,放入湿盒在暗室中孵育60min。PBS冲洗2次;
(4)待组织周围变干后,加入50μL Converter-AP盖上盖玻片,放于湿盒中,37℃孵育30min。PBS冲洗3次;
(5)加入50μL NBT/BCIP,15-25℃暗室中孵育10min,PBS冲洗3次;
(6)封片同前;
(7)统计: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。
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15A-图15D所示,为与对照组相比,低剂量WT-161(50mg/kg)治疗后未显著抑制NCI-H460 WT异位移植瘤的生长,而在高剂量WT-161(75mg/kg)治疗后则略有抑制,抑瘤率为36.93%。然而,两种剂量的WT-161均显著抑制了NCI-H460 DNMT3A KO异位移植瘤的生长,抑瘤率分别为56.06%和75.23%,呈现剂量依赖性的抑制作用。综上表明,WT-161可选择性抑制DNMT3A敲除肺癌异位移植瘤的生长。图16A-图16B显示,与溶剂对照组相比,给予WT-161治疗后,初期裸鼠体重略微下降,随后体重稳定在一定范围,偶有回升。各组裸鼠体重比较均无统计学意义。同时,与溶剂对照组相比,各组心、肝、脾、肺、肾指数比较均无统计学意义(P>0.05)。综上表明,WT-161在选定的两个剂量下安全性较好,毒性较小。图17A-图17B显示,低剂量WT-161(50mg/kg)治疗后未显著抑制NCI-H460 WT异位移植瘤中Ki67的表达,而在高剂量WT-161(75mg/kg)治疗后则略有抑制,Ki67阳性率相比于对照组降低了49.27%。相比之下,两种剂量的WT-161均显著抑制了NCI-H460 DNMT3A KO异位移植瘤中Ki67的表达,Ki67阳性率相比于对照组分别降低了65.01%和93.83%。进一步证实HDAC6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18A-图18D所示,给予WT-161治疗后,显著增加了DNMT3A缺失肿瘤中凋亡细胞的数量,且呈剂量依赖性。同时,免疫印迹结果显示,给予WT-161治疗后,显著增加了DNMT3A缺失肿瘤中PARP蛋白的裂解。综上表明,HDAC6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。
4.结论
与野生型相比,HDAC6抑制剂WT-161显著抑制了NCI-H460/H1299DNMT3A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。WT-161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,以及PARP蛋白的裂解。
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.组蛋白去乙酰化酶6(HDAC6)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、子宫内膜癌、黑色素瘤、胃癌、胶质瘤、宫颈癌、结肠癌、食管癌、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HDAC6抑制剂是从转录或翻译水平上抑制HDAC6基因表达的物质,优选地,所述HDAC6抑制剂是小分子HDAC6抑制剂或者对HDAC6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HDAC6抑制剂选自以下一种或多种:WT-161、tubastatin A、tubacin、M344、ACY-63、ACY-216、ACY-241(citarinostat)、ACY-251、ACY-257、ACY-738、ACY-775、ACY-1215(ricolinostat)、ISOX、ST-3-06、ST-2-92、nexturastat A、nexturastat B、HPOB、CAY10603、BRD9757、TCS HDAC6 20b、伏立诺他(vorinostat)、HPB、mTOR/HDAC6-IN-1、HDAC6-IN-3、CG347B、QTX125、QTX125 TFA、AES-135、ACY-1083、SW-100、J22352、丁苯羟酸(bufexamac)、BRD73954、SS-208、MPT0G211、MPT0G211甲磺酸盐、KA2507、KA2507盐酸盐、RTS-V5、CRA-026440、NN-390、droxinostat(NS41080)、SKLB-23bb、MPI_5a、resminostat、SR-4370、PTACH(NCH-51)、LMK-235、米罗地辛(romidepsin)(FK 228)、XP5、AES-350、依布硒林氧化物、MPT0E028、MC1742、FNDR-20123或FNDR-20123游离碱。
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HDAC6抑制剂抑制DNMT3A基因缺失引起的癌细胞的恶性表型,优选地,所述癌细胞的恶性表型包括癌细胞的增殖。
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HDAC6基因或HDAC6蛋白,其中所述HDAC6基因或所述HDAC6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、子宫内膜癌、黑色素瘤、胃癌、胶质瘤、宫颈癌、结肠癌、食管癌、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)制备DNMT3A基因缺失的癌细胞;
(2)向步骤(1)中制备得到的癌细胞中加入候选药物,待所述候选药物与所述癌细胞孵育一段时间后,检测所述癌细胞中DNMT3A基因缺失细胞与配对野生型细胞的细胞成活率;和
(3)如果所述DNMT3A基因缺失细胞对药物更敏感,则认为所述候选药物是所述DNMT3A基因缺失细胞选择性杀伤药物。
10.根据权利要求9所述的药物筛选方法,其特征在于:所述癌细胞选自以下一种或多种:肺癌、白血病、子宫内膜癌、黑色素瘤、胃癌、胶质瘤、宫颈癌、结肠癌、食管癌、肝癌或肉瘤。
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