CN111214482B - 一种靶向linc00467基因的siRNA在白血病耐药性中的应用 - Google Patents

一种靶向linc00467基因的siRNA在白血病耐药性中的应用 Download PDF

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CN111214482B
CN111214482B CN202010108441.9A CN202010108441A CN111214482B CN 111214482 B CN111214482 B CN 111214482B CN 202010108441 A CN202010108441 A CN 202010108441A CN 111214482 B CN111214482 B CN 111214482B
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饶春宝
陆小梅
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Dongguan Institute Of Pediatrics
Dongguan Eighth People's Hospital (dongguan Children's Hospital)
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Abstract

本发明涉及生物医药技术领域，具体涉及具体涉及一种靶向linc00467基因的siRNA在白血病耐药性中的应用，本发明针对linc00467基因的核苷酸序列设计合成siRNA,该siRNA转入人白血病细胞K562之后能高效抑制linc00467基因的表达并显著降K562细胞耐药性，从而达到抗肿瘤的目的。因此，本发明设计的linc00467 siRNA分子可用于制备治疗白血病的的药物，且具有广阔的应用前景。

Description

一种靶向linc00467基因的siRNA在白血病耐药性中的应用
技术领域
本发明涉及生物医药技术领域，具体涉及一种靶向linc00467基因的siRNA在白血病耐药性中的应用。
背景技术
白血病是一种侵袭性血液学恶性肿瘤，是15～29岁人群中最常见的癌症死亡原因，是由血液和骨髓中未成熟的白血病细胞积累引起的。目前的标准治疗方案是以大剂量的常规化疗进行诱导缓解，待取得完全缓解(complete remission，CR)后再进行后续治疗，以达到强化和维持治疗的作用，但此种治疗方法的预后及生存率仍不理想。白血病耐药是指白血病细胞对所用化疗药物不敏感或具有抵抗性。当某一患者虽经几个疗程的联合化疗，骨髓内的白血病细胞百分比无明显下降，或是一时减少，但在短期休疗后很快又增长至化疗前水平，这种情况即可视为白血病耐药。白血病细胞产生耐药性是白血病治疗失败的主要原因之一。
小干扰RNA(Small interfering RNA；siRNA)是一种长20到25个核苷酸的双链RNA，在生物学上有许多不同的用途。目前已知siRNA主要参与RNA干扰(RNAi)现象，以带有专一性的方式调控基因的表达，这使siRNA成为研究基因功能与药物靶标的一项重要工具。
发明内容
为了克服现有技术中存在的缺点和不足，本发明的目的在于提供一种靶向linc00467基因的siRNA在白血病耐药性中的应用。
本发明的目的通过下述技术方案实现：一种靶向linc00467基因的siRNA在白血病耐药性中的应用。
优选的，所述靶向linc00467基因的siRNA序列为：si-linc00467-F：5'-CAGGGAGGUUUAAUAGACATT-3'，si-linc00467-R：5'-UGUCUAUUAAACCUCCCUGTT-3'。
一种靶向linc00467基因的siRNA在制备用于治疗白血病的药物的应用。
优选的，所述靶向linc00467基因的siRNA序列为：si-linc00467-F：5'-CAGGGAGGUUUAAUAGACATT-3'，si-linc00467-R：5'-UGUCUAUUAAACCUCCCUGTT-3'。
一种用于治疗白血病的药物，所述药物包括靶向linc00467基因的siRNA。
优选的，所述靶向linc00467基因的siRNA序列为：si-linc00467-F：5'-CAGGGAGGUUUAAUAGACATT-3'，si-linc00467-R：5'-UGUCUAUUAAACCUCCCUGTT-3'。
一种用于检测靶向linc00467基因的siRNA的试剂盒，所述试剂盒包括靶向linc00467基因的siRNA序列和阴性对照siRNA序列。
优选的，所述靶向linc00467基因的siRNA序列为：si-linc00467-F：5'-CAGGGAGGUUUAAUAGACATT-3'，si-linc00467-R：5'-UGUCUAUUAAACCUCCCUGTT-3'。
优选的，所述阴性对照siRNA序列为：si-NC-F：5'-UUCUCCGAACGUGUCACGUTT-3'，si-NC-R：5'-ACGUGACACGUUCGGAGAATT-3'。
本发明的有益效果在于：本发明针对linc00467基因的核苷酸序列设计合成siRNA，该siRNA转入人白血病细胞K562之后能高效抑制linc00467基因的表达并显著降K562细胞耐药性，从而达到抗肿瘤的目的。因此，本发明设计的linc00467 siRNA分子可用于制备治疗白血病的的药物，且具有广阔的应用前景。
附图说明
图1是linc00467在白血病初发组、难治性和普通型中的表达量检测；
图2是Linc00467表达水平与白血病患者预后的关系；
图3是si-linc00467干扰效率的检测；
图4是干扰linc00467的表达显著降低K562细胞对阿霉素的耐药性(**代表P<0.01)。
具体实施方式
为了便于本领域技术人员的理解，下面结合实施例及附图1-4对本发明作进一步的说明，实施方式提及的内容并非对本发明的限定。
实施例1长链非编码RNA linc00467在白血病患者中的表达水平研究
1、研究对象：
研究对象包括16例初发组白血病患者，14例白血病普通患者，11例难治性白血病患者以及白血病细胞系K562。
2、骨髓单个核细胞的采集与储存：
(1)每个患者常规抽取骨髓液4ML于肝素抗凝管管中。
(2)取一空管，加入淋巴细胞分离液3ML，沿管壁缓慢加入等量骨髓标本，1500rpm离心20分钟。吸取分离界面层的单个核细胞于1MLEP管中，加入1mL0.84％氯化铵，混匀，静置10分钟以充分裂解残余红细胞，1500rpm离心5分钟。
(3)弃上清，加入1ML生理盐水，将细胞洗涤一次，1500rpm离心5分钟。
(4)重复步骤(3)1次。弃上清，储存于-80℃或者直接提取总RNA。
3、细胞总RNA的提取和cDNA合成：
细胞总RNA的提取采用柱式法Total RNA小量提取试剂盒(Takara，货号9767)，步骤根据试剂盒说明书进行。
cDNA合成采用PrimeScriptTM IV 1st strand cDNA Synthesis Mix(Takara，货号6215)，步骤根据试剂盒说明书进行。
4、荧光定量PCR检测linc00467的表达：
荧光定量PCR采用PowerUpTM SYBRTM Green(Thermo Fisher，货号A25741)，步骤根据试剂盒说明书进行。Linc00467表达在白血病初发组中表达量最低，难治性白血病患者中最高，普通型中表达量中等(实验结果如图1所示)。
实验结果表明，长链非编码RNA linc00467在白血病患者初发组中表达量低，经治疗后表达量升高，难治性白血病患者表达量高于普通患者。(难治性患者定义为至少经过两个诱导周期后未达到完全缓减(complete remission，CR)，或在CR的12个月内复发(<5％骨髓胚细胞)以及对诱导反应有抵抗力的人。
实施例2长链非编码RNA linc00467的表达水平与白血病患者预后的相关性研究
查阅肿瘤数据库TGCA中白血病患者linc00467表达与预后相关信息(如图2所示)，发现linc00467表达水平与白血病患者预后正相关，高表达linc00467的患者生存期明显短于低表达linc00467的患者。
实施例3白血病细胞K562中利用siRNA干扰linc00467的干扰效率研究
将siRNA以20nM的剂量转染培养的K562细胞，48小时后提取细胞总RNA，逆转录成cDNA，荧光定量PCR检测linc00467表达水平(实验结果如图3所示)，可以看出，该siRNA对linc00467具有约80％的干扰效率。靶向linc00467基因的siRNA序列为：si-linc00467-F：5'-CAGGGAGGUUUAAUAGACATT-3'，si-linc00467-R：5'-UGUCUAUUAAACCUCCCUGTT-3'；阴性对照siRNA序列为：si-NC-F：5'-UUCUCCGAACGUGUCACGUTT-3'，si-NC-R：5'-ACGUGACACGUUCGGAGAATT-3'。
实施例4白血病细胞K562中利用siRNA干扰linc00467的表达对细胞耐药性的影响
在转染siRNA48小时后，培养液中添加不同浓度的化疗药物阿霉素(Adriamycin)，24小时后通过CCK-8试剂盒检测细胞存活率(实验结果如图4所示)，可以看出，通过siRNA干扰linc00467的表达能显著降低K562细胞对阿霉素的耐药性。
上述实施例为本发明较佳的实现方案，除此之外，本发明还可以其它方式实现，在不脱离本发明构思的前提下任何显而易见的替换均在本发明的保护范围之内。

Claims (1)

1.一种靶向linc00467基因的siRNA在制备用于改善患者对白血病耐药性的药物中的应用，其特征在于：所述耐药性是由化疗药物阿霉素产生的，所述靶向linc00467基因的siRNA序列为：si-linc00467-F：5'-CAGGGAGGUUUAAUAGACATT-3'，si-linc00467-R：5'-UGUCUAUUAAACCUCCCUGTT-3'。
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