CN107267518B - 特异性抑制CLDN8基因表达的siRNA及其重组载体和应用 - Google Patents
特异性抑制CLDN8基因表达的siRNA及其重组载体和应用 Download PDFInfo
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Abstract
本发明公开了一种特异性抑制CLDN8基因表达的siRNA及其重组载体和它在逆转卵巢癌紫杉醇耐药中应用,属于分子生物学与生物医药技术领域。所述siRNA,包括正义链和反义链,正义链:5’‑UAUAGAUUUUUGUCAUUUGCA‑3’;反义链:5’‑CAAAUGACAAAAAUCUAUAUU‑3’。本发明提供的siRNA可以特异性、高效地抑制CLDN8基因的mRNA和蛋白表达,减少细胞增殖,增加细胞凋亡,降低细胞迁移和侵袭能力,并能有效逆转卵巢癌细胞对紫杉醇的耐药。本发明还提供了所述siRNA及其重组载体在制备治疗卵巢癌、结直肠癌、前列腺癌、骨肉瘤、胃癌或逆转卵巢癌耐药的药物中的应用。
Description
技术领域
本发明涉及分子生物学与生物医药技术领域,具体为一种特异性抑制CLDN8基因表达的siRNA及其重组载体和应用。
背景技术
RNA干扰(RNA interference,RNAi)是动植物中广泛存在的序列特异性转录后基因沉默机制。1998年美国科学家Andrew Fire在秀丽新小杆线虫C.Elegans体内首次发现正义链和反义链的混合物(即dsRNA)所产生的基因沉默效应至少10倍于反义核苷酸的抑制作用,而且能在子代中诱导出同样的基因抑制现象。对RNAi现象的机理研究表明,微量siRNA即能通过转录后基因沉默使大量靶RNA沉默,而这种高效、特异降解同源RNA从而导致序列特异性基因沉默的关键性分子是长为21-23碱基的小双链寡核苷酸,也称作小干扰RNA(siRNA)。进一步研究发现短于21bp或者长于25bp的双链RNA均不能有效启动RNAi,而且其中只要有一个碱基错配,基因沉默的效应就明显减退甚至消失,充分体现了siRNA作用的特异性。
显示出强大基因沉默效能的siRNA作为近年来生物技术的重大发现而备受瞩目,正是因为它具有特异性和高效性阻断同源基因的基因抑制功能,是基因功能研究强有力的工具。siRNA具有许多传统方法无法比拟的优势和特点。目前虽然已有一些抑制特定基因表达的方法,如反义RNA、基因敲除(knockout)等,但siRNA显示出了明显优于这些技术的优势:与反义RNA相比,它具有更高的特异性和持续性;与复杂耗时的基因敲除相比,siRNA是更简便有效的手段。目前利用siRNA作为基因沉默的方法被广泛用于恶性肿瘤机制的研究,研究的关键点主要集中在如何提高基因沉默效能,包括靶基因位点选择、导入系统的优化和对宿主细胞功能的影响等,并由此推动了以该技术为基础的肿瘤特异高效靶向治疗策略的研发进程。
卵巢癌是最常见的妇科恶性肿瘤之一,其病死率居各类妇科肿瘤的首位。卵巢癌治疗最大的障碍是肿瘤细胞耐药性的产生,特别是出现多药耐药。随着铂类/泰素药物联合化疗的实施,卵巢癌的初次化疗反应率可达80%。但大多数患者在2~3年内复发。对复发后卵巢癌,即使再采用作用机制完全不同的化疗药物,也产生耐药,从而使肿瘤治疗后生存率得不到显著的提高。晚期卵巢癌的五年生存率始终徘徊在20~30%。高复发率和复发后的高耐药率是卵巢癌高死亡率最主要的原因之一,寻找卵巢癌化疗耐药的解决方案是目前肿瘤研究的重要课题。
分子靶向药物和化疗联合应用是目前提高恶性肿瘤患者生存率最有前景的治疗方法。CLDN8(Claudin 8)基因位于染色体21q22.11,作为一种参与构成紧密连接的跨膜蛋白,通过形成膜内原纤维,进而构成紧密连接的骨架,其COOH末端的PDZ结构能与其他紧密连接蛋白分子相互作用,从而固定在紧密连接复合体上,在维持紧密连接的结构和功能中发挥重要作用。近年来陆续有一些研究在结直肠癌、前列腺癌、骨肉瘤、胃癌中都检测到CLDN8的异常表达,且往往与细胞癌变以及紧密连接功能缺失密切相关。研究显示在结直肠癌组织中CLDN8表达异常增高,在肿瘤细胞浸润和肝转移中发挥了重要作用;CLDN8通过调控雄激素受体及其下游信号通路,促进前列腺癌细胞的增殖;骨肉瘤细胞中CLDN8不仅能促进细胞增殖,还能抑制骨肉瘤细胞的凋亡。虽然到目前为止CLDN8在肿瘤发生中的具体功能及其作用机制尚不清楚,但CLDN8参与了肿瘤的发展进程已经被逐步认识。
我们先期的研究表明CLDN8在卵巢癌紫杉醇耐药细胞株A2780/Taxol中有异常升高的现象,但该基因是否与卵巢癌发病和卵巢癌耐药有关还有待进一步研究。因此,采用RNAi技术对卵巢癌耐药细胞株中CLDN8基因表达进行干扰,将是对卵巢癌发病机制的重要补充,是对卵巢癌及其耐药治疗的重要探索和应用。
发明内容
本发明的目的在于提供一种特异性抑制CLDN8基因表达的siRNA,用于卵巢癌发病机制研究。本发明的另一个目的在于提供该siRNA在制备治疗卵巢癌和逆转紫杉醇耐药的药物中的应用。
为实现上述目的,本发明采用如下技术方案:
本发明设计、合成3对特异性抑制CLDN8基因表达的siRNA,转染到卵巢癌紫杉醇耐药细胞株A2780/Taxol中,结果发现S1抑制CLDN8基因表达的干扰效果最明显。
本发明提供了一种特异性抑制CLDN8基因表达的siRNA(S1),包括正义链和反义链,
所述正义链:5’-UAUAGAUUUUUGUCAUUUGCA-3’(SEQ ID NO.1);
所述反义链:5’-CAAAUGACAAAAAUCUAUAUU-3’(SEQ ID NO.2)。
作为优选,所述正义链和反义链的5’和3’端的3个碱基进行2’-甲氧基修饰。本发明研究证明,2’-甲氧基修饰后的siRNA(S1)稳定性增加,能提高其在体内抵抗核糖酶的水解的能力,降低免疫刺激反应,延长siRNA干扰基因表达下调的作用时间,使其作用具有高效性、特异性。
本发明提供了一种包含编码所述siRNA的DNA序列的RNA干扰试剂盒。所述试剂盒中包含克隆了所述siRNA的DNA质粒载体,应用时,该质粒载体在真核细胞中转录表达所述的siRNA,进而沉默CLDN8基因的表达。
本发明提供了一种含有编码所述siRNA的DNA序列的重组载体。作为优选,采用的原始载体为慢病毒载体pLKO.1puro。
本发明还提供了重组载体的构建方法,包括:
(1)合成CLDN8-S1片段,选择Age I和EcoR I两个酶切位点,根据S1的序列,设计其shRNA序列,序列如下:
正义链:
5’-CCGGAATATAGATTTTTGTCATTTGCATTCAAGAGATGCAAATGACAAAAATCTATATTTTTTTTGGTACC-3’(SEQ ID NO.7);
反义链:
5’-AATTGGTACCAAAAAAAATATAGATTTTTGTCATTTGCATCTCTTGAATGCAAATGACAAAAATCTATATT-3’(SEQ ID NO.8);
(2)退火得到CLDN8-S1的DNA片段;
(3)用慢病毒载体pLKO.1puro构建pLKO.1-CLDN8-sh1重组载体。
本发明提供的siRNA能够高效特异性抑制卵巢癌细胞CLDN8基因的表达,减少细胞增殖,增加细胞凋亡,降低细胞迁移和侵袭能力,因此,所述siRNA和重组载体作为CLDN8基因表达抑制剂可以应用于肿瘤疾病发病机制的研究当中。
本发明提供了所述siRNA和重组载体在制备CLDN8基因表达抑制剂中的应用。
本发明提供了所述siRNA和重组载体在制备治疗卵巢癌、结直肠癌、前列腺癌、骨肉瘤或胃癌药物中的应用。
本发明研究表明,紫杉醇耐药株A2780/Taxol转染所述siRNA后,该细胞株对紫杉醇的敏感性明显提高,逆转指数为5.73,说明本发明提供的siRNA对紫杉醇耐药株A2780/Taxol的耐药具有非常显著的逆转效果,因此,所述siRNA对逆转卵巢癌紫杉醇耐药的治疗具有潜在应用价值。
本发明提供了所述的siRNA和重组载体在制备逆转卵巢癌紫杉醇耐药的药物中的应用。
本发明具备的有益效果:
本发明提供的siRNA能够特异性、高效地抑制肿瘤细胞中CLDN8基因的mRNA和蛋白表达,减少肿瘤细胞增殖,增加肿瘤细胞凋亡,降低肿瘤细胞迁移和侵袭能力,并能有效逆转卵巢癌细胞对紫杉醇的耐药,将其应用于肿瘤发病机制研究以及制备肿瘤治疗及逆转卵巢癌耐药治疗的药物中,具有重要意义。
附图说明
图1为qRT-PCR检测A2780细胞和A2780/Taxol细胞中CLDN8mRNA表达,其中A为A2780细胞,AR为A2780/Taxol细胞。
图2为Western Blotting检测A2780细胞和A2780/Taxol细胞中CLDN8蛋白表达,其中A为A2780细胞,AR为A2780/Taxol细胞。
图3为qRT-PCR检测S1,S2,S3转染48h后A2780/Taxol细胞中CLDN8mRNA表达。
图4为Western Blotting检测S1,S2,S3转染72h后A2780/Taxol细胞中CLDN8蛋白表达。
图5为pLKO.1-CLDN8-sh1重组质粒及插入酶切位点示意图。
图6为小发卡shRNA示意图。U6启动子指导下游小发卡shRNA的转录;包括23个S1正义链碱基,23个S1反义链碱基。
图7为Western Blotting检测转染pLKO.1-CLDN8-sh1后A2780/Taxol细胞中CLDN8蛋白表达。
图8为相差显微镜观察转染pLKO.1-CLDN8-sh1后A2780/Taxol细胞数量和形态变化。
图9为溴标法检测转染pLKO.1-CLDN8-sh1后A2780/Taxol细胞的增殖。
图10为Caspase3活性检测转染pLKO.1-CLDN8-sh1后A2780/Taxol细胞的凋亡。
图11为细胞划痕实验检测转染pLKO.1-CLDN8-sh1后A2780/Taxol细胞迁移能力。
图12为Transwell检测转染pLKO.1-CLDN8-sh1后A2780/Taxol细胞迁移能力(A)和侵袭能力(B)。
图13为转染pLKO.1-CLDN8-sh1后A2780/Taxol细胞对紫杉醇耐药的逆转。
具体实施方式
下面结合实施例对本发明作进一步说明。以下的实施例中采用的方法目的是更好地理解本发明,但并不限于本发明。如无特殊说明,实施例中涉及的实验方法均为常规方法,所用的实验材料均为常规试剂公司购买。
采用SPSS 18.0统计分析软件,各样本数据以均数±标准差表示,两组之间的差异用T检验(Independent-Sample T Test),多组之间的检验用单因素方差分析(One-Way ANOVA),IC50使用probit回归分析,P<0.05有统计学差异。
卵巢癌细胞株A2780及卵巢癌紫杉醇耐药细胞株A2780/Taxol由浙江省女性生殖健康研究重点实验室细胞库保存;
兔抗人CLDN8一抗(Cat.ab110050)购自abcam公司;鼠抗人GAPDH一抗(Cat.60004-1-Ig)、辣根过氧化物酶标记山羊抗小鼠IgG(H+L)二抗(Cat.SA00001-1)、辣根过氧化物酶标记山羊抗兔IgG(H+L)二抗(Cat.SA00001-2)均购自Proteintech公司;
Western Blotting Luminol Reagent检测试剂盒(Cat.sc-2048)购自Santa Cruz公司;
cDNA逆转录试剂盒PrimeScriptTMRT Master Mix(Cat.RR036A)、荧光定量PCR检测试剂盒SYBR Premix Ex Taq(perfect Real time,Cat.DRR041A)购自TaKaRa公司;Lipofectamine3000转染试剂盒(Cat.L3000008)购自Invitrogen公司;
真核表达载体选择RNAi载体pLKO.1puro,源于全球科学家质粒共享非盈利组织Addgene;
限制性内切酶Age I(Cat.R0552S)、EcoR I(Cat.R0101S)、Kpn I(Cat.R0142S)购自NEB公司;T4连接酶(Cat.2011A)、DNA片段纯化试剂盒(Cat.9761)、DNA凝胶回收试剂盒(Cat.9762)、质粒DNA小量纯化试剂盒(Cat.9760)均购自TaKaRa公司;
siRNA由TaKaRa公司合成;PCR引物及克隆用DNA由上海生工生物工程公司合成;
预染蛋白Marker(Cat.26616)购自Fermentas公司;
溴标法细胞增殖检测试剂盒Cell Proliferation ELISA,BrdU(colorimetric,Cat.11647229001)购自Roche公司;
CaspACE Assay System(colorimetric,Cat.G7351)购自Promega公司;
细胞迁移、侵袭模型Transwell Permeable Supports(Cat.3428)购自Corning公司;
Lab-Tek II Chamber Slide System—Lab-Tek腔室玻片系统购自Nunc公司(Cat.154526);
BD MatrigelTMBasement Membrane Matirx基质膜(Cat.356234)购自BD公司;
siRNA阴性对照AllStars Negative Control SiRNA(Cat.1027281)购自QIAGEN公司;
SDS-PAGE凝胶配置试剂盒(Cat.CW0022M)购自康为世纪公司;
0.45um PVDF膜(Cat.IPVH00010)购自Millipore公司;
紫杉醇(Cat.P106868)购自Aladdin公司。
实施例1.卵巢癌细胞株A2780及其紫杉醇耐药细胞株A2780/Taxol中CLDN8表达差异的研究
一、实时荧光定量RT-PCR(qRT-PCR)检测CLDN8基因mRNA表达
培养48h后吸弃6孔板中的培养基,用PBS洗涤两次后用Trizol抽提总RNA,ThermoNano Drop2000分光光度仪测定RNA浓度,并按SYBR Premix Ex Taq(perfect Real time)试剂盒说明书操作。第一步RNA变性,反应体系:RNA0.5ug,去RNA酶DEPC水补足至6.8ul;反应条件:70℃孵育10min后置于冰上。第二步逆转录,反应体系:按照PrimeScript RTMaster Mix试剂盒说明书进行逆转录;反应条件:42℃孵育60min,85℃灭活5min后,-20℃保存。
取1ul逆转录产物进行荧光定量PCR反应。PCR引物序列:
5’-TGGCTCTTTCTCCGGACCTA-3’;
5’-GATGATGGCATTGGCAACCC-3’,产物长度:224bp;
反应条件:95℃10s,95℃5s,6℃30s,共40个循环。
采用2-△CT法计算各组样本中CLDN8mRNA的表达量。
结果:如图1所示,A2780/Taxol细胞和它的亲本细胞A2780相比,CLDN8mRNA表达增高了60.68%(P<0.05),表明在紫杉醇耐药细胞株中CLDN8mRNA表达显著增高。
二、Western Blotting检测CLDN8蛋白表达
培养72h后吸弃6孔板中的培养基,PBS洗涤3次,加入RIPA蛋白裂解液(100ul/孔),吹打数次,冰上孵育5min,使之充分裂解,4℃,12000转离心5分钟,收集上清,分装-20℃贮存;每个样品95℃变性5min后取10ul上样,8%SDS-PAGE电泳,200V,10min;100V,100min;转至PVDF膜:110V,120min;用含5%脱脂奶粉的TBS封闭液封闭60min;一抗孵育:CLDN8一抗(1:500)、GAPDH一抗(1:5000)室温下孵育2h;TBS洗膜10min×3次;二抗孵育:辣根过氧化物酶标记山羊抗小鼠IgG(H+L)二抗(1:10000)、辣根过氧化物酶标记山羊抗兔IgG(H+L)二抗(1:10000)孵育1h;TBST洗膜10min×3次,TBS洗膜10min×1次;ECL显影后,用Image QuantLAS4000mini(GE Healthcare)对图象扫描处理。
结果:如图2所示,A2780/Taxol细胞和它的亲本细胞A2780相比,CLDN8蛋白表达也显著增高(P<0.05)。
实施例2.CLDN8siRNA设计合成
在Genebank中查获CLDN8基因mRNA序列(NM_199328.2),用siDirect Ver2.0软件(http://sidirect2.rnai.jp/)在线设计获得3对siRNA序列(如SEQ ID NO.1-SEQ IDNO.6)所示。设计过程中选择同时满足文献报道的三种算法(Ui-Tei×Reynolds×Amarzguioui)的序列,并选择siRNA作用特异性最高的23nt长度片段,该设计可避免将来体内实验时发生干扰素样免疫反应,选择起始密码子后100nt,避开5’和3’端UTR区,GC含量控制在30-70%。共选择3对23nt长度的siRNA作为实验筛选干扰片段,结构特征表现为正义链和反义链3’端各有两个碱基外挂,结构特点如下
随后用BLASTN(https://blast.ncbi.nlm.nih.gov/Blast.cgi)在线进行同源性搜索,排除有同源性的序列,尽可能避免非特异性片段对siRNA特异性作用效应的影响。
最后在化学合成时对正义链和反义链的5’和3’端连续3个嘌呤(嘧啶)碱基进行2’-OMe(2’-甲氧基)修饰,增加siRNA分子在细胞内的化学稳定性,延长siRNA干扰基因表达下调的时间和效应。最终的序列和修饰如表1和式(Ⅰ)如下:
表1
实施例3.三对CLDN8siRNA在卵巢癌紫杉醇耐药株A2780/Taxol中对CLDN8基因干扰效果的检测和筛选
一、实验分组:
1.A2780/Taxol正常组(不转染siRNA),以下称作AR;
2.A2780/Taxol阴性对照组(转染阴性对照siRNA),以下称作AR-N;
3.A2780/Taxol实验组(转染S1),以下称作AR-S1;
4.A2780/Taxol实验组(转染S2),以下称作AR-S2;
5.A2780/Taxol实验组(转染S3),以下称作AR-S3。
二、分组转染
为确保转染效率,降低细胞毒性,我们采用Lipofectamine3000转染试剂进行siRNA转染。转染前一天,胰酶消化细胞并计数,细胞铺板在六孔板中,使其在转染日密度0.5×106/ml,细胞融合至70-90%。每孔用125μl无血清OPTI-MEM培养基稀释5ulLipofectamine 3000试剂并充分混匀;制备siRNA预混液,用125μl无血清OPTI-MEM培养基稀释siRNA至终浓度为50nM,并充分混匀;在已稀释的Lipofectamine 3000试剂中加入siRNA预混液(1:1),室温孵育5min;最后将siRNA-脂质体复合物加入细胞中,37℃,5%的CO2中继续培养。48h后检测CLDN8mRNA表达,72h后检测CLDN8蛋白表达。
三、实时荧光定量RT-PCR(qRT-PCR)检测CLDN8基因mRNA表达
检测步骤同前,采用2-△CT法计算各组样本中CLDN8mRNA的表达量。
结果:如图3所示,A2780/Taxol细胞分别转染S1、S2、S3后,CLDN8mRNA的表达均有明显下降,其中S1干扰效果与阴性对照组比较,CLDN8mRNA下调了82.20%,与S2(47.91%)和S3(39.27%)相比,有显著差异(P<0.05)。结果表明,S1对CLDN8有最好的干扰效果。
四、Western Blotting检测CLDN8蛋白表达
检测步骤同前,ECL显影后,用Image Quant LAS4000mini(GE Healthcare)对图象扫描处理。
结果:如图4所示,与阴性对照相比,S1转染后,A2780/Taxol细胞中CLDN8蛋白表达显著下降(P﹤0.05),且与S2和S3相比有显著差异(P﹤0.05)。结果表明,在紫杉醇耐药株A2780/Taxol中,S1对CLDN8的蛋白表达具有最好的干扰效果。故经过筛选,S1被选择作为后续研究的siRNA。
实施例4.真核载体pLKO.1-CLDN8-sh1的构建及对CLDN8基因表达的干扰效果检测
一、实验分组:
1.A2780/Taxol正常组(不转染任何载体),以下称作AR;
2.A2780/Taxol阴性对照组(转染pLKO.1puro空载体),以下称作AR-N;
3.A2780/Taxol实验组1(转染pLKO.1-CLDN8-sh1),以下称作AR-sh1;
二、合成CLDN8-S1片段
选择Age I和EcoR I两个酶切位点,根据S1的序列,设计其shRNA序列并构建到真核表达载体pLKO.1puro中。序列如下:
正义链:
5’-CCGGAATATAGATTTTTGTCATTTGCATTCAAGAGATGCAAATGACAAAAATCTATATTTTTTTTGGTACC-3’(SEQ ID NO.7);
反义链:
5’-AATTGGTACCAAAAAAAATATAGATTTTTGTCATTTGCATCTCTTGAATGCAAATGACAAAAATCTATATT-3’(SEQ ID NO.8);
三、真核载体pLKO.1-CLDN8-sh1的构建
用真核表达载体pLKO.1puro构建pLKO.1-CLDN8-sh1重组表达载体(图5和6),具体方法参见美国冷泉港出版社《分子克隆实验指南》。
将CLDN8-S1正义链和反义链经退火程序(95℃变性2min;缓慢冷却退火至25℃),4℃保存。Age I和EcoR I完全酶切载体pLKO.1puro,37℃过夜;酶切产物用DNA凝胶回收试剂盒回收DNA。退火产物与载体酶切回收片段进行连接反应,反应体系(10ul):T4连接酶1ul,T4连接酶缓冲液1ul,退火产物与载体酶切回收片段混合物(摩尔比3:1);去离子水补至10ul,16℃连接过夜;取5ul连接产物置于100ul JM109感受态细菌中,冰浴30min,42℃热休克90s,冰浴5min,加LB培养基1000ul,37℃摇床培养30min,5000rpm离心5min,弃上清,将细菌均匀涂布于LB平板上(含50ug/ml氨苄青霉素),37℃倒置培养过夜;挑选若干独立菌落接种于含相应抗性的LB培养基中,37℃震荡过夜扩菌;收集细菌,用质粒DNA纯化试剂盒抽提获得质粒DNA,Kpn I酶切鉴定,获得pLKO.1-CLDN8-sh1重组质粒。
四、pLKO.1-CLDN8-sh1重组质粒转染细胞后观察干扰效果
将pLKO.1-CLDN8-sh1转染入对数生长期的A2780/Taxol细胞中。转染参照Lipofectamine3000操作说明书,每孔用125μl无血清OPTI-MEM培养基稀释5ulLipofectamine 3000试剂并充分混匀;在125μl无血清OPTI-MEM培养基中加入5ug重组质粒DNA,加入P3000试剂10ul,充分混匀,制备重组质粒预混液;在已稀释的Lipofectamine3000试剂中加入重组质粒预混液(1:1),室温孵育5mim;最后将重组质粒-脂质体复合物250ul加入细胞中,37℃,5%的CO2中继续培养。72h后检测CLDN8蛋白表达。
如图7所示,转染pLKO.1-CLDN8-sh1重组质粒后,与阴性对照(转染空质粒)相比,A2780/Taxol细胞中CLDN8蛋白表达显著下降(P﹤0.05),结果表明,转染pLKO.1-CLDN8-sh1重组质粒能有效干扰CLDN8的蛋白表达。
实施例5.转染pLKO.1-CLDN8-sh1特异性阻断CLDN8表达后对肿瘤细胞增殖、凋亡、迁移和侵袭的影响
一、实验分组:
1.A2780/Taxol正常组(不转染任何载体),以下称作AR;
2.A2780/Taxol阴性对照组(转染pLKO.1puro空载体),以下称作AR-N;
3.A2780/Taxol实验组(转染pLKO.1-CLDN8-sh1),以下称作AR-sh1。
二、分组转染
转染步骤同前,转染后继续培养细胞,用于检测细胞的增殖、凋亡、迁移和侵袭。
三、细胞增殖试验
细胞在96孔板中转染pLKO.1-CLDN8-sh1后继续培养72h,每孔加入10ulBrdU标记液至BrdU终浓度为10uM,37℃孵育2h;吸除BrdU标记液,每孔加入200ul FixDenat,20℃孵育30min;吸除FixDenat,每孔加入100ul anti-BrdU-POD,20℃孵育90min;每孔200ulWashing Solution洗涤3次;加入100ul/孔底物溶液,20℃孵育20min,检测波长370nm(参考波长492nm)测吸光度(A),细胞增殖的能力用A实验组/A对照组表示。
结果如图8和图9所示,在A2780/Taxol细胞中,转染pLKO.1-CLDN8-sh1后,相差显微镜下观察可见,实验组细胞数量显著减少;溴标法测试细胞增殖结果显示:与阴性对照相比,实验组细胞增殖能力下降了81.88%,有显著性差异(P<0.05)。说明特异性阻断CLDN8的表达后,能抑制肿瘤细胞增殖。
四、Caspase3活性检测细胞凋亡
转染72h后收集细胞,裂解液调整细胞密度为1×108/ml,冰上裂解15min,15000g×20min,收集上清。按照CaspACE Assay System(colorimetric)说明书同时制备阳性和阴性对照样品,测定并调整各组蛋白浓度相同。96孔板中每孔加入Caspace AssayBuffer32ul,DMSO2ul,100nM DTT 10ul,去离子水调整体积为98ul,加入2ul DEVD-pNA底物,37℃孵育4h,检测波长405nM测吸光度,用ΔA法计算每组样品Caspase3活性。
结果如图10所示,A2780/Taxol细胞转染pLKO.1-CLDN8-sh1后,Caspase3活性增加了3.75倍,与阴性对照比较,有显著性差异(P<0.05),说明特异性阻断CLDN8的表达后,能促进肿瘤细胞凋亡。
五、细胞划痕试验检测细胞迁移能力
在六孔板背后用直尺均匀划横线,约0.5cm划一道,横穿过孔,每孔至少有6条横线。细胞转染pLKO.1-CLDN8-sh1后,继续培养24h细胞融合成单层状态时,在选定区域用200ul的枪头在六孔板中垂直划痕,PBS洗3次去除划下的细胞,加入无血清培养基继续培养。0h,24h,48h时间点拍照,随机选取6条水平线,计算细胞间距离均值。
结果如图11所示,pLKO.1-CLDN8-sh1转染细胞24h和48h后,A2780/Taxol细胞间距离显著大于阴性对照组,细胞划痕后愈合能力显著下降,说明特异性阻断CLDN8的表达后,能抑制肿瘤细胞的迁移。
六、Transwell试验检测细胞迁移能力
细胞转染48h后,用胰酶消化收集,用无血清培养基重悬,调整细胞密度为5×105/ml,上室加2ml细胞悬液,下室加10%FBS完全培养基2ml,继续培养24h,取出小室,PBS洗3次,用棉签小心去除上室上层表面的细胞,倒置晾干,95%乙醇固定25min,苏木素染色,显微镜下观察、计数、拍照。每个小室计数10个视野,取平均值统计并分析细胞迁移能力的改变。
细胞迁移实验结果如图12A所示,转染pLKO.1-CLDN8-sh1后,A2780/Taxol实验组与阴性对照组穿透小室的细胞数量分别是68±15与279±29,两者有统计学差异(P<0.05);该结果表明,特异性阻断CLDN8的表达后,能抑制肿瘤细胞的迁移。
七、Transwell试验检测细胞侵袭能力
将-20℃保存的基质胶先在4℃复温液化,取基质胶与OPTI-MEM以1:6冰上混匀稀释,包被小室底部膜的上室面,37℃固化30min,吸除小室内析出的液体。基质胶包被后其余步骤同上,每个小室计数10个视野,取平均值统计并分析细胞侵袭能力的改变。
细胞侵袭实验结果如图12B所示,A2780/Taxol实验组与阴性对照组穿透小室的细胞数量分别是43±10与188±32,两者有统计学差异(P<0.05);该结果表明,特异性阻断CLDN8的表达后,能抑制肿瘤细胞浸润。
实施例6.pLKO.1-CLDN8-sh1特异性阻断CLDN8表达后对卵巢癌耐药的逆转作用
一、实验分组:
1.A2780正常组(不转染任何载体),以下称作A;
2.A2780/Taxol正常组(不转染任何载体),以下称作AR;
3.A2780/Taxol阴性对照组(转染pLKO.1puro空载体),以下称作AR-N;
4.A2780/Taxol实验组(转染pLKO.1-CLDN8-sh1),以下称作AR-sh1。
二、分组转染
转染步骤同前,转染后继续培养细胞24h。
三、转染pLKO.1-CLDN8-sh1后细胞对紫杉醇敏感性的检测
各组取对数生长期细胞,胰酶消化后细胞重悬,细胞计数并调整细胞悬液的密度为1×105/ml,接种到96孔板继续培养24h。次日,各组中加入紫杉醇,浓度梯度分别设200ug/ml,100ug/ml,50ug/ml,25ug/ml,12.5ug/ml,6.25ug/ml,3.125ug/ml,0ug/ml,作用24h后,用溴标法在波长370nm(参考波长492nm)测吸光度(A),计算紫杉醇对每组细胞的抑制率,抑制率=A实验组/A阴性对照组。每个浓度设3个复孔,取平均值。
抑制率为50%时的药物浓度为半数抑制浓度(IC50);
耐药株A2780/Taxol的IC50与其亲本细胞株A2780的IC50的比值为耐药倍数(ResistantFolder,RF);
耐药株A2780/Taxol的IC50与其转染pLKO.1-CLDN8-sh1(逆转剂)后的IC50的比值为耐药逆转指数(Reversal Index,RI)。
结果如表2、表3、图13所示,A2780/Taxol对紫杉醇的IC50(36.82±5.27ug/ml)显著高于亲本A2780对紫杉醇的IC50(1.29±0.26ug/ml),耐药倍数高达28.54,提示A2780/Taxol对紫杉醇的敏感性显著低于亲本细胞A2780,高度耐药。而当A2780/Taxol转染pLKO.1-CLDN8-sh1之后,对紫杉醇的敏感性明显提高(6.71±1.32ug/ml),转染pLKO.1-CLDN8-sh1对A2780/Taxol紫杉醇耐药的逆转效果非常明显,逆转指数为5.73。
表2.A2780及A2780/Taxol对紫杉醇的药物敏感性
表3.转染pLKO.1-CLDN8-sh1后A2780/Taxol对紫杉醇药物敏感性的逆转
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Claims (3)
1.特异性抑制CLDN8基因表达的siRNA在制备逆转卵巢癌紫杉醇耐药的药物中的应用,其特征在于,所述siRNA包括正义链和反义链,所述正义链的核苷酸序列如SEQ ID NO.1所示,反义链的核苷酸序列如SEQ ID NO.2所示。
2.如权利要求1所述的应用,其特征在于,所述正义链和反义链的5’和3’端的3个碱基进行2’-甲氧基修饰。
3.一种含有编码特异性抑制CLDN8基因表达的siRNA的DNA序列的重组载体在制备逆转卵巢癌紫杉醇耐药的药物中的应用,其特征在于,所述siRNA包括正义链和反义链,所述正义链的核苷酸序列如SEQ ID NO.1所示,反义链的核苷酸序列如SEQ ID NO.2所示。
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