CN105412140B - 小分子RNA hsa-miR-874在制备治疗胶质母细胞瘤药物中的应用 - Google Patents
小分子RNA hsa-miR-874在制备治疗胶质母细胞瘤药物中的应用 Download PDFInfo
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Abstract
本发明属于生物技术研究领域,涉及小分子RNAhsa‑miR‑874在杀伤GBM中的干细胞及非干细胞肿瘤等方面的用途。主要是在制备治疗胶质母细胞瘤药物、制备治疗胶质母细胞瘤干细胞药物及制备抑制胶质母细胞瘤细胞生长的药物中的应用。本发明利用qRT‑PCR技术,发现与正常神经干细胞相比,hsa‑miR‑874在GBM干细胞中表达下调;与正常脑组织相比,GBM肿瘤组织中hsa‑miR‑874表达也下调;miR‑874可抑制肿瘤细胞的生长,引起肿瘤细胞的凋亡,并对肿瘤细胞的侵袭力,肿瘤干细胞的自我更新有抑制作用,靶基因STAT3在mRNA和蛋白水平均表达下调;miR‑874通过其靶基因STAT3参与了GBM的发生发展过程,它可作为GBM发生的调控分子,从而为临床治疗提供理论和实验依据。
Description
技术领域
本发明属于生物技术研究领域,涉及制备抗胶质母细胞瘤药物的miRNA,特别涉及与胶质母细胞瘤相关的小分子RNA hsa-miR-874在杀伤GBM中的干细胞及非干细胞肿瘤等方面的用途。
背景技术
胶质母细胞瘤(glioblastoma,GBM)是恶性度最高,无法治愈的脑肿瘤[1-3]。近年来的研究证实胶质母细胞瘤中存在一些具有干细胞特性的肿瘤细胞—胶质瘤干细胞(gliomastem cell,GSC)[4-6],它们是胶质母细胞瘤难以治愈的主要原因[7,8]。胶质瘤干细胞具有极强的成瘤性,对常规放化疗有更强的耐受性,其所携带的基因突变和一般肿瘤细胞有所不同[7-10]。因此开发针对胶质瘤干细胞的药物或疗法已经成为当前胶质母细胞瘤研究的重点;发现既对胶质瘤干细胞,又对胶质瘤细胞起作用的分子对于胶质母细胞瘤的治疗研究具有重要意义。
在胶质瘤干细胞药物研究中,近年来微RNA(microRNA或miRNA)逐渐被众多研究者所重视[11,12]。miRNA成为研究热点的主要原因是:1)可以调控上百个基因,能在系统水平上对细胞进行调控;2)只有21–23个核苷酸,可以作为易吸收的小分子药物;3)更加容易合成,作为药物的成本较低;4)细胞本身就具有的一种小分子,可以避免潜在的免疫(排斥)反应;5)可以由细胞释放入血液,更加易于血液检测和诊断。
miRNA是一种非编码RNA,由细胞核内的基因转录而来,最后在细胞质内形成大小约为22个核苷酸的单链RNA。miRNA可以和mRNA的3'端非翻译区(3'UTR)结合,从而达到减少蛋白翻译的作用[13-15]。目前已经探明miRNA在哺乳动物中调控超过60%的编码RNA(能最终翻译成蛋白)。这些蛋白参与细胞活动的各个环节,如发育、增殖、命运决定、生长控制、凋亡[16,17]。由于miRNA靠5'端约7个核苷酸(称为种子区,seed region)识别靶mRNA的3'UTR,理论上一个miRNA可以与几百个mRNA结合;一个mRNA的3'UTR上也可以有多个与miRNA种子区互补的序列。因此作为药物靶点,少量miRNA就可以在细胞的系统水平(systemic level)上控制细胞的多个信号通路及活动。
国内外的一些研究组已经开展miRNA与肿瘤的基础和应用研究,如miR-137 在脑胶质瘤中低表达,在胶质瘤细胞中过表达miR-137可以抑制细胞生长,降低细胞侵袭能力[18];miR-21能增强脑胶质瘤对卡莫司汀的耐药性[19];miR-10b在多种肿瘤中过表达,抑制miR-10b可以有效地治疗乳腺癌的转移[20,21];miR-124往往在胶质母细胞瘤中低表达,人为过表达miR-124能降低胶质母细胞瘤的侵袭力 [22,23]。已有生物医学公司开始研发miR-10b的拮抗剂用于胶质母细胞瘤的治疗。上述事实表明:1)miRNA做为药物治疗肿瘤有着切实可行的应用基础和研究价值;2)miRNA已进入大医药公司的研发计划,开发新miRNA做为胶质母细胞瘤的治疗药物具有良好的市场前景。
发明内容
发明人在研究中发现在胶质瘤干细胞中,miR-874的表达显著低于其在正常神经干细胞中的表达;在胶质母细胞瘤组织中,miR-874的表达显著低于其在正常脑组织中的表达。miR-874在多种肿瘤中低表达,被认为是一种抑癌因子(tumor suppressor),目前关于miR-874的研究还没有将其应用于胶质瘤的治疗,因此可以根据miR-874的差异表达设计针对胶质瘤干细胞和胶质瘤细胞的治疗研究,这对于治疗胶质母细胞瘤药物的研究具有重要意义。
针对现有技术,本发明对小分子RNAhsa-miR-874在GBM的调控作用及其用法进行了研究,研究发现,其可作为抑制GBM细胞生长的调控分子,引起GBM细胞的凋亡,抑制GBM细胞的侵袭/转移,抑制GBM细胞中肿瘤干细胞的干性,从而可以用于制备治疗GBM的药物、制备治疗GBM干细胞的药物、抑制GBM细胞生长的药物、抑制GBM侵袭/转移的药物。
本发明第一个目的是请求保护小分子RNAhsa-miR-874在制备治疗胶质母细胞瘤药物中的应用。
本发明第二个目的是请求保护小分子RNA hsa-miR-874在制备治疗胶质母细胞瘤干细胞药物中的应用。
本发明第三个目的是请求保护小分子RNA hsa-miR-874在制备抑制胶质母细胞瘤细胞生长的药物中的应用。
本发明所涉及的的小分子RNAhsa-miR-874其核苷酸序列为:SEQ ID NO.15’-cugcccuggcccgagggaccga-3’。
制备上述药物的方法具体为:有效量的小分子RNAhsa-miR-874与一种或多 种药学上可接受的载体,制备抗胶质母细胞瘤药物。
本发明利用qRT-PCR技术发现与正常神经干细胞相比,hsa-miR-874在GBM干细胞中表达下调;同时,与正常脑组织相比,GBM肿瘤组织中hsa-miR-874表达也下调。
本发明利用生物信息学方法确定了hsa-miR-874的靶基因为STAT3,并构建了hsa-miR-874靶基因STAT3的报告载体,命名为psiCheck-STAT3。双荧光检测结果表明STAT3的确是hsa-miR-874的靶基因。
本发明体外转染hsa-miR-874,利用细胞生长曲线观察到miR-874可抑制GBM肿瘤细胞及肿瘤干细胞的生长;利用Tunnel染色法观察到miR-874可引起GBM肿瘤细胞及肿瘤干细胞的凋亡;利用细胞Boyden小室法可观察到miR-874抑制细胞迁移与侵袭;利用细胞球形成可观察到miR-874抑制GBM干细胞的干性。
本发明体外转染hsa-miR-874后,对靶基因进行RT-PCR和Wstern检测,其结果表明,靶基因STAT3在mRNA和蛋白水平均表达下调。
因此,本发明miR-874可能通过其靶基因STAT3参与了GBM干细胞与肿瘤细胞的癌症发生发展过程,研究miR-874与GBM的关系意义重大。
有益效果:
本发明利用qRT-PCR技术发现与正常神经干细胞相比,hsa-miR-874在GBM干细胞中表达下调;同时,与正常脑组织相比,GBM肿瘤组织中hsa-miR-874表达也下调,利用生物信息学方法和双荧光检测确定了hsa-miR-874的靶基因为STAT3;miR-874可抑制肿瘤细胞的生长,引起肿瘤细胞的凋亡,并对肿瘤细胞的侵袭力,肿瘤干细胞的自我更新有抑制作用,靶基因STAT3在mRNA和蛋白水平均表达下调;miR-874通过其靶基因STAT3参与了GBM的发生发展过程,它可作为GBM发生的调控分子,从而为临床治疗提供理论和实验依据。
附图说明
图1:qRT-PCR检测hsa-miR-874在GBM干细胞和肿瘤细胞中的表达,其中,A:miR-874在神经干细胞(NSC)与GBM干细胞(GSC)中的表达差异,NSC和GSC分别使用3个细胞系(line1-3);B:miR-874在正常脑组织(Normal)与GBM病人脑组织(GBM)中的表达差异;
图2:miR-874与其推测的靶基因STAT3的双荧光报告检测;
图3:miR-874引起GBM干细胞的凋亡;
图4:miR-874引起GBM细胞的凋亡;
图5:miR-874抑制GBM干细胞的生长;
图6:靶基因STAT3表达差异的检测,其中,A:RT-PCR检测靶基因在mRNA水平上的表达差异;B:Western检测靶基因在蛋白水平上的表达差异。
具体实施方式
下面结合实施例对本发明做进一步说明,但不用于限制本发明的保护范围,下述实施例中如无特殊说明,所采用的实验方法均为常规方法,所用材料、试剂等均可从生物或化学公司购买。
实施例1:hsa-miR-874在GBM干细胞及GBM组织中表达水平的检测
Trizol法分别提取GBM干细胞和GBM组织细胞的总RNA。用PBS将细胞洗涤2遍,每瓶加入1mlTrizol,吹打混匀,室温孵育5min;将细胞液转移入DEPC处理过的1.5ml离心管中,每管加入0.2ml氯仿,剧烈振荡15s混匀,冰上静置3~5min使分层;4℃,12000rpm,离心15min;小心吸取上层液体,转移到另一个DEPC处理过的1.5ml离心管中,加入等体积已在4℃预冷的异丙醇,上下颠倒轻轻混匀15~30s,冰上(室温)静置5~10min;4℃,12000rpm,离心15min;弃上清,加入1ml 75%乙醇,轻轻洗涤沉淀2次;4℃,7500rpm离心10min,弃上清,EP管倒置晾干,加入DEPC水30ul溶解RNA。紫外吸收测定法和琼脂糖凝胶电泳检测总RNA的浓度和纯度。
利用Invitrogen的逆转录试剂盒将上述所提总RNA进行cDNA的合成。反应体系(20ul)如下:总RNA 100pg-lug,5×逆转录反应缓冲液4ul,10×SuperScript逆转录酶2ul,EDPC水补足到20ul。将上述体系混匀,PCR仪上进行逆转录,反应条件:42℃30min,95℃5min,4℃保存。
再以cDNA为模板,利用特异性hsa-miR-874TaqMan引物,在StepOne实时定量PCR仪上进行Real-time PCR。反应体系(20ul)如下:20×TaqMan MicroRNA Assays 1ul,Universal Master Mix 10ul,cDNA 5ng,加DEPC水至20ul。反应条件:95℃10min;95℃15s,60℃1min,40个循环。以U6小RNA为内参对照,采用2-ΔΔCt相对定量法处理数据。
Real-time PCR结果表明,与正常神经干细胞相比,hsa-miR-874在GBM干细胞中表达下调,下调5倍左右(如图1A);与正常脑组织相比,hsa-miR-874在GBM组织中表达下调,下调2.5倍左右(如图1B)。
实施例2:hsa-miR-874靶基因的预测
通过生物信息学方法,利用网上数据库(TargetScan:http://www.targetscan.org/;Pictar:http://pictar.bio.nyu.edu/;miRanda:http://microrna.sanger.ac.uk/)预测hsa-miR-874相关的靶基因,暂确定hsa-miR-874的靶基因为STAT3。
实施例3:hsa-miR-874靶基因报告载体的构建
依据GenBank中STAT3基因的序列,选取包含与hsa-miR-874匹配的一段序列设计引物,(STAT3-F和STAT3-R),并在STAT3-F的5′端悬垂内切酶KpnI的酶切位点和保护碱基,在STAT3-R 5′端悬垂另一内切酶BglIII的酶切位点和保护碱基。然后进行PCR扩增,PCR反应体系(50ul)如下:2×反应缓冲液25ul,10uM上游引物1ul,10uM下游引物1ul,DNA2ul(小于1ug),ddH2O 21ul。反应条件:94℃3min;94℃30s,55℃40s,72℃40s,32个循环;72℃延伸5min;4℃保存。扩增后对PCR产物进行双酶切,酶切体系为(50ul):10×T Buffer5ul,KpnI2.5ul,BglIII 2.5ul,PCR产物15ul,ddH2O 25ul。酶切反应在37℃水浴中进行,3h后利用凝胶回收试剂盒回收酶切产物,产物最终溶于10μLBuffer EB中。
再对载体psiCheck control进行双酶切,酶切体系为(50ul):10×T Buffer 5ul,KpnI 2.5ul,BglIII 2.5ul,psiCheck control 10ul,ddH2O 30ul。酶切反应在37℃水浴中进行,3h后利用凝胶回收试剂盒回收酶切产物,产物最终溶于10μL Buffer EB中。
将上述酶切产物进行连接,连接体系为(20ul):10×连接Buffe 2μL,载体片段6μL,目的基因片段10μL,T4DNA连接酶2μL。连接反应在4℃下进行,16~20h后,70℃水浴10min灭活连接酶,终止连接反应。
连接产物转化大肠杆菌DH5α,涂菌平皿于37℃孵箱内培养12~16h。然后挑选阳性克隆,分别进行PCR扩增,酶切和测序验证,获得正确的hsa-miR-874表达载体,命名为pS-STAT3-874。
实施例4 hsa-miR-874靶基因报告载体的体外转染
用Invitrogen公司的Lipofectamine 2000转染试剂将pS-STAT3-874转染至GBM干细胞,转染步骤简述如下:转染前一天,以每孔4×105个细胞的浓度接种于6孔板;常规培养20h后,细胞生长至对数增殖期,稀释适当体积的pS-STAT3-874(或psiCheck-control)于100ul OPTI-MEM培养基中,轻柔混合,使其终浓度为20ug/ml;Lipofectamine 2000:pS-STAT3-874(或psiCheck-control)按4∶2的比例在96孔板中配制转染混合物,剧烈震荡5-10s;静置15min后,将转染混合物缓慢加到含GBM干细胞和OPTI-MEM培养基的培养孔中,轻摇30s;在37℃5%的CO2孵育6h后,更换成含GBM干细胞培养基,继续培养48-72h后检测转染水平以及后续实验。
实施例5 hsa-miR-874的体外转染
从Invitrogen公司购买用于转染的hsa-miR-874,用Invitrogen公司的Lipofectamine 2000转染试剂将pS-STAT3-874转染至GBM干细胞,转染步骤同实施例4。
实施例6:双荧光报告的检测
GBM干细胞接种24小时后进行转染。转染方法同实施例4,然后进行双荧光报告的检测。双荧光素酶报告系统的检测,采用Promega的Dual-luciferase荧光素酶报告基因检测系统,按试剂盒说明操作,简要过程如下:转染GBM干细胞48h后用PBS洗一遍细胞,然后每孔用被动裂解液100ul裂解细胞,取15ul裂解产物加入50ul Luciferase反应底物,然后用荧光测量仪测定Firefly荧光,再加入StopGlo试剂50ul,马上测量Renilla荧光。分析检查得到Firefly荧光素酶和Renilla荧光素酶的荧光值,用Renilla荧光素酶荧光值对Firefly荧光素酶荧光值进行归一化。每组实验至少重复3次,取平均值。
双荧光报告检测结果显示,当hsa-miR-874靶基因报告载体pS-miR-29c与hsa-miR-874共转染GBM干细胞后,荧光素酶的荧光值降低了5倍多,表明STAT3的确是hsa-miR-874的靶基因(如图2)。
实施例7:miR-874引起GBM干细胞的凋亡
miR-874体外转染到GBM干细胞中,方法如实施例4和实施例5,转染后24h,将细胞于玻璃切片上用4%多聚甲醛固定20min,在冰上将细胞用渗透液中浸泡2min,用滤纸小心吸去载玻片上组织周围的多余液体,立即在切片上加50ul Tunnel反应液,置湿盒中于37C反应1hr,在荧光显微镜下观察细胞染色可见经miR-874转染的GBM干细胞有较多的凋亡细胞(如附图3所示,其中红色为凋亡细胞)。
实施例8:miR-874引起GBM组织细胞的凋亡
将于原代细胞培养条件下(含血清)培养的GBM细胞用miR-874进行体外转染,再进行Tunnel染色,方法如实施例7,结果显示miR-874可引起GBM细胞的凋亡(如附图4所示)。
实施例9:miR-874抑制GBM干细胞生长。
将体外转染miR-874的GBM干细胞与转染对照miRNA的GBM干细胞分别进行培养,可见转染了miR-874的GBM干细胞随着时间的增加数量逐渐较少,而转染了对照miRNA的GBM干细胞则呈现出正常的细胞增殖(图5)。
实施例10:靶基因表达差异的检测
(1)RT-PCR检测靶基因在mRNA水平上的表达差异
hsa-miR-874体外转染GBM干细胞,转染方法同实施例5,转染后48h,利用Trizol法提取细胞的总RNA。提取方法同实施例1,紫外吸收测定法和琼脂糖凝胶电泳检测总RNA的浓度和纯度后,利用试剂盒将所提总RNA进行cDNA的合成。
反应体系(20ul)如下:总RNA 100pg-lug,5×逆转录反应缓冲液4ul,10×SuperScript逆转录酶2ul,EDPC水补足到20ul。将上述体系混匀,PCR仪上进行逆转录,反应条件:42℃30min,95℃5min,4℃保存。
再以cDNA为模板,对靶基因进行RT-PCR。反应体系(20ul)如下:2×反应缓冲液10ul,10uM上游引物0.4ul,10uM下游引物0.4ul,cDNA 1ul,DEPC水8.2ul。反应条件:94℃2min;94℃30s,55℃30s,72℃40s,30个循环;72℃5min,4℃保存。以β-actin为靶基因的内参对照。
RT-PCR结果表明,与对照相比,转染了hsa-miR-874的靶基因在mRNA水平表达降低。检测结果见图6A。
(2)Western检测靶基因在蛋白水平上的表达差异
利用Western检测靶基因在蛋白水平上的表达差异,检测步骤简述如下:收集实施例5中经处理的各组细胞至Ep管中,PBS洗涤两次。各管中加入100μL细胞 蛋白裂解液,冰上孵育30min(每隔5min轻弹混匀)。12000rpm,4℃离心5min,取上清至新Ep管中(20μL/管),-80℃保存备用。用洗洁精清洗玻璃板,流水冲净,再用酒精棉球擦洗,晾干。安装好灌胶的装置,在100mL小烧杯内配制分离胶(10%),沿高玻璃板倒胶至矮玻璃板的大约3/4(注意不要有气泡),接着加满双蒸水。待30min后分离胶凝聚,用吸水纸吸净上层水,再灌积层胶(5%),插入梳子。待积层胶凝聚后,将凝胶放入电泳槽中安装好,矮玻璃板在内,高玻璃板在外,加入1×SDS电泳缓冲液,液面超出矮玻璃。小心拔出梳子,注意不要有气泡。取各组细胞的裂解液100μL,各加入20μL 6×SDS上样缓冲液,混匀。低分子量蛋白质Marker 10μL中加入2μL 6×SDS上样缓冲液,混匀。沸水中煮沸3~5min,12000rpm离心1min,冰上放置,上样。用缓冲液冲洗梳孔后,按顺序上样(对照组、miR-874组)每组样品设3个孔,7μL/孔。在胶的两侧分别加入3uL蛋白质分子量Marker(彩色)。稳压电泳,积层胶中电流为60V,进入分离胶,电压增加至100V。电泳结束后,取出电泳装置,小心剥下凝胶,切下包含所有样品的三分之一胶,经考马斯亮兰染色,检测是否有目的蛋白条带,并切下剩余三分之二胶中各目的条带所在范围的凝胶。根据凝胶的大小剪4块Whatman滤纸和1块PVDF膜(两者的大小不可超过凝胶),将剪好的滤纸、PVDF膜(提前在甲醇中浸泡5-6min)、凝胶及转膜装置中的海绵置于转移缓冲液中浸泡3~5min。在海绵上依次放置:两层滤纸、凝胶、PVDF膜、两层滤纸。加紧海绵,放入转移槽内。(注意:凝胶一侧靠近负极,PVDF膜一侧靠近正极)。电流400mA,稳流,4℃转移1h。转移结束后,取出塑料支架,依次去掉各层,用铅笔在膜的上缘做好标记,切下Marker上样孔的所对应的膜的1/2,用丽春红染色3s,而后用水清洗,检查转膜是否完全。将其余的PVDF膜放在大小合适的杂交袋中,根据膜和袋的大小加入适量的封闭液,封口,室温封闭1h。在杂交袋上剪一小口,小心取出回收封闭液,不洗膜。在不同杂交袋内分别加入以封闭液按1∶1000稀释的小鼠抗人Tnfaip3抗体和按1∶2000稀释的小鼠抗人β-actin抗体,37℃杂交1h,4℃杂交过夜。从杂交袋中小心取出PVDF膜,用洗膜液振荡洗涤3次,10min/次。再以TBS洗涤1次,10min。将PVDF膜分别与用TBST按1∶10000稀释的羊抗鼠二抗室温孵育2h。从杂交袋中小心取出PVDF膜,用洗膜液振荡洗涤3次,10min/次。在EP管中中加入适量ECL显色液A、B(1∶1)配制好的溶液,均匀加到PVDF 膜上,立即在暗室内显影。观察目的条带的变化趋势。
Weatern结果表明,与对照相比,转染了hsa-miR-874的靶基因在蛋白水平表达明显降低。检测结果见图6B。
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Claims (3)
1.小分子RNAhsa-miR-874在制备治疗胶质母细胞瘤药物和治疗胶质母细胞瘤干细胞药物中的应用。
2.小分子RNAhsa-miR-874在制备抑制胶质母细胞瘤细胞生长的药物中的应用。
3.根据权利要求1或2所述的应用,其特征在于,所述的小分子RNA hsa-miR-874其核苷酸序列为:SEQ ID NO.15’-cugcccuggcccgagggaccga-3’。
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