CN102617538A - 对微小核糖核酸具广谱调控作用的山酮类小分子及其合成方法和应用 - Google Patents
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Abstract
Description
技术领域
本发明涉及山酮类化合物,具体涉及可以作为微小核糖核酸(miRNA)功能抑制剂的山酮类化学小分子化合物及其制备方法和应用。
背景技术
微小核糖核酸(miRNA),是一类长约19-23个核苷酸的单链非编码核糖核酸分子,文献报道其可以调控约30%的基因表达,因此微小核糖核酸与许多的生理活动如生长发育、细胞增殖与分化、细胞凋亡等密切相关。近来的研究也表明miRNA不仅可以作为多种疾病的生物标志物,也与许多疾病如病毒感染、癌症等的发生、发展有着紧密的联系。因为已经发现一些源癌基因和肿瘤抑制因子是miRNA的靶标,如果调控这些的miRNA本身发生了功能性紊乱,那么就有可能会引发肿瘤的发生。因此对miRNA进行调控就显得尤为重要,并且对miRNA的调控具有潜在的治疗与miRNA相关疾病的应用。
化学小分子以其独特的优势如分子小、穿透力强、易于修饰等吸引了化学生物学家的目光,而且目前普遍采用的miRNA抑制剂是miRNA的反义核苷酸,用化学小分子对其进行调控的则很少。本发明人以用化学小分子对miRNA进行调控展开研究,通过合成及筛选发现了一类山酮类化学小分子是通用性的miRNA功能的抑制剂。
发明内容
解决的技术问题:本发明提供一种山酮类化合物及其合成方法与应用,该类化合物可以普遍地对miRNA进行调控。
技术方案:对微小核糖核酸具广谱调控作用的山酮类小分子,结构如下:
其中R1-R8为氢、含有一至五个碳的:烷基、羟基、烯丙氧基、炔丙氧基、酰基或酯基;
X为氧或硫。
优选的山酮类小分子结构式如下:
上述对微小核糖核酸具广谱调控作用的山酮类小分子的制备方法,步骤为:按比例,1.3g芒果苷元和0.605g烯丙基溴溶解于20mL无水DMSO中,在100℃下回流2h,冷却至室温后,倾入200mL冰水中,用冰醋酸调节pH=6~7,150mL乙酸乙酯提取,用300-400目的硅胶为固定相,乙酸乙酯-石油醚混合溶剂为流动相柱层析,得到上述山酮类小分子。
上述山酮类小分子在制备微小核糖核酸功能抑制剂的应用。
上述山酮类小分子在制备微小核糖核酸与Ago2蛋白复合过程的抑制剂的应用。
上述山酮类小分子在制备促进癌细胞凋亡药物的应用。
本发明采用现代细胞生物学和分子生物学方法研究化学小分子对miRNA的影响及其作用机制。在细胞水平上,通过对化学小分子的筛选,提出山酮类化合物1及其类似物可以作为miRNA的通用抑制剂,而它的作用机制是通过影响了miRNA与RISC的结合,从而导致了miRNA无法与其靶mRNA作用。
(一)山酮类化合物的合成
以上山酮类化合物可以通过芒果苷元与其他相应的取代基缩合得到,或者是通过各种芳香性的酚与芳香性的酸缩合得到,反应的一般方法为:
在圆底烧瓶中加入芒果苷元和相应的溴代物(R-Br)并使之溶于溶解于无水DMSO中,加热回流使之反应完全。反应液用水后处理后用乙酸乙酯提取出粗产物,柱层析纯化分离后得到最终产物山酮类化合物(TM)。
(二)小分子的筛选
首先针对miR-133a(肌肉组织特异性miRNA),采用荧光素酶分析(Luciferase assay)对化学小分子进行筛选。通过小分子刺激后的细胞的luciferase信号强度可以判断小分子对miRNA-133a的作用强弱,信号强度加强证明小分子对miR-133a有抑制作用,加强程度越高抑制作用越好。实际流程如下:
(1)构建含有miR-133a结合位点的luciferase报告质粒;
(2)将该质粒转染到骨骼肌细胞C2C12中;
(3)用化学小分子对转染后的细胞进行刺激;
(4)测定luciferase信号。
转入不含miRNA结合位点的荧光素酶报告质粒到细胞,证明荧光素酶信号的变化是因为小分子对miRNA的影响,而不是小分子对荧光素酶直接产生了作用。
(三)小分子的特异性检测
同样采用荧光素酶分析检测最终筛出来的小分子对其他miRNA,例如miR-1(另一肌肉组织特异性),miR-122(肝组织特异性)和miR-21(癌细胞特异性)的抑制作用。实际流程如下:
(1)分别构建含有miR-1,miR-122或miR-21结合位点的luciferase报告质粒;
(2)将构建好的质粒分别转染到肌肉细胞(C2C12 cell),肝癌细胞(HepG2 cell)以及子宫颈癌细胞(HeLa cell)。
(3)用候选化学小分子对转染后的细胞进行刺激;
(4)测定luciferase信号。
(四)小分子的作用机制研究
本发明通过real-time qPCR方法对小分子的作用机制进行研究。其中所述real-time qPCR方法包括以下步骤:
(1)从小分子刺激后的细胞中提取总RNA,通过RNA逆转录反应得到cDNA样品;
(2)用微小核糖核酸设计引物;
(3)加入荧光探针进行PCR反应;
(4)检测并比较刺激和未被刺激细胞内的miRNA表达量的变化。
有益效果:荧光素酶结果显示化合物1及其类似物对miR-133a,miR-1,miR-122和miR-21的功能都有一定的抑制作用,其中化合物1的作用为最明显。所述的miRNA分别是肌肉组织,肝癌组织以及子宫颈癌特异性miRNA,这说明了这类化合物非特异性的抑制了所有miRNA的作用,它可以作为一个通用的miRNA抑制剂。在另一方面,RT-qPCR结果显示化合物1不影响成熟体miR-133a,miR-1,miR-122和miR-21的表达量。miRNA功能被抑制而表达量不变说明化合物1不是直接与miRNA作用的,而是在miRNA成熟体与其靶mRNA结合的途径起抑制作用的。
附图说明
图1为山酮类化合物1的核磁图谱;
图2为山酮类化合物2的核磁图谱;
图3为山酮类化合物5的核磁图谱;
图4为山酮类化合物6的核磁图谱;
图5为荧光素酶显示不同化合物(化合物1-6)刺激48小时后,C2C12细胞内的荧光素 酶活性;
图6为荧光素酶显示化合物1刺激48小时后,不同细胞内的荧光素酶活性;
图7为化合物1刺激48小时后,不同miRNA成熟体的表达量变化;
图8a为化合物1刺激48小时后,Ago2蛋白在不同细胞内的表达;
图8b为图8a蛋白表达分析;
图9化合物1显著抑制microRNA与Ago2蛋白的结合。
具体实施方式
一、山酮类化合物合成:
实施例1
芒果苷元(1.3g,5mmol)和烯丙基溴(0.605g,5mmol)溶解于无水DMSO(20mL)中,在100℃下回流2h,冷却至室温后,倾入冰水(200mL)中,用冰醋酸调节pH=6~7,乙酸乙酯提取(50mL×3),用300-400目的硅胶为固定相,乙酸乙酯-石油醚混合溶剂为流动相梯度淋洗柱层析,所述石油醚类型为(60-90℃),得到上述山酮衍生物1-4,产率分别为21%,3%,9.8%,10%。
实施例2
在一个100mL的圆底烧瓶中分别加入氯化锌(2.66g,20mmol),5-甲基-1,3苯二酚(1.24g,10mmol),邻羟基苯甲酸(2.76g,20mmol),用油浴加热到140℃,反应5小时,反应结束后用约150mL乙酸乙酯充分溶解,滤去固体,蒸去溶剂,柱层析分离后重结晶得到化合物5(361mg,16%)。
实施例3
在一个100mL的圆底烧瓶中分别加入氯化锌(2.66g,20mmol),3,5-二羟基苯硫酚(1.42g,10mmol),间羟基苯甲酸(2.76g,20mmol),用油浴加热到140℃,反应5小时,反应结束后用约150mL乙酸乙酯充分溶解,滤去固体,蒸去溶剂,柱层析分离后重结晶得到化合物6(490mg,20%)。
二、对miRNA抑制作用的研究:
实施例4:
荧光素酶分析检测化合物1-6对细胞内的miR-133a,miR-1,miR-122和miR-21的抑制能力。具体步骤为:
(1)构建荧光素酶报告质粒
合成一段miRNA靶标序列,然后通过DNA酶连接反应将其插入到荧光素酶报告基因的3’非编码区,之后把构建得到的荧光素酶报告质粒转化进入感受态大肠杆菌中,再挑选单克隆菌落进行测序验证构建得到的荧光素酶报告质粒的正确性。具体操作为:
I)利用HindIII和SpelI内切酶酶切(37℃,4小时)荧光素酶报告质粒的3’非编码区,再从琼脂糖中回收目的酶切产物。
II)通过T4DNA连接酶分别催化miR-133a,miR-1,miR-122和miR-21靶标序列与酶切产物的连接(16℃,过夜)。miR-133a,miR-1,miR-122和miR-21靶标序列是由invitrogen公司合成提供,其序列分别是:
CAGCTGGTTGAAGGGGACCAAA;
GGGTACATAAAGAAGTATGTGC;
CAAACACCATTGTCACACTCCA;
TCAACATCAGTCTGATAAGCTA。
III)把构建好的荧光素酶报告质粒转化进入感受态大肠杆菌中然后铺在含有100μg/mL抗生素(Amp)固体培养基板上37℃过夜培养。
IV)挑选单克隆菌落,扩大培养,再从中提取重组质粒进行测序验证。
实验中所用质粒用Endo-free Midi Kit(OMEGA BIO-TEK)试剂盒提取,提取步骤按照说明书步骤进行,最后一步以相同体积三蒸水代替缓冲液洗下质粒。
(2)质粒转染
利用lipofectamine2000(从invitrogen公司购得)及按照厂商提供的标准试验流程将荧光素酶报告质粒转染到细胞膜内。具体操作为:
分别将C2C12,HepG2和HeLa细胞种入48孔板中,以含有10%(v/v)牛血清和1%
(v/v)青霉素-链霉素(10000单位/mL)的DMEM-High Glucose培育。在其生长密度达到70%时,给细胞换液,将正常细胞培养液换为Opti-MEM I培养基(每孔200μL),再往C2C12细胞加入50μL含有miR-133a-荧光素酶报告质粒(0.25μg),β-gal(0.15μg)质粒和lipofectamine2000(1μL)的Opti-MEM I混合液或含有miR-1荧光素酶报告质粒,β-gal质粒和lipofectamine2000的Opti-MEM I混合液;往HepG2细胞加入miR-122荧光素酶报告质粒,β-gal质粒和lipofectamine2000混合液;往HeLa细胞加入miR-21荧光素酶报告质粒,β-gal质粒和lipofectaine2000混合液,然后摇匀,在37℃,5%(v/v)CO2的培养箱条件下,培育6小时。
(3)化学小分子刺激
将C2C12细胞转染培养液换为含有2%(v/v)马血清和1%(v/v)青霉素-链霉素(10000单位/mL)的DMEM-High Glucose培养液,诱导分化。另一方面将HeLa,HepG2细胞转染液换为含有2%(v/v)牛血清和1%(v/v)青霉素-链霉素(10000单位/mL)的DMEM-High Glucose培养液,使其生长缓慢。同时,每孔加入终浓度为10μM的化合物(化合物预溶解在DMSO、培养基中DMSO最终含量为0.1%(v/v))刺激48小时后观察化合物对细胞的影响。
(4)收细胞及荧光素酶活性检测
主要分为两个部分:(1)目的蛋白-荧光素酶活性检测(2)内参-β-gal的测定。具体检测方法为:
(1)荧光素酶活性检测:每孔加入50μL 1×Cell Culture Lysis Reagent(由5×稀释而来),剧烈摇动10min。收集溶液到0.2mL离心管中,液氮冻融两次。取20μL细胞溶液到1.5mL离心管中,加入100μL荧光底物(Promega公司的luciferase assay system中溶剂与荧光物质混合而成),混匀后立即放入检测仪Modulus single tube multimode reader检测,读数记录。
(2)β-gal测定:溶液体系组成为1.5μL 100×Mg2+溶液,33μL 1×ONPG,100.5μL磷酸钠溶液,15μL细胞溶液。将混合溶液在37℃放置30min,至黄色出现,加入250μL 1M碳酸钠溶液终止反应。吸取100μL溶液加入96孔细胞培养板,在420nm处测定吸光度。
注:为了证实化合物只是对细胞内miRNA起作用而不是对荧光素酶质粒起作用,不含miRNA靶标序列的荧光素酶质粒(又称空载质粒)被转染到细胞内,然后利用小分子刺激,48小时后收细胞及检测荧光素酶信号。有关实际操作步骤如上。
(5)数据分析
数据分析为相对比较方法,b-gal为内参。实验结果显示化合物1刺激细胞后,细胞内的荧光素酶信号都是上升的(图6),说明它都分别对miR-133a,miR-1,miR-122和miR-21都有抑制作用的。所述的miRNA分别是肌肉组织,肝癌组织以及子宫颈癌特异性miRNA,这说明了它非特异性的抑制了所有miRNA的作用,它可以作为一个通用的miRNA抑制剂。
实施例5:
RT-qPCR检测化合物1刺激后细胞内成熟miR-133a,miR-1,miR-122和miR-21的表达。具体步骤为:
(1)总RNA的提取
使用Trizol试剂(Invitrogen公司)提取化合物1刺激48小时后细胞内总RNA;
(2)制备cDNA样品
通过提出到的RNA进行逆转录反应制备cDNA样品.逆转录的反应体系包括2μL 5×AMV buffer、1μL 10mM each dNTP mixture(Takara公司)、0.5μL AMV(Takara公司),2μL RNA(1μg/μL)以及0.5μL miRNA特异性反向引物混和物.总体积为10μL,反应步骤为16℃孵育30分钟,42℃反应30分钟,85℃孵育5分钟;
(3)RT-qPCR
采用的是探针法PCR。首先取1μLcDNA,加入0.3μL Taq酶(Takara公司),0.33μLmiRNA或基因特异性探针引物,1.2μL 25mM MgCl2,0.4μL 2.5mM each dNTP mixture(Takara公司),2μL 10×PCR buffer,14.77μLH2O,共20μL体系进行PCR。仪器使用的是ABI Prism 7300荧光定量PCR仪,反应条件为95℃、5分钟进行1个循环→95℃、15秒,60℃、1分钟进行40个循环。每个样品进行三副重复孔。miR-133a,miR-1,miR-122和miR-21分别利用了miR-133a引物探针,miR-1引物探针,miR-122引物探针以及miR-21引物探针扩增定量,U6作为内参。
(4)数据分析
数据处理方法为相对比较法,也被认为是ΔΔCT法。CT设为反应达到域值时的循环数,而ΔCT=CT样品-CT内参,采用的内参是U6。每个样品miRNA(被化合物刺激后)相对于对照miRNA(未被化合物刺激的)的表达量可以用方程2-ΔΔCT表示,其中ΔΔCT=ΔCT 样品-ΔCT对照,采用的内参是U6。实验结果显示化合物1对细胞内的miR-133a,miR-1,miR-122和miR-21的表达量都没有显著影响(图7)。
实施例6:
蛋白印迹法(Western Blot)检测化合物1对AGO2蛋白水平的影响。具体步骤:
(1)总蛋白的提取与处理
使用RIPA细胞裂解液提取化合物刺激48小时后细胞内总蛋白,然后进行Bradfold比色法测定蛋白浓度。根据浓度加入5×SDS-PAGE蛋白上样缓冲液,100℃浴热5分钟使蛋白变性。
(2)上样,SDS-PAGE胶电泳,转膜和封膜
把30μg的蛋白样品上样到10%SDS-PAGE胶加样孔中,在80V恒压下电泳。当目的蛋白Ago2适当分离后,停止电泳,然后取出SDS-PAGE胶,在300mA,把胶上的蛋白转到PVDF膜上,转膜时间为1.5小时。转膜后把膜取出,标记正面,放在丽春红中染色观察蛋白是否转上,然后把目的范围条带剪下,用1×TBST把丽春红洗脱,再用5%无脂 牛奶-TBST液室温摇动1小时封膜。
(3)一抗孵育
目的蛋白:AGO2抗体(Abcam公司)以1∶1000比例用5%无脂牛奶-TBST液稀释后,4℃过夜孵育封闭后的膜。之后用TBST在侧摆摇床上洗涤15分钟4次。
内参:GAPDH抗体(SantaCruz公司)以1∶3000比例用5%无脂牛奶-TBST液稀释后,4℃过夜孵育封闭后的膜。之后用TBST在侧摆摇床上洗涤15分钟4次。
(4)二抗孵育
目的蛋白:羊抗兔抗体以1∶3000比例用5%无脂牛奶-TBST液稀释后,室温摇床孵育膜一小时。
内参:羊抗鼠抗体以1∶5000比例用5%无脂牛奶-TBST液稀释后,室温摇床孵育膜一小时。
(5)蛋白检测
使用荧光检测试剂曝光检测蛋白。结果显示化合物1对C2C12细胞,HepG2细胞和HeLa细胞内的AGO2蛋白水平都没有影响的(图8a,图8b,图9)。
实施例7:
免疫共沉淀法(immunoprecipitation)检测化合物1刺激细胞后,细胞里与AGO2结合的miRNA量。具体步骤为:
(1)蛋白提取
用细胞裂解液裂解化合物1刺激48小时后的C2C12,HepG2和HeLa细胞。然后进行Bradfold比色法测定蛋白浓度。
(2)AGO2蛋白免疫共沉淀
各取300ug的蛋白到不同的1.5mL的离心管里,再往里加入4μL AGO2免疫共沉淀抗体(Abcam公司),4℃摇晃过夜。隔天再往里加入50μL的磁珠(beads),4℃继续摇晃4小时,3000rpm离心后弃上清。接着用1mL的0.2%NP40-PBS洗涤4次。
(3)总RNA的提取
使用Trizol试剂(Invitrogen公司)提取免疫共沉淀的AGO2内的总RNA。
(4)制备cDNA样品
通过提出到的RNA进行逆转录反应制备cDNA样品。逆转录的反应体系包括2μL5×AMV buffer、1μL 10mM each dNTP mixture(Takara公司)、0.5μL AMV(Takara公司),2μL RNA以及0.5μL miRNA特异性反向引物混和物.总体积为10μL,反应步骤为16℃孵育30分钟,42℃反应30分钟,85℃孵育5分钟.
(5)RT-qPCR
采用的是探针法PCR。首先取1μL cDNA,加入0.3μL Taq酶(Takara公司),0.33μLmiRNA或基因特异性探针引物,1.2μL 25mM MgCl2,0.4μL 2.5mM each dNTP mixture (Takara公司),2μL 10×PCR buffer,14.77μLH2O,共20μL体系进行PCR。仪器使用的是ABI Prism 7300荧光定量PCR仪,反应条件为95℃、5分钟进行1个循环→95℃、15秒,60℃、1分钟进行40个循环。C2C12细胞的miR-133a和miR-1,HepG2细胞的miR-122和HeLa细胞的miR-21分别利用了miR-133a引物探针,miR-1引物探针,miR-122引物探针以及miR-21引物探针扩增定量。
(6)数据分析
数据处理方法为相对比较法。CT设为反应达到域值时的循环数,每个样品miRNA(被化合物刺激后与AGO2结合的)相对于对照miRNA(未被化合物刺激的)的表达量可以用方程2-ΔCT表示,其中ΔCT=CT样品-CT对照。实验结果显示化学小分子1刺激细胞后,AGO2蛋白内的miRNA表达量都降低了(图9),说明化合物1能抑制或干扰了miRNAs与AGO2蛋白的结合,从而使miRNAs的功能降低。
序列表
<110> 南京大学
<120> 对微小核糖核酸具广谱调控作用的山酮类小分子及其合成方法和应用
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<170> PatentIn version 3.3
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<213> 人工序列
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Claims (6)
1.对微小核糖核酸具广谱调控作用的山酮类小分子,其特征在于结构如下:
其中R1-R8为氢、含有一至五个碳的:烷基、羟基、烯丙氧基、炔丙氧基、酰基或酯基;X为氧或硫。
3.权利要求1或2所述对微小核糖核酸具广谱调控作用的山酮类小分子的制备方法,其特征在于步骤为:按比例,1.3g芒果苷元和0.605g烯丙基溴溶解于20mL无水DMSO中,在100℃下回流2h,冷却至室温后,倾入200mL冰水中,用冰醋酸调节pH=6~7,150mL乙酸乙酯提取,用300-400目的硅胶为固定相,乙酸乙酯-石油醚混合溶剂为流动相柱层析,得到上述山酮类小分子。
4.权利要求1或2所述山酮类小分子在制备微小核糖核酸功能抑制剂的应用。
5.权利要求1或2所述山酮类小分子在制备微小核糖核酸与Ago2蛋白复合过程的抑制剂的应用。
6.权利要求1或2所述山酮类小分子在制备促进癌细胞凋亡药物的应用。
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CN104557841A (zh) * | 2014-11-27 | 2015-04-29 | 中国科学院南海海洋研究所 | 两个色酮类化合物及其制备方法和在制备抗肿瘤药物中的应用 |
CN114105943A (zh) * | 2020-09-01 | 2022-03-01 | 深圳有为技术控股集团有限公司 | 3-位取代硫杂蒽酮化合物及其制备方法和光聚合体系应用 |
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CN103159729A (zh) * | 2012-10-22 | 2013-06-19 | 南昌大学 | α-,γ-倒捻子素衍生物及其在制备抗癌药物中的应用 |
CN104557841A (zh) * | 2014-11-27 | 2015-04-29 | 中国科学院南海海洋研究所 | 两个色酮类化合物及其制备方法和在制备抗肿瘤药物中的应用 |
CN104557841B (zh) * | 2014-11-27 | 2017-05-24 | 中国科学院南海海洋研究所 | 两个色酮类化合物及其制备方法和在制备抗肿瘤药物中的应用 |
CN114105943A (zh) * | 2020-09-01 | 2022-03-01 | 深圳有为技术控股集团有限公司 | 3-位取代硫杂蒽酮化合物及其制备方法和光聚合体系应用 |
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