CN113736783A - 一种抑制Notch和NF-κB信号激活的嵌合诱骗寡核苷酸及其应用 - Google Patents
一种抑制Notch和NF-κB信号激活的嵌合诱骗寡核苷酸及其应用 Download PDFInfo
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- CN113736783A CN113736783A CN202110887267.7A CN202110887267A CN113736783A CN 113736783 A CN113736783 A CN 113736783A CN 202110887267 A CN202110887267 A CN 202110887267A CN 113736783 A CN113736783 A CN 113736783A
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Abstract
本发明涉及一种抑制Notch和NF‑κB信号激活的嵌合诱骗寡核苷酸及其应用,该嵌合诱骗寡核苷酸是由2个RBPJ特异性识别位点、1个NF‑κB特异性识别位点组成的单链,其序列为:5’‑TCGTGGGAATTTCCCACGCTAGTTTTTCTAGCGTGGGAAATTCCCACGA‑3’,序列两端进行硫代修饰,退火后形成发卡样双链结构。所述嵌合诱骗寡核苷酸进入细胞后能竞争结合Notch信号的关键转录因子RBPJ,抑制Notch信号的激活,同时还能竞争结合p65/NF‑κB,抑制NF‑κB信号的激活,因此可应用于体外和体内研究Notch/NF‑κB信号对细胞增殖、分化和凋亡等的影响,以及治疗Notch和NF‑κB信号同时异常激活的急性T淋巴细胞白血病等恶性肿瘤。
Description
技术领域
本发明涉及诱骗寡核苷酸及其应用,尤其是一种抑制Notch和NF-κB信号激活的嵌合诱骗寡核苷酸及其应用,具体是转录因子免疫球蛋白Kappa J区重组信号结合蛋白(以下简称RBPJ)和B细胞Kappa轻链基因增强子结合核因子(以下简称NF-κB)嵌合诱骗寡核苷酸及其在抑制Notch和NF-κB信号激活中的应用,属于医药技术领域。
背景技术
Notch信号途径是进化上高度保守的信号途径,介导相邻细胞间直接的信号转导,广泛参与细胞命运决定、细胞增殖、分化和凋亡等,在血管生成、肿瘤发生发展等方面发挥重要作用。Notch信号途径由配体、受体、下游信号转导分子和核内应答过程组成。目前在哺乳动物中共发现了5种Notch配体(Jagged1、Jagged2、Delta-like 1、Delta-like 3和Delta-like4)、4种Notch受体(Notch1-4)。当相邻细胞间配体与受体结合后,通过γ分泌酶复合物酶切,释放胞内段NICD(Notch的活性形式),它能进入细胞核,与转录因子RBPJ结合,使RBPJ由转录抑制状态变成活化状态,募集共激活因子Mastermind-like等,结合特异的DNA序列(C/TGTGGGAA),激活下游Hes1等基因的表达。RBPJ是Notch四种受体的共同下游转录因子,RBPJ的阻断或抑制能有效阻断经典的Notch信号(Artavanis-Tsakonas S等,Science,1999,284:770-6;Borggrefe T等,Cell Mol Life Sci,2009,66:1631-46;HighFA等,Nat Rev Genet,2008,9:49-61)。
NF-κB信号广泛参与调控免疫和炎症反应、细胞存活、增殖、分化和凋亡等,在多种疾病中NF-κB信号持续激活,如肿瘤、关节炎、哮喘、神经退行性疾病和心脏病等。目前哺乳动物中NF-κB家族成员主要包括RelA(p65),RelB,c-Rel,NF-κB1(p50/p105)和NF-κB2(p52/p100),它们的N末端都存在一个高度保守的Rel同源结构域,两种相同或不同的NF-κB成员组成同二聚体或异二聚体,其中p65与p50形成的异二聚体最为常见。静息状态时NF-κB与其抑制蛋白IκB结合,以无活性的形式位于胞浆中,而当细胞受到刺激后IκB被其激酶(IKK)磷酸化,并快速发生泛素化-降解,NF-κB与IκB解离并暴露核定位信号,进入细胞核与染色体DNA上的特异序列GGGRNWYYCC(R:A/G,W:A/T,Y:C/T)结合,激活下游多种靶基因的表达,如Bcl2,Bcl-Xl,c-Myc,IL8,VEGF等(Zhang Q等,Cell,2017,168:37-57;Hayden MS等,Cell,2008,132:344-62)。
Notch信号与NF-κB信号存在交互作用,在多种肿瘤中两者常同时激活,如急性T淋巴细胞白血病(T-ALL)、乳腺癌、黑色素瘤、卵巢癌、结肠癌、胰腺癌等,在肿瘤的发生、发展过程中发挥重要作用(DiDonato JA等,Immunol Rev,2012,2461:379-400;NtziachristosP等,Cancer Cell,2014,25:318-34)。以急性T淋巴细胞白血病为例,Weng AP等发现50%以上的人T-ALL都有Notch1激活型突变(Science,2004,306:269-71),而近来研究表明许多T-ALL样本中NF-κB信号亦存在异常活化,其机制可能是异常激活的Notch信号通过促进IKK的表达而激活NF-κB信号,而且NF-κB信号抑制剂bortezomib能显著抑制T-ALL细胞的生长(Vilimas T等,Nat Med,2007,13:70-7)。Espinosa L等亦发现T-ALL中Notch信号能通过Hes1抑制NF-κB信号的负调节因子Cyld的表达,从而导致T-ALL中NF-κB信号的激活(CancerCell,2010,18:268-81)。Xiu等发现Notch信号和NF-κB信号共同激活能促进B细胞发生恶性转化(Blood,2020,135:108-120)。
转录因子诱骗(Transcription Factor Decoy,TFD)策略是近年发展起来的一种阻断基因转录的有效方法,其原理是体外合成寡核苷酸,其序列与转录因子特异识别位点一致,将其导入细胞后能够竞争性抑制特定转录因子与基因组DNA序列上的顺式元件结合,从而阻止靶基因的转录。转录因子诱骗技术有以下突出特点:①序列短,易合成,易导入细胞;②特异性高,容易识别靶蛋白;③属于DNA序列,与小干扰RNA(SiRNA)相比更稳定;④在转录前和转录水平发挥抑制作用,作用效果强大(Hecker M等,Biochem Pharmacol,2017,144:29-34)。目前针对核转录因子如E2F、AP1、NF-κB等合成的双链诱骗寡核苷酸已被用作基因功能研究的工具,并有大量临床前和临床研究,因此转录因子诱骗策略具有良好的应用前景。目前未见有同时针对RBPJ和NF-κB的嵌合诱骗寡核苷酸的报道。
发明内容
本发明的目之一是提供一种抑制Notch和NF-κB信号激活的嵌合诱骗寡核苷酸,它是一种同时针对转录因子RBPJ和NF-κB的诱骗寡核苷酸,此嵌合诱骗寡核苷酸能竞争结合Notch信号的关键转录因子RBPJ,有效抑制Notch信号的激活,同时还能竞争结合p65/NF-κB,抑制NF-κB信号的激活。
本发明的目之二是一种抑制Notch和NF-κB信号激活的嵌合诱骗寡核苷酸的应用。
为了实现上述目的,本发明采用如下的技术方案:一种抑制Notch和NF-κB信号激活的嵌合诱骗寡核苷酸,它是由2个RBPJ特异性识别位点和1个κB特异性识别位点组成的单链,退火后形成发卡样双链结构,其RBPJ/NF-κB嵌合诱骗寡核苷酸序列如SEQ.ID.NO.1所示。
进一步,所述序列5’和3’端3个核苷酸进行硫代修饰。
进一步,所述诱骗寡核苷酸能抑制Notch信号和NF-κB信号的激活。
进一步,所述的一种抑制Notch和NF-κB信号激活的嵌合诱骗寡核苷酸在制备抗恶性肿瘤药物中的应用。
进一步,所述恶性肿瘤为急性T淋巴细胞白血病、乳腺癌、黑色素瘤、卵巢癌或结肠癌。
由于采用上述技术方案,本发明具有以下有益效果:
1.RBPJ/NF-κB嵌合诱骗寡核苷酸可以作为同时抑制Notch信号和NF-κB激活的候选分子,特异性抑制Notch信号下游和NF-κB信号下游所有靶基因的转录表达;
2.上述RBPJ/NF-κB嵌合诱骗寡核苷酸是单链DNA分子,能直接退火形成稳定性强的发卡样双链结构,且序列两端经过硫代修饰,在体内体外稳定性强;
3.根据RBPJ/NF-κB嵌合诱骗寡核苷酸的原理可知,它在Notch和NF-κB信号都有异常激活的恶性肿瘤,包括但不限于急性T淋巴细胞白血病、乳腺癌、黑色素瘤、卵巢癌、结肠癌、胰腺癌等有应用前景。
上述说明仅是本发明技术方案的概述,为了能够更清楚的了解本发明的技术手段,并可依照说明书的内容予以实施,下面结合附图对本发明进行详细描述,使本发明的目的和优点将变得更加显而易见。
附图说明
图1RBPJ/NF-κB嵌合诱骗寡核苷酸的序列结构和发卡样双链结构形成示意图;
图2RBPJ/NF-κB嵌合诱骗寡核苷酸抑制Notch和NF-κB信号激活的机制示意图;
图3RBPJ/NF-κB嵌合诱骗寡核苷酸对Notch报告基因的影响;
图4RBPJ/NF-κB嵌合诱骗寡核苷酸对NF-κB报告基因的影响;
图5实时定量PCR检测RBPJ/NF-κB嵌合诱骗寡核苷酸对Notch下游靶基因Hes1和Hey1,NF-κB下游靶基因Bcl2和c-Myc表达的影响;
图6免疫印迹检测RBPJ/NF-κB嵌合诱骗寡核苷酸对Notch下游靶基因Hes1和Hey1,NF-κB下游靶基因Bcl2和c-Myc表达的影响;
图7染色质共沉淀检测RBPJ/NF-κB嵌合诱骗寡核苷酸对RBPJ与Hes1启动子区结合的影响,对p65与Bcl2启动子区结合的影响;
图8RBPJ/NF-κB嵌合诱骗寡核苷酸对Jurkat细胞生长的影响;
图9Annexin V/PI染色检测RBPJ/NF-κB嵌合诱骗寡核苷酸对Jurkat细胞凋亡的影响;
图10免疫印迹检测Jurkat细胞凋亡分子Cleaved-Casp3的表达;
图11TUNEL法检测RBPJ/NF-κB嵌合诱骗寡核苷酸对Jurkat细胞凋亡的影响。
具体实施方式
根据文献(Tun T et al.Recognition sequence of a highly conserved DNAbinding protein RBP-Jκ.Nucleic Acids Research,1994,22:965-971)研究报道的转录因子RBPJ特异识别序列C/TGTGGGAA,根据综述文献(Zhang Q,et al.30Years of NF-κB:ABlossoming of Relevance to Human Pathobiology.Cell.2017,168:37-57)报道的NF-κB特异识别序列GGGRNWYYCC(R:A/G,W:A/T,Y:C/T),设计合成了同时以RBPJ和NF-κB为靶点的嵌合诱骗寡核苷酸,由2个RBPJ特异识别位点和1个NF-κB特异识别位点组成的单链,其序列为:SEQ.ID.NO.1:RBPJ/NF-κB嵌合诱骗寡核苷酸序列:5’-TCGTGGGAATTTCCCACGCTAGTTTTTCTAGCGTGGGAAATTCCCACGA-3’,所述序列5’和3’端3个核苷酸进行硫代修饰,退火后形成发卡样双链结构。
上述RBPJ/NF-κB嵌合诱骗寡核酸用于抑制经典Notch和NF-κB信号的激活,将体外合成经过硫代修饰、退火的嵌合诱骗寡核苷酸转染细胞后,竞争性抑制转录因子RBPJ和NF-κB与基因组DNA序列上的相应的顺式元件结合,从而阻止Notch和NF-κB信号下游靶基因的转录,达到抑制Notch和NF-κB信号激活的目的。
实施例
1.RBPJ/NF-κB诱骗寡核苷酸(以下称为Decoy ODN)的设计与合成
根据转录因子RBPJ特异识别序列C/TGTGGGAA和NF-κB特异识别序列GGGRNWYYCC(R:A/G,W:A/T,Y:C/T),设计RBPJ/NF-κB诱骗寡核苷酸,对特异识别序列进行突变,作为对照寡核苷酸(Ctrl),经Genbank检索未查到与其它基因有高度同源性。
SEQ.ID.NO.2:Decoy ODN序列:
5’TCGTGGGAATTTCCCACGCTAGTTTTTCTAGCGTGGGAAATTCCCACGA3’
SEQ.ID.NO.3:Ctrl序列:
5’TCGTTTTAATTTAAAACGCTAGTTTTTCTAGCGTTTTAAATTAAAACGA3’
序列中划线部分为突变序列,序列两端3个核苷酸进行硫代修饰,由北京奥科生物技术有限公司合成、纯化。
合成的寡核苷酸以双蒸水溶解,配置成20μM浓度,通过在水浴中的煮沸10分钟-自然冷却、反复3次的方法退火形成发卡样双链结构。
2.细胞培养
HEK293细胞:是人胚胎肾细胞系,由空军军医大学生化教研室馈赠,用DMEM培养基(含10%胎牛血清和1%青/链霉素)在5%CO2的37℃恒温孵箱中进行常规传代培养。
HUVEC细胞:是人脐静脉内皮细胞,由空军军医大学生化教研室馈赠,用Sciencell公司内皮细胞专用培养基ECM(含5%胎牛血清、1%青/链霉素和1%的ECGS)进行细胞培养。
Jurkat细胞:是急性T淋巴细胞白血病细胞系,购于武汉普诺赛生命科技有限公司,以RPMI-1640培养基(含10%胎牛血清和1%青/链霉素)进行常规传代培养。
3.报告基因实验
报告基因质粒pGa9816(启动子区含3个RBPJ结合位点)、pEFBOS-NICD(Notch活化形式NICD)质粒、报告基因质粒3×κB-Luc(启动子区含3个NFκB结合位点)、pCMV-p65质粒和内参质粒phRL-TK来源于空军军医大学生化教研室韩骅教授馈赠。
1)转染前一天接种HEK293细胞1.5×104于96孔板,每4孔为一组,转染当天70%铺满。
2)混合质粒和寡核苷酸(寡核苷酸配置成20μM浓度),每孔加入DNA如下,Notch信号报告基因:
阴性对照组:pGa9816 50ng,pEFBOS 50ng,phRL-TK 5ng
对照组1:pGa9816 50ng,pEFBOS-NICD 50ng,Ctrl 0.125μL,phRL-TK5ng
对照组2:pGa9816 50ng,pEFBOS-NICD 50ng,Ctrl 0.25μL,phRL-TK5ng
对照组3:pGa9816 50ng,pEFBOS-NICD 50ng,Ctrl 0.5μL,phRL-TK5ng
实验组1:pGa9816 50ng,pEFBOS-NICD 50ng,Decoy ODN 0.125μL,phRL-TK 5ng
实验组2:pGa9816 50ng,pEFBOS-NICD 50ng,Decoy ODN 0.25μL,phRL-TK 5ng
实验组3:pGa9816 50ng,pEFBOS-NICD 50ng,Decoy ODN 0.5μL,phRL-TK5ng
NF-κB信号报告基因:
阴性对照组:3×κB-Luc 50ng,pCMV 50ng,phRL-TK 5ng
对照组1:3×κB-Luc 50ng,pCMV-p65 50ng,Ctrl 0.125μL,phRL-TK5ng
对照组2:3×κB-Luc 50ng,pCMV-p65 50ng,Ctrl 0.25μL,phRL-TK5ng
对照组3:3×κB-Luc 50ng,pCMV-p65 50ng,Ctrl 0.5μL,phRL-TK5ng
实验组1:3×κB-Luc 50ng,pCMV-p65 50ng,Decoy ODN 0.125μL,phRL-TK 5ng
实验组2:3×κB-Luc 50ng,pCMV-p65 50ng,Decoy ODN 0.25μL,phRL-TK 5ng
实验组3:3×κB-Luc 50ng,pCMV-p65 50ng,Decoy ODN 0.5μL,phRL-TK5ng
3)按每孔0.5μl脂质体加入无血清的DMEM,孵育不超过5min。
4)混合DNA与脂质体,室温孵育20-30min,将DNA/脂质体混合物加入细胞。
5)24-48h后弃培养基,PBS轻轻洗涤,弃上清。
6)配制1×passive lysis,每孔加入20μl,室温摇床裂解15min。
7)吸取裂解产物于新离心管,4℃,12000rpm离心30s。
8)吸取5μl上清,加入20μl LARⅡ和20μl stop&Glo,以报告基因检测仪检测。
4.诱骗寡核苷酸细胞转染实验
利用QIAGEN公司Hiperfect转染试剂转染Jurkat和HUVEC细胞。
Jurkat细胞转染:Jurkat细胞接种于24孔板,每孔400μl完全培养基接种2×105细胞,在100μl无血清1640培养基中加入2.5μl诱骗寡核苷酸(储存液浓度20μM)和6μlHiperfect,Vortex混匀,室温孵育10min,加入培养细胞中培养24-48h。
HUVEC细胞转染与Notch信号激活:HUVEC细胞接种于24孔板,转染当天80%的铺满,每孔换400μl完全培养基ECM,在100μl无血清ECM培养基中加入2.5μl诱骗寡核苷酸(储存液浓度20μM)和3μl Hiperfect,Vortex混匀,室温孵育10min,加入培养细胞中培养24h。同时在另一24孔板中加入Dll4-Fc蛋白(购于Sino biology公司,储存液浓度0.25mg/ml,每孔加入250μl PBS溶液和1μl Dll4-Fc),4℃孵育12h。转染24h后,胰酶消化HUVEC,接种于Dll4-Fc包被的培养孔中,再培养24h刺激HUVEC的Notch信号激活。
5.实时定量聚合酶链式反应(Real-time PCR)检测Notch和NF-κB信号下游基因的表达
1)提取细胞总RNA
Trizol法常规提取细胞中总RNA。步骤如下:收集24孔板中培养Jurkat细胞或HUVEC细胞,每孔加入0.5ml TRIzol,室温静置5min;加入0.15ml氯仿,剧烈震荡15s,室温静置3min,12000rpm,4℃离心15min,吸取上层水相,转入新1.5ml离心管中;加入0.25ml异丙醇,震荡混匀,室温静置10min,12000rpm,4℃离心10min,弃上清;加入0.5ml 75%乙醇(以无RNA酶的水配制)洗涤,12000rpm,4℃离心5min,弃上清;室温干燥后溶于20μl DEPC处理的水中;以分光光度计进行定量;A260/280比值监测RNA的纯度。
2)RNA反转录
反转录试剂盒(PrimeScript RT Master Mix Perfect Real Time)购于大连Takara公司。
20μl反应体系:
5×PrimeScript RT Master Mix 4μl
RNA 1μg
RNase Free dH2O 至20μl
反应条件:37℃15min,85℃5s终止反应。
3)实时定量PCR引物设计和合成
交由北京奥科公司设计和和合成实时定量PCR引物。引物序列如表1:
4)实时定量PCR
采用Takara公司TB GreenTM Premix EX TaqTM II(Tli RNaseH plus)试剂盒进行实时定量PCR,其20μl反应体系为:
实时定量PCR反应条件:95℃预变性30s,95℃变性30s,60℃延伸34s,共进行40个循环。同一样本三复孔,以β-actin作为内参照。
6.免疫印迹(Western blot)
RIPA裂解液常规提取细胞蛋白,BCA法定量。进行SDS-PAGE电泳,稳流转膜到PVDF膜上,以含5%脱脂奶粉PBST溶液封闭PVDF膜,室温孵育2h。以封闭液稀释一抗,4℃过夜。一抗抗Hes1抗体(购于Cell Signaling Technology),1:1000稀释;一抗Hey1(购于proteintech公司),1:1000稀释;一抗Cleaved-Casp3(购于Cell Signaling Technology公司),1:1000稀释;一抗Bcl2(购于Cell Signaling Technology公司),1:1000稀释;一抗c-Myc(购于Cell Signaling Technology公司),1:1000稀释;一抗内参抗β-Actin抗体1:5000稀释(购于proteintech公司),一抗4℃孵育过夜。PBST洗膜3次,每次15min。二抗购于proteintech公司:抗小鼠IgG-HRP 1:5000稀释,抗兔IgG-HRP 1:5000稀释。PBST洗膜3次,每次15min。ECL显色发光记录。
7.染色质共沉淀实验(ChIP)
利用Cell Signaling Technology公司的酶处理染色体共沉淀试剂盒进行相关实验,按说明书操作,其具体步骤如下:
1)每皿接种1×107Jurkat细胞,使用Hiperfect进行转染诱骗寡核苷酸(同比扩大转染体系);
2)蛋白与DNA交联:每10ml培养基需要270μl 37%的新鲜甲醛溶液,室温放置10分钟,加入1ml 10×甘氨酸溶液稍加转动使之混匀,室温孵育5分钟,终止固定反应,收集固定后的细胞到50ml锥底管中,4℃,1500rpm离心5分钟沉淀细胞后,用冰冷的PBS漂洗两次;
3)细胞核处理和染色体剪切:每1×107细胞重悬到预冷的1ml 1×缓冲液A+DTT+PIC中,冰上孵育10分钟,每3分钟颠倒混匀一次,4℃ 2000g离心5分钟以沉淀细胞核,弃上清,重悬在1ml冰冷的缓冲液B+DTT中,再次离心,弃上清,并将沉淀用100μl缓冲液B+DTT再次重悬,加入0.5μl微球菌核酸酶,颠倒混匀数次,37℃孵育20min,每隔3-5分钟颠倒混匀一次,将DNA消化成长度约为150-900bp的片段,加入10μl 0.5M EDTA终止消化,4℃ 16000g离心1分钟,沉淀细胞核,重悬于100μl 1×ChIP缓冲液+PIC中,冰上孵育10分钟,超声波破碎细胞核(60W,20s/次,3次),4℃ 9400g离心10分钟,除去样品中的细胞核碎片,将上清转移到一个新管子中,即为交联的染色质片段样品;
4)染色质免疫沉淀:每管对应5-10μg的染色质DNA,每个沉淀反应需要用稀释后的1×ChIP缓冲液补齐到500μl,将稀释后的ChIP染色质,每个样品管吸取10μl样品并转移到一个新的离心管中,作为2%样品输入对照(Input),在各个样品管中加入对应的抗RBPJ抗体或抗p65抗体(购于Cell Signaling Technology公司,1:50稀释),在每组阴性对照管中加入IgG,4℃的转子孵育过夜,重悬混匀蛋白G磁珠,取30μl加入到每个免疫沉淀反应中,4℃的转子上孵育2小时,将离心管放在磁性分离架上,将蛋白G磁珠吸附至管壁,小心地吸走上清,加入1ml低盐漂洗液,4℃转动并孵育5分钟,重复漂洗3次,加入1ml高盐漂洗液漂洗1次;
5)洗脱并解交联:每份ChIP免疫沉淀样品中分别加入150μl 1×ChIP洗脱缓冲液,用涡旋混合器轻轻震荡(1200rpm),65℃孵育30分钟将染色质从抗体-蛋白G微球上洗脱下来,将离心管放在磁性分离架上吸附蛋白G磁珠,小心地将每个样品管中洗脱下来的染色质上清转移到一个新的离心管中,在所有管中,包括第一步的2%样品输入对照(2%Input)管,都加入6μl 5M NaCl和2μl蛋白酶K,65℃孵育2小时;
6)离心柱纯化DNA:向每个DNA样品中添加750μl DNA结合缓冲液,转移到离心柱中,室温18500g离心30秒,倒掉废液,每个离心柱中加入750μl的DNA漂洗缓冲液18500g离心30秒,倒掉废液,再次18500g离心30秒,取出离心柱,插入到一个干净的1.5ml离心管中,在每个离心柱中加入50μl DNA洗脱缓冲液,室温静置2分钟,18500g离心30秒洗脱DNA。
7)进行实时定量PCR反应
Hes1和Bcl2启动子区的ChIP引物交由北京奥科公司合成。
Hes1序列为:
Forward:5’-ATTGGCCGCCAGACCTTG-3’(SEQ.ID.NO.14),
Reverse:5’-GCTCGTGTGAAACTTCCCAAAC-3’(SEQ.ID.NO.15);
Bcl2序列为:
Forward:5’-CTTTAACCTTTCAGCATCACAGAGG-3’(SEQ.ID.NO.16)
Reverse:5’-CTTTGCATTCTTGGACGAGGG-3’(SEQ.ID.NO.17)
8.细胞增殖实验
取生长期Jurkat细胞,计数后重悬2×105细胞于24孔板,按上述诱骗寡核苷酸转染步骤进行转染,每组三复孔,连续3天每天以台盼蓝染色后进行活细胞计数。
9.TUNEL法检测细胞凋亡
TUNEL试剂盒购于Promega公司,操作步骤如下:
1)玻片预处理:多聚赖氨酸(Gibco公司,50μg/ml)37℃孵育12h,无菌水洗涤3次,晾干;
2)常规接种Jurkat细胞,按上述诱骗寡核苷酸转染步骤进行转染,培养3天;
3)生理盐水浸泡5min,PBS洗涤5min,4%多聚甲醛浸泡15min,PBS洗涤5min,重复一次;
4)PBS稀释蛋白酶K至20μg/ml,覆盖样本8-10min,PBS洗涤5min,4%多聚甲醛浸泡5min,再次以PBS洗涤5min;
5)100μl Equi Buffer覆盖样本5-10min;
6)冰上配制rTdT Buffer,
50μl体系45μl Equi Buffer
5μl NucleMix
1μl rTdTE;
7)rTdT Buffer覆盖样本,37℃避光孵育60min;
8)去离子水稀释20×SSC 10倍变为2×SSC,40ml,2×SSC避光浸泡15min;
9)PBS洗涤3次,每次5min;
10)Hochest 1:5000稀释,染色15min;
11)去离子水洗涤3次,50%甘油封片,荧光显微镜下采图;
10.Annexin V/PI法检测细胞凋亡
1)细胞按上述诱骗寡核苷酸转染步骤进行转染,培养3天,收集细胞,300g离心5min,弃上清,PBS洗涤,重悬细胞并计数;
2)取2×105重悬的细胞,加入500μL稀释的1×Annexin V Binding Buffer工作液重悬细胞;
3)细胞悬液中加入5μL的Annexin V-APC染色液和5μL PI细胞核DNA染色液,轻柔涡旋混匀后,室温避光孵育15-20min;
4)FACS检测。
11.统计学处理
实验重复3次以上,数据以GraphPad Prism 8软件分析,两两比较采用t检验,P<0.05具有统计学意义,*<0.05,**<0.01,***<0.001,****<0.0001。
下面结合附图对实验结果进行进一步说明。
如图1所示,图1显示了RBPJ/NF-κB嵌合诱骗寡核苷酸(Decoy ODN)的序列,含有2个RBPJ特异识别位点(黄色方框)和1个NF-κB特异识别位点(绿色方框),通过煮沸-自然冷却退火的方法形成发卡样结构,在序列两端3个核苷酸位点进行的硫代修饰。
如图2所示,图2是RBPJ/NF-κB嵌合诱骗寡核苷酸抑制Notch信号和NF-κB信号激活的机制示意图:退火形成的发卡样Decoy ODN转染进入细胞后能竞争性结合Notch信号关键转录因子RBPJ,抑制RBPJ与染色体DNA的结合,抑制Notch下游Hes1,Hey1等靶基因的转录表达;Decoy ODN能竞争性结合p65/NF-κB,抑制RBPJ与染色体DNA的结合,抑制NF-κB信号下游Bcl2,c-Myc等靶基因的转录表达。
如图3所示,为了验证RBPJ/NF-κB嵌合诱骗寡核苷酸是否能抑制Notch信号进行了报告基因实验:报告基因质粒pGa9816启动子区含3个RBPJ特异的结合位点,当Notch信号激活时(转染Notch活化形式NICD),RBPJ由抑制状态变为激活转态,激活荧光素酶的表达,而转染Decoy ODN后能显著降低其荧光素酶的表达,且随着Decoy ODN的浓度增加,抑制效果增强。
如图4所示,为了验证RBPJ/NF-κB嵌合诱骗寡核苷酸是否能抑制NF-κB信号进行报告基因实验:报告基因质粒3×κB-Luc启动子区含3个NF-κB特异的结合位点,NF-κB信号激活(转染活化形式p65)能促进荧光素酶的表达,而转染Decoy ODN后能显著降低其荧光素酶的表达,且随着Decoy ODN的浓度增加,抑制效果增强。
如图5所示,为了验证RBPJ/NF-κB嵌合诱骗寡核苷酸是否能抑制Notch信号和NF-κB信号,A)通过实时定量PCR检测Notch信号下游靶基因Hes1和Hey1的mRNA水平,在Jurkat细胞中转染Decoy ODN(100nM)能抑制Hes1和Hey1的表达,而以hDll4激活HUVEC的Notch信号,发现此时转染Decoy ODN(100nM)亦能降低Hes1和Hey1的mRNA水平;B)通过实时定量PCR检测NF-κB信号下游靶基因Bcl2和c-Myc的mRNA水平,Jurkat细胞中转染Decoy ODN(100nM)能抑制其Bcl2和c-Myc的表达。
如图6所示,为了验证RBPJ/NF-κB嵌合诱骗寡核苷酸是否能抑制Notch和NF-κB信号,采用免疫印迹的方法检测Notch和NF-κB信号下游蛋白质表达,A)在HUVEC细胞中转染Decoy ODN(50,100nM),Hes1和Hey1的蛋白质水平降低;B)Jurkat细胞中转染Decoy ODN(50,100nM)能降低Hes1、Hey1、Bcl2和c-Myc的蛋白质表达。
如图7所示,通过染色质共沉淀的方法确定RBPJ/NF-κB嵌合诱骗寡核苷酸是否影响RBPJ与Hes1启动子的结合(A和B),是否影响NF-κB与Bcl2启动子的结合(C和D),结果显示Decoy ODN明显削弱了RBPJ与Hes1启动子区RBPJ结合位点的结合,明显削弱了p65与Bcl2启动子区NF-κB结合位点的结合。
如图8所示,Jurkat细胞转染Decoy ODN(50,100nM)、Ctrl(100nM)后不同时间进行细胞计数,发现Decoy ODN能抑制Jurkat细胞的生长,且Decoy ODN浓度100nM组细胞数目最少,提示其对Jurkat细胞生长的抑制效果优于50nM组。
如图9所示,通过FACS检测Jurkat细胞的AnnexinV/PI染色情况,结果显示转染Decoy ODN 50nM和100nM后Jurkat细胞AnnexinV阳性比例增加,提示细胞凋亡增加,且100nM组细胞凋亡比50nM组多。
如图10所示,通过免疫印迹的方法检测Jurkat细胞凋亡分子Cleaved-Casp3的表达,结果显示转染Decoy ODN 50nM和100nM后Jurkat细胞后Cleaved-Casp3表达增加,且100nM组Cleaved-Casp3高于50nM组。
如图11所示,通过TUNEL染色法检测细胞凋亡,结果显示Decoy ODN能促进Jurkat细胞凋亡(绿色为阳性),且100nM组细胞凋亡数目最多。
实验结果表明:
1.RBPJ/NF-κB嵌合诱骗寡核苷酸能同时抑制Notch信号和NF-κB信号的激活;
2.RBPJ/NF-κB嵌合诱骗寡核苷酸能抑制体外培养Jurkat细胞的生长;
3.RBPJ/NF-κB嵌合诱骗寡核苷酸能促进体外培养的Jurkat细胞发生细胞凋亡。
本发明所述嵌合诱骗寡核苷酸进入细胞后能竞争结合Notch信号的关键转录因子RBPJ,抑制Notch信号的激活,同时还能竞争结合p65/NF-κB,抑制NF-κB信号的激活,因此可应用于体外和体内研究Notch/NF-κB信号对细胞增殖、分化和凋亡等的影响,以及治疗Notch和NF-κB信号同时异常激活的急性T淋巴细胞白血病等恶性肿瘤药物中的应用。恶性肿瘤包括但不限于急性T淋巴细胞白血病、乳腺癌、黑色素瘤、卵巢癌、结肠癌、胰腺癌等。
本实施例没有详细叙述的部分和英文缩写属本行业的公知常识,在网上可以搜索到,这里不一一叙述。
序列表
<110> 何飞
<120> 一种抑制Notch和NF-κB信号激活的嵌合诱骗寡核苷酸
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Claims (5)
1.一种抑制Notch和NF-κB信号激活的嵌合诱骗寡核苷酸,其特征是:它是由2个RBPJ特异性识别位点和1个κB特异性识别位点组成的单链,退火后形成发卡样双链结构,其RBPJ/NF-κB嵌合诱骗寡核苷酸序列如SEQ.ID.NO.1所示。
2.根据权利要求1所述的一种抑制Notch和NF-κB信号激活的嵌合诱骗寡核苷酸,其特征是:所述序列5’和3’端3个核苷酸进行硫代修饰。
3.根据权利要求1或2所述的一种抑制Notch和NF-κB信号激活的嵌合诱骗寡核苷酸,其特征是:所述诱骗寡核苷酸能抑制Notch信号和NF-κB信号的激活。
4.根据权利要求1-3任意一项所述的一种抑制Notch和NF-κB信号激活的嵌合诱骗寡核苷酸在制备抗恶性肿瘤药物中的应用。
5.根据权利要求4所述的一种抑制Notch和NF-κB信号激活的嵌合诱骗寡核苷酸在制备抗恶性肿瘤药物中的应用,其特征是:所述恶性肿瘤为急性T淋巴细胞白血病、乳腺癌、黑色素瘤、卵巢癌或结肠癌。
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