CN103405786A - Nsmce2的反义核苷酸序列在制备抑制结直肠癌细胞生长药物中的应用 - Google Patents
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Abstract
本发明公开了非SMC元件2同源物NSMCE2的反义核苷酸序列在制备抑制结直肠癌细胞生长药物中的应用,属于肿瘤生物治疗技术领域。在本发明中,设计合成了一条非SMC元件2同源物NSMCE2的反义核苷酸序列,如SEQ ID NO.1所示。本发明针对NSMCE2基因高扩增和过表达的结直肠癌细胞,将NSMCE2反义核苷酸序列应用于结直肠癌细胞生长抑制药物制备中,利用NSMCE2反义核苷酸序列通过RNAi技术干扰其表达从而有效抑制了结直肠癌细胞生长,降低了结直肠癌细胞恶性程度,因此,本发明的提出为结直肠癌的治疗提供了一种新的靶向生物治疗策略。
Description
技术领域
本发明涉及非SMC元件2同源物(non-SMC element2homolog,NSMCE2)的反义核苷酸序列的新用途,具体的,本发明涉及NSMCE2的反义核苷酸序列在制备抑制卵巢癌细胞生长药物中的应用,属于肿瘤生物治疗技术领域。
背景技术
非SMC元件2同源物(non-SMC element2homolog,NSMCE2),又名NSE2、MMS21,属于E3SUMO连接酶的Siz/PIAS(SP)家族成员,在该基因的C末端有一个SP的指环结构。该基因通过参与SMC5-SMC6复合体的形成而发挥生物学作用。染色体结构维持(structural maintenance of chromosomes,SMC)蛋白是一类普遍存在于生物界的、高度保守的染色体ATP酶,以SMC蛋白为核心可形成三种多蛋白复合体,其中一种为SMC5-SMC6复合体,该复合体以SMC分子中央的绞链(Hinge)区为中点自行折叠,自折叠的SMC5和SMC6两个分子通过绞链区相互结合,形成V型结构,而NSE2与SMC5的coiled-coiled区域结合,之后其他NSE分子再与此复合体结合。SMC5-SMC6复合体主要参与DNA的复制与重组修复。在DNA双链断裂的损伤应答的多个步骤中都起到作用。该复合体与HR修复通路相关,在MRN依赖的修复途径中,该复合体被招募到DNA双链断裂处,而参与到DNA修复过程中。SMC5-SMC6复合体突变还可以导致X DNA的产生,而该类型的DNA与DNA损伤和DNA复制抑制相关。SMC5-SMC6复合体还参与染色体结构的维持,还在姐妹染色单体联合中起作用。而将该复合体中的NSMCE2基因沉默之后会引起细胞对不同类型的DNA损伤(羟基脲、离子射线、甲基甲磺酸盐、紫外线)的敏感性增加。
双微体是1962年在肺癌恶性胸水细胞中发现的染色体外成对存在、着色与染色质相似或较浅、可自由复制的细胞遗传学结构,常含有扩增基因,参与细胞增殖,或为细胞生长提供选择优势。多种肿瘤的演进过程往往伴随着癌基因的扩增和过表达,癌基因拷贝数的增加是导致癌基因激活的重要机制之一。我们的前期工作发现并确认人结直肠腺癌细胞NCI-H716中有NSMCE2基因的扩增和过表达,而且NSMCE2以双微体形式扩增,提示其过表达在结直肠癌的形成及演进过程中可能起到重要的作用。
在结直肠癌的手术治疗后,辅助化疗是结肠癌综合治疗的一个重要组成部分。辅助化疗的机理在于用化疗控制减灭根治术后体内的残留病灶。但因癌症化疗的副作用较大,往往会引起很多毒副反应和合并症、后遗症,从而为广大结直肠癌患者带来了较大伤害。
针对肿瘤细胞的特点进行特异性治疗可以提高肿瘤治疗的敏感性和特异性,有的放矢。本发明针对与结直肠癌细胞发生发展过程中起重要作用的NSMCE2,用其反义核苷酸序列进行干扰,通过抑制NSMCE2的表达从而控制结直肠癌细胞的生长,针对这一与结直肠癌细胞双微体形成密切相关的重要靶点进行干预,通过减少肿瘤细胞中双微体数目,从而控制肿瘤细胞的演进,进而抑制结直肠癌细胞生长,达到预防或治疗结直肠癌的目的。
发明内容
本发明针对目前结直肠癌化疗效果欠佳的情况,提供一种通过NSMCE2反义核苷酸序列干扰NSMCE2表达,降低双微体的数目,从而抑制结直肠癌细胞生长,起到预防或治疗结直肠癌的作用。
本发明的技术方案是特异性的针对NSMCE2扩增以其反义核苷酸序列进行RNA干扰抑制,从而抑制结直肠癌细胞的增殖及侵袭能力,起到抑制结直肠癌细胞生长的作用。
为了达到上述目的,具体的,本发明采用了以下技术方案:
1、检测肿瘤细胞中NSMCE2扩增情况
选择含有双微体的人结直肠癌细胞系NCI-H716为研究对象。
(1)常规细胞培养;
(2)通过PCR和RT-PCR技术从DNA及mRNA水平检测细胞中NSMCE2扩增和表达情况。
2、构建pSuper-shRNA-NSMCE2基因真核表达载体抑制NSMCE2表达。
(1)构建并鉴定pSuper-shRNA-NSMCE2表达载体;
(2)将构建成功的载体转染入NCI-H716细胞中,用Western Blotting验证NSMCE2蛋白水平沉默的效果;
(3)建立NCI-H716稳定转染pSuper-shRNA-NSMCE2细胞系。
3、抑制NSMCE2表达对NCI-H716细胞生物学行为的影响。
(1)中期核型分析检测抑制NSMCE2表达后的双微体数目的变化;
(2)绘制细胞生长曲线检测抑制NSMCE2表达后细胞增殖能力的变化;
(3)流式细胞术检测抑制NSMCE2表达后的细胞周期变化;
(4)侵袭实验检测抑制NSMCE2表达后细胞侵袭能力的变化。
在此研究的基础上,本发明首先提出了非SMC元件2同源物(NSMCE2)的反义核苷酸序列在制备抑制结直肠癌细胞生长药物中的应用。
在本发明中,优选的,所述的非SMC元件2同源物(NSMCE2)的反义核苷酸序列如SEQ ID NO.1所示。
其次,本发明还提出了一种非SMC元件2同源物(NSMCE2)的反义核苷酸序列,其特征在于所述的反义核苷酸序列如SEQ ID NO.1所示。
含有所述的核苷酸序列的表达载体以及含有所述表达载体的宿主细胞也应在本发明的保护范围之内。
在本发明中,优选的,所述的表达载体是以载体pSUPER.retro.puro为基础载体构建得到的。
在本发明中,更优选的,所述的表达载体是通过以下方法构建得到的:
(1)合成以下含有非SMC元件2同源物(NSMCE2)的反义核苷酸序列的寡核苷酸序列:
hNSMCE2-SiR+:
GATCCCCAAGAATTCTGATGCAGACTTTTTCAAGAGAAAAGTCTGCAT CAGAATTCTTTTTTTA(SEQ ID NO.2)
hNSMCE2-SiR-:
AGCTTAAAAAAAGAATTCTGATGCAGACTTTTCTCTTGAAAAAGTCTG CATCAGAATTCTTGGG(SEQ ID NO.3);
(2)寡核苷酸退火:
反应条件:95℃5min;80℃10min;70℃10min;60℃10min;50℃10min;37℃10min;获得含有非SMC元件2同源物(NSMCE2)的反义核苷酸序列的寡核苷酸序列;
(3)pSUPER.retro.puro载体线性化:
用BglⅡ、HindⅢ两种内切酶对pSUPER.retro.puro进行双酶切,获得线性质粒载体;
(4)步骤(2)得到的寡核苷酸片段分别与线性化的表达质粒pSUPER.retro.puro连接重组,获得pSuper-shRNA-NSMCE2重组质粒;
(5)分别将pSuper-shRNA-control和pSuper-shRNA-NSMCE2重组质粒转化至感受态DH5a细胞中,涂于含氨苄平板培养基上培养后挑取阳性菌落摇菌培养,提取重组质粒DNA,即得。
进一步的,本发明还提出了所述的表达载体在制备抑制结直肠癌细胞生长药物中的应用。
本发明针对NSMCE2高扩增和高表达的结直肠癌细胞,利用NSMCE2反义核苷酸序列,通过RNAi干扰的方法减少NSMCE2的表达,从而有效减少了双微体数目,抑制了结直肠癌细胞的生长,降低了其恶性程度,将NSMCE2的反义核苷酸序列应用于结直肠癌细胞生长抑制药物的制备中,能够为结直肠癌的生物治疗提供新的药物制备和靶向性治疗方案,提高治疗的特异性。
附图说明
图1为ShRNA-NSMCE2重组质粒酶切鉴定结果;
图2为Western Blot检测NCI-H716转染前后NSMCE2蛋白表达;
图3为NSMCE2基因沉默后NCI-H716细胞中期核型(箭头所指为双微体);
图4为NSMCE2基因沉默后NCI-H716细胞双微体数目(DMs)统计图;
(**:p<0.01转染后细胞与未转染细胞相比,双微体数目存在显著差异)
图5为NSMCE2基因沉默后NCI-H716细胞生长曲线;
图6为NSMCE2基因沉默后流式细胞仪检测NCI-H716细胞周期;
图7为NSMCE2基因沉默后NCI-H716细胞的基底膜侵袭实验结果(100×);
图8为NSMCE2基因沉默后NCI-H716细胞侵袭细胞数比较的统计学分析。
(*:p<0.05转染后细胞与未转染细胞相比,侵袭细胞数存在差异)
具体实施方式
下面结合具体实施例来进一步描述本发明,本发明的优点和特点将会随着描述而更为清楚。但实施例仅是范例性的,并不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换,但这些修改和替换均落入本发明的保护范围内。
实施例1、NSMCE2的反义核苷酸序列在制备抑制结直肠癌细胞生长药物中的应用
1、检测肿瘤细胞中NSMCE2扩增情况
(1)常规细胞培养:选择含有双微体的人结直肠癌细胞系NCI-H716(购自美国菌种保藏中心ATCC)为研究对象,用含10%胎牛血清的RPMI-1640培养基,置于CO2孵箱中于37℃条件下进行培养。
(2)DNA及mRNA水平检测NCI-H716细胞中NSMCE2扩增及表达情况:DNA水平,以正常人外周血DNA为对照,检测NSMCE2在NCI-H716中扩增情况;mRNA水平,以正常人结直肠组织cDNA为对照,检测NSMCE2在NCI-H716中扩增情况。
按照QIAamp DNA Extract Kit(QIAGEN,德国)说明书操作提取正常人外周血DNA及NCI-H716细胞DNA。采用TRIzol Reagent(Invitrogen,美国)总RNA提取试剂,按说明书提供的方法提取正常人结直肠组织总RNA及人结直肠癌细胞NCI-H716的总RNA。依测定浓度调整各组样品总RNA量,采用Transcriptor FirstStand cDNA System Kit(Roche,瑞士)逆转录试剂盒,按照说明书提供的方法逆转录合成cDNA第一链。反应条件50℃60min;85℃5min;16℃+∞。-20℃保存cDNA第一链。检索NCBI Gene数据库,获得NSMCE2基因序列,应用Primer5.0software设计并合成基因特异性引物,所用引物由Invitrogen公司合成,序列如下表1和表2所示:
表1DNA引物
NSMCE2基因的PCR扩增反应条件为:
95℃ 5min
95℃ 30s,60℃30s,72℃30s,30个循环
72℃ 10min
16℃ +∞
1.5%琼脂糖凝胶电泳检测结果。
2、构建pSuper-shRNA-NSMCE2基因真核表达载体抑制NSMCE2表达。
(1)构建并鉴定pSuper-shRNA-NSMCE2表达载体:
合成以下含有非SMC元件2同源物(NSMCE2)的反义核苷酸序列(AAGAATTCTGATGCAGACTTTT,SEQ ID NO.1所示)的寡核苷酸序列(Oligos):
hNSMCE2-SiR+:
GATCCCCAAGAATTCTGATGCAGACTTTTTCAAGAGAAAAGTCTGCAT CAGAATTCTTTTTTTA(SEQ ID NO.2)
hNSMCE2-SiR-:
AGCTTAAAAAAAGAATTCTGATGCAGACTTTTCTCTTGAAAAAGTCTG CATCAGAATTCTTGGG(SEQ ID NO.3);
(2)寡核苷酸退火:
溶解oligos的浓度至100nmol/mL,反应体系:
反应条件:95℃5min;80℃10min;70℃10min;60℃10min;50℃10min;37℃10min;分别获得含有非SMC元件2同源物(NSMCE2)的反义核苷酸序列的寡核苷酸序列;
(3)pSUPER.retro.puro载体线性化:
pSUPER.retro.puro为OligoEngine公司产品,质粒全长为6349bp。
环状siRNA表达质粒载体pSUPER.retro.puro含有BglⅡ、HindⅢ限制性酶切位点,用这两种内切酶对pSUPER.retro.puro进行双酶切,获得线性质粒载体,且两端为所需酶切位点粘性末端。
酶切体系如下:
37℃水浴2h;1%琼脂糖凝胶电泳;按照PCR产物回收步骤进行回收。
(4)寡核苷酸片段与线性化的SiRNA表达质粒pSUPER.retro.puro连接重组:
反应体系:
16℃反应过夜。
(5)pSuper-shRNA-control和pSuper-shRNA-NSMCE2重组质粒转化至感受态DH5a:从-80℃冰箱中取出感受态,立即插入冰内,待其自然融化。加入10μL连接产物,轻轻混匀后冰上放置30min。42℃水浴60s,再放入冰上5min,加入500μL LB培养液(不含氨苄),置于37℃振荡培养箱中培养,150rpm,2h。离心,3500rpm,2min。弃400μL上清液,剩余100μL液体轻轻混匀,均匀涂布于AMP(+)LB培养皿上,37℃培养过夜。
挑选阳性克隆:在含Amp的平皿培养基上接种后培养16~20小时后,可见散在分布的菌落,从中挑选数个单克隆菌落,分别接种于2.5mL LB培养液中(含氨苄),37℃振荡培养16~20小时,180rpm。
根据QIAGEN公司提供的说明书进行操作快速提取pSuper-shRNA-control和pSuper-shRNA-NSMCE2重组质粒DNA,双酶切法鉴定ShRNA-NSMCE2与pSUPER.retro.puro质粒是否连接成功,图1是1%琼脂糖凝胶电泳结果图,表示NSMCE2的sh-RNA的真核表达载体构建成功。所获得的质粒经上述方法初步验证后送Invitrogen公司测序。将测序所得到的序列使用NCBI数据库进行BLAST同源性分析比对。
3、将构建成功的载体转染入NCI-H716细胞中,建立NCI-H716稳定转染pSuper-shRNA-NSMCE2细胞系,以Western Blotting验证NSMCE2蛋白水平沉默的效果;
pSuper-shRNA-NSMCE2的转染:分组为实验组:pSuper-shRNA-NSMCE2;对照组:pSuper-shRNA-control。
作为对照的核苷酸序列为:CTGGCATCGGTGTGGATGA(SEQ ID NO.4),用于构建pSuper-shRNA-control的寡核苷酸序列(Oligos):
hcontrol-SiR+:
GATCCCCCTGGCATCGGTGTGGATGATCAAGAGTCATCCACACCGATGCCAGTTTTTA(SEQ ID NO.5)
hcontrol-SiR-:
AGCTTAAAAACTGGCATCGGTGTGGATGACTCTTGATCATCCACACCGATGCCAGGGG(SEQ ID NO.6)
将上述质粒分别转染入NCI-H716细胞,转染过程按照LipofectinTM2000转染试剂说明书操作。在转染24h之后,加入puro(嘌呤霉素)进行稳定筛选,2~3天更换含相同puro浓度的培养基。7天后,空白对照组细胞基本都已经死亡,而转染组的细胞有散在的细胞克隆,待6孔板内细胞生长至70~80%时转移至培养瓶中继续培养。得到稳定转染的细胞组和对照组培养至铺满培养瓶。之后,分别提取pSuper-shRNA-control和pSuper-shRNA-NSMCE2的总蛋白。并将细胞的总蛋白进行Western印迹实验,以判断RNA干涉效果,图2是Western Blot图表示转染pSuper-shRNA-NSMCE2后NCI-H716中NSMCE2蛋白水平表达下降因此可以应用转染细胞系做后续实验。
4、抑制NSMCE2表达对NCI-H716细胞生物学行为的影响。
(1)将处于对数生长期的pSuper-shRNA-control和pSuper-shRNA-NSMCE2两种细胞中加入终浓度为0.02μg/mL的秋水仙素,37℃继续培养1h,将细胞用吸管吹打下来,将悬液装入离心管中1000r/min离心5min,并用PBS冲洗两次,加入37℃预热的0.075mol/L的KCl,在37℃水浴中低渗作用13min,逐滴加入1mL新鲜固定液(甲醇:冰醋酸=3:1)混匀,1500r/min离心7min,弃上清加入10mL固定液,轻轻混匀,室温固定30min,1500r/min离心7min,重复一次,弃上清,加入适当固定液混匀,将细胞悬液以一定高度滴于预冷的载玻片上,室温晾干。Giemsa染液染色7min,流水冲洗,晾干。置于显微镜下观察,统计双微体数目,结果如图3和图4所示。图3是中期核型分析检测双微体数目图,图4是中期核型分析统计图,从图3和图4结果均可以看出转染后细胞中双微体数目下降。
(2)绘制细胞生长曲线检测抑制NSMCE2表达后细胞增殖能力的变化;
MTT法绘制细胞生长曲线:接种细胞,取对数生长期对照组细胞pSuper-shRNA-control和实验组细胞pSuper-shRNA-NSMCE2以细胞浓度为4×104个/mL,接种于96孔板,每孔接种100μL,每种细胞每个观察时间点均设计为3个复孔。培养24h后,每孔加入AQueous One Solution Reagent20μL,继续培养4h。选择490nm波长,酶联免疫检测仪测定各孔的吸光值,并记录结果。以时间为横轴,取3个相同生长天数及携带相同载体的光吸收值均值作为该类细胞的某天吸光值,并作为纵轴绘制细胞生长曲线,结果如图5所示。从图5结果可以看出抑制NSMCE2表达后NCI-H716细胞增殖能力降低。
(3)流式细胞术检测抑制NSMCE2表达后的细胞周期变化;
将培养好的pSuper-shRNA-control和pSuper-shRNA-NSMCE2两种细胞经0.25%胰酶消化、全培养基终止。离心,3500rpm,5min。PBS洗涤细胞2次后,加入3~5mL预冷的75%乙醇,4℃固定过夜。次日将细胞取出再次用PBS洗涤2次,用PBS配成1×106个/mL的细胞悬液,后续按照cycle TESTTM PLUS DNA试剂盒提供的操作步骤进行,行流式细胞术检测细胞周期的改变,结果如图6所示。从图6结果可以看出转染后细胞G1期较高,S期较低。
(4)侵袭实验检测抑制NSMCE2表达后细胞侵袭能力的变化。
应用BD BioCoatTM MatrigelTM Invasion Chamber对pSuper-shRNA-control和pSuper-shRNA-NSMCE2两种细胞进行重组基底膜侵袭实验。从-20℃取出侵袭小室放入24孔板中,室温放置。在24孔板和小室内各加入500μL热的(37℃)无血清1640培养基,37℃孵箱中水化2h。水化后,小心地将液体移出,不要碰到matrigel基底膜。将pSuper-shRNA-control和pSuper-shRNA-NSMCE2两种细胞用胰酶消化,全培养基终止消化,细胞计数,将细胞稀释到10万/mL。24孔板内加入750μL全培养基,之后,将小室放入24孔板中,注意小室膜下不要产生气泡。迅速加入500μL细胞悬液(5万个细胞/孔),每组细胞设3个复孔。镜下观察,吹打均匀。37℃孵箱中,培养24h。取出24孔板,棉签蘸无血清培养基擦干净膜的内表面,去掉未发生侵袭的细胞。膜在无水甲醇中固定1min,蒸馏水洗2遍;苏木素染色5min,水洗干净(至无色);伊红染色20s,水洗干净。将膜完全晾干。刀切膜,中性树胶将膜粘到载玻片上,盖上盖玻片。光镜下观察,计数,拍照。图7是细胞基底膜侵袭实验图,图8是细胞基底膜侵袭统计图,表明转染后细胞侵袭能力下降。
综上可见,利用NSMCE2反义核苷酸序列通过RNAi技术干扰其表达能够有效减少细胞中双微体数目,抑制结直肠癌细胞生长,从而降低结直肠癌细胞恶性程度,因此,本发明提出了一种新的结直肠癌的靶向生物治疗策略。
Claims (8)
1.非SMC元件2同源物(NSMCE2)的反义核苷酸序列在制备抑制结直肠癌细胞生长药物中的应用。
2.如权利要求1所述的应用,其特征在于所述的非SMC元件2同源物(NSMCE2)的反义核苷酸序列如SEQ ID NO.1所示。
3.非SMC元件2同源物(NSMCE2)的反义核苷酸序列,其特征在于所述的反义核苷酸序列如SEQ ID NO.1所示。
4.一种表达载体,其特征在于含有权利要求3所述的核苷酸序列。
5.如权利要求4所述的表达载体,其特征在于所述的表达载体是以载体pSUPER.retro.puro为基础载体构建得到的。
6.如权利要求5所述的表达载体,其特征在于所述的表达载体是通过以下方法构建得到的:
(1)合成以下含有非SMC元件2同源物(NSMCE2)的反义核苷酸序列的寡核苷酸序列:
hNSMCE2-SiR+:
GATCCCCAAGAATTCTGATGCAGACTTTTTCAAGAGAAAAGTCTGCATCAGAATTCTTTTTTTA
hNSMCE2-SiR-:
AGCTTAAAAAAAGAATTCTGATGCAGACTTTTCTCTTGAAAAAGTCTGCATCAGAATTCTTGGG;
(2)寡核苷酸退火:
反应条件:95℃5min;80℃10min;70℃10min;60℃10min;50℃10min;37℃10min;获得含有非SMC元件2同源物(NSMCE2)的反义核苷酸序列的寡核苷酸序列;
(3)pSUPER.retro.puro载体线性化:
用BglⅡ、HindⅢ两种内切酶对pSUPER.retro.puro进行双酶切,获得线性质粒载体;
(4)步骤(2)得到的寡核苷酸片段分别与线性化的表达质粒pSUPER.retro.puro连接重组,获得pSuper-shRNA-NSMCE2重组质粒;
(5)分别将pSuper-shRNA-NSMCE2重组质粒转化至感受态DH5a细胞中,涂于含氨苄平板培养基上培养后挑取阳性菌落摇菌培养,提取pSuper-shRNA-NSMCE2重组质粒DNA,即得。
7.一种宿主细胞,其特征在于含有权利要求4-6任一项所述的表达载体。
8.权利要求4-6任一项所述的表达载体在制备抑制结直肠癌细胞生长药物中的应用。
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