CN111575283A - Dkc1特异小干扰rna及其应用 - Google Patents
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Abstract
本发明提供了一种DKC1特异小干扰RNA,其靶向的DKC1位点的核苷酸序列如SEQ ID NO.1或SEQ ID NO.2所示。本发明还提供了一种DKC1特异小干扰RNA,其核苷酸序列如SEQ ID NO.3或SEQ ID NO.5所示。本发明还提供了所述DKC1特异小干扰RNA在制备肿瘤治疗药物中的应用。本发明提供的DKC1特异小干扰RNA能够明显抑制细胞内DKC1的表达,从而抑制肿瘤细胞的增殖,进而提高肿瘤患者的生存率。
Description
技术领域
本发明属于医药领域,具体涉及一种DKC1特异小干扰RNA及其应用。
背景技术
DKC1(Dyskeratosis Congenita 1,DKC1)基因编码角化不良蛋白(dyskerin),该蛋白是H/ACA小核仁核糖核蛋白(small nucleolar ribonucleoprotein,snoRNP)的重要组成部分,并参与多种细胞事件,如蛋白表达、细胞生长及细胞增殖。除此之外,角化不良蛋白(dyskerin)也是端粒酶核糖核蛋白复合物的一部分,对维持端粒酶的活性及端粒大小具有重要的意义。研究报道,DKC1作为原癌基因,在多种肿瘤细胞中高表达,如肝癌、肠癌、肺癌、乳腺癌、前列腺癌和口腔鳞状细胞癌等。DKC1具有促进肿瘤增殖、转移及侵袭的作用,其高表达预示不良预后。因此,DKC1具有作为肿瘤治疗靶标的潜力。
结直肠癌及肺癌均为全球范围内最常见的肿瘤,其发病率及死亡率均位列前三。结直肠癌是一种遗传性的疾病,目前发现其主要的基因突变有:APC、TP53、KRAS、PIK3CA、SMAD4、BRAF等。随着早期筛查的普及及结直肠癌生物靶标的发现及应用,结直肠癌的治疗取得的重大的进步,因此,进一步研究结直肠癌新的治疗靶标将有效提高结直肠癌患者的生存率。绝大多数肺癌为非小细胞肺癌。目前针对非小细胞肺癌的治疗手段已由姑息化疗手段发展到靶向主要原癌基因的精准治疗手段,这大大提高了患者的生存期。当前已发现的靶向驱动基因有:EGFR、ALK、ROS1、BRAF、MET。然而,仍有很大一部分非小细胞肺癌的患者未找到其明确的驱动基因,因此,寻找新的可能的治疗靶点对提高肺癌患者的生存率至关重要。
发明内容
本发明的目的是针对以上要解决的技术问题,提供一种能够有效治疗肿瘤的技术方案。
为了实现以上目的,本发明提供了一种DKC1特异小干扰RNA,其靶向的DKC1位点的核苷酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
本发明还提供了一种DKC1特异小干扰RNA,其正向核苷酸序列如SEQ ID NO.3或SEQ ID NO.5所示。
根据本发明所述的DKC1特异小干扰RNA,其中所述DKC1特异小干扰RNA降低DKC1mRNA的表达。
根据本发明所述的DKC1特异小干扰RNA,其中所述DKC1特异小干扰RNA的靶向的DKC1位点的核苷酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
另一方面,本发明还提供了所述DKC1特异小干扰RNA在制备肿瘤治疗药物中的应用。
优选地,本发明的所述肿瘤意指机体在各种致瘤因子作用下,局部组织细胞增生所形成的新生物,包括但不限于黑色素瘤、肺癌、非小细胞肺癌、肾癌、淋巴瘤、头颈鳞癌、膀胱癌、结直肠癌、肝癌、胃癌、前列腺癌、骨肉瘤,特别是结直肠癌或非小细胞肺癌。
另一方面,本发明还提供了一种重组载体,其含有根据本发明所述的DKC1特异小干扰RNA。
另一方面,本发明还提供了一种宿主细胞,其含有根据本发明所述的重组载体。优选地,该重组载体以pLKO.1-TRC克隆载体为基础构建得到。
另一方面,本发明还提供了一种试剂盒,其含有根据本发明所述的DKC1特异小干扰RNA。
另一方面,本发明还提供了一种肿瘤治疗方法,其使用根据本发明所述的DKC1特异小干扰RNA来治疗肿瘤。
本发明以DKC1高表达的结直肠癌细胞系(HCT 116和DLD-1)及非小细胞肺癌细胞系(A549和PC-9)为研究对象,并将DKC1特异小干扰RNA转入细胞内稳定表达,结果发现,小干扰RNA可以明显抑制DKC1的表达,与对照细胞相比,结直肠癌细胞及非小细胞肺癌细胞的增殖明显受到抑制。
本发明通过前期的筛选,发现新的结直肠癌及非小细胞肺癌的治疗靶标DKC1,利用DKC1特异的小干扰RNA,降低DKC1的表达,从而抑制结直肠癌及非小细胞肺癌细胞的增殖,进而提高肿瘤患者的生存率,促进精准医学治疗在肿瘤治疗中的应用,提高结直肠癌及非小细胞肺治疗的有效性。
附图说明
图1是shDKC1-1测序结果。
图2是shDKC1-2测序结果。
图3示出了Western Blot检测稳定干扰DKC1表达的HCT 116细胞中DKC1的表达水平。
图4示出了Western Blot检测稳定干扰DKC1表达的DLD-1细胞中DKC1的表达水平。
图5示出了Western Blot检测稳定干扰DKC1表达的A549细胞中DKC1的表达水平。
图6示出了Western Blot检测稳定干扰DKC1表达的PC-9细胞中DKC1的表达水平。
图7示出了稳定干扰DKC1表达的HCT 116细胞生长曲线。
图8示出了稳定干扰DKC1表达的DLD-1细胞生长曲线。
图9示出了稳定干扰DKC1表达的A549细胞生长曲线。
图10示出了稳定干扰DKC1表达的PC-9细胞生长曲线。
图11示出了稳定干扰DKC1表达的HCT 116细胞平板克隆形成。
图12示出了稳定干扰DKC1表达的DLD-1细胞平板克隆形成。
图13示出了稳定干扰DKC1表达的A549细胞平板克隆形成。
图14示出了稳定干扰DKC1表达的PC-9细胞平板克隆形成。
具体实施方式
下面结合具体实施例和附图,对本发明的技术方案作进一步的详述,但本发明的保护范围并不限于以下实施例。
值得注意的是,出于简要清楚的目的,以下实施例中对一些常规的技术操作步骤、试剂、仪器并未进行细致的描述,但应理解,如未特别说明,这些常规技术操作步骤、试剂、仪器对本领域普通技术人员而言是显而易见的。
下面结合具体实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:构建稳定干扰DKC1表达的慢病毒载体
(1)分别合成两种DKC1特异小干扰RNA(DKC1-shRNA),命名为shDKC1-1和shDKC1-2,其核苷酸序列如下:
shDKC1-1正向:
5'-CCGGTATGTTGACTACAGTGAGTCTCTCGAGAGACTCACTGTAGTCAACATATTTTTG-3'(SEQID NO.3)
shDKC1-1反向:
5'-AATTCAAAAATATGTTGACTACAGTGAGTCTCTCGAGAGACTCACTGTAGTCAACATA-3'(SEQID NO.4)
shDKC1-2正向:
5'-CCGGGCTCAGTGAAATGCTGTAGAACTCGAGTTCTACAGCATTTCACTGAGCTTTTTG-3'(SEQID NO.5)
shDKC1-2反向:
5'-AATTCAAAAAGCTCAGTGAAATGCTGTAGAACTCGAGTTCTACAGCATTTCACTGAGC-3'(SEQID NO.6)
以上两种DKC1特异小干扰RNA分别靶向位于DKC1上的以下序列:
SEQ ID NO.1:TATGTTGACTACAGTGAGTCT;
SEQ ID NO.2:GCTCAGTGAAATGCTGTAGAA。
(2)shDKC1-1正向和反向序列、以及shDKC1-2正向和反向序列分别进行退火之后,连接到pLKO.1-TRC克隆载体(Addgene,货号#10878)上,连接产物转化,并挑取单克隆菌落,挑取的单克隆菌落扩大培养之后,提取质粒,质粒经过酶切鉴定之后,送至测序公司进行sanger测序。
测序结果如图1和图2所示,表明稳定干扰DKC1的表达载体构建成功。
实施例2:构建稳定稳定干扰DKC1表达的结直肠癌及非小细胞肺癌细胞系
(1)包装慢病毒
首先在10cm培养皿中接种2.5×106的HEK293T细胞,第二天利用转染试剂PEImax,将病毒包装载体与上述实施例1构建好的重组表达载体共同转染HEK293T细胞,转染8小时之后换液,换液之后48小时后收集慢病毒上清液。72小时之后再收集一次慢病毒上清液。
(2)慢病毒感染并筛选稳定细胞株
将步骤(1)中包装的慢病毒感染结直肠癌细胞系HCT 116和DLD-1及非小细胞肺癌细胞系A549和PC-9,感染48小时之后,加入嘌呤霉素进行筛选,筛选7天之后得到稳定干扰DKC1表达的细胞。
(3)稳定细胞株鉴定
收集上述筛选得到的稳定干扰DKC1表达的细胞株,提取蛋白,采用免疫印迹的方法检测DKC1敲降效率。
具体操作流程如下:收集的细胞加入RIPA裂解液,置于冰上裂解半小时,然后12,000rpm离心10分钟取上清,用BCA的方法进行蛋白定量,然后分别取30ug总蛋白进行Western Blot分析。
鉴定结果如图3、图4、图5和图6所示。与对照细胞(shCTL,未经处理的同种细胞)相比,在两种结直肠癌细胞系HCT 116和DLD-1及两种非小细胞肺癌细胞系A549和PC-9中,两条DKC1shRNA(shDKC1-1和shDKC1-2)均可有效敲降DKC1表达,其中GAPDH为内参蛋白。
实施例3:DKC1敲降对细胞生长曲线的影响
将上述得到的稳定干扰DKC1表达的细胞株,以每孔2,500个细胞接种到96孔板,并用CCK8的方法检测细胞增殖,连续检测5天。三次生物学重复计算平均值,Prism软件进行生物学统计,绘制细胞生长曲线。
结果如图7、图8、图9和图10。结果表明,敲降DKC1的表达可以明显抑制两种结直肠癌细胞系HCT 116和DLD-1及两种非小细胞肺癌细胞系A549和PC-9的细胞增殖。
实施例4:DKC1敲低对细胞克隆形成能力的影响
将上述得到的稳定干扰DKC1表达的细胞株,以每孔1,000个细胞接种到6孔板,培养7天之后,用4%的多聚甲醛固定细胞,进行结晶紫染色,染色拍照之后,ImageJ软件计算克隆数目。三次生物学重复计算平均值,Prism软件进行生物学统计,并绘制相应的克隆数柱状图。
结果如图11、图12、图13和图14所示。结果表明,敲降DKC1的表达可以明显抑制两种结直肠癌细胞系HCT 116和DLD-1及两种非小细胞肺癌细胞系A549和PC-9的细胞的克隆形成能力。
序列表
<110> 中山大学肿瘤防治中心(中山大学附属肿瘤医院、中山大学肿瘤研究所)
<120> DKC1特异小干扰RNA及其应用
<160> 6
<170> SIPOSequenceListing 1.0
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<213> 人工序列(Artificial Sequence)
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<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<213> 人工序列(Artificial Sequence)
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aattcaaaaa tatgttgact acagtgagtc tctcgagaga ctcactgtag tcaacata 58
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<212> DNA
<213> 人工序列(Artificial Sequence)
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aattcaaaaa gctcagtgaa atgctgtaga actcgagttc tacagcattt cactgagc 58
Claims (10)
1.一种DKC1特异小干扰RNA,其靶向的DKC1位点的核苷酸序列如SEQ ID NO.1或SEQ IDNO.2所示。
2.一种DKC1特异小干扰RNA,其正向核苷酸序列如SEQ ID NO.3或SEQ ID NO.5所示。
3.根据权利要求2所述的DKC1特异小干扰RNA,其特征在于,所述DKC1特异小干扰RNA降低DKC1 mRNA的表达。
4.根据权利要求2所述的DKC1特异小干扰RNA,其特征在于,所述DKC1特异小干扰RNA的靶向的DKC1位点的核苷酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
5.根据权利要求1至4任一项所述的DKC1特异小干扰RNA在制备肿瘤治疗药物中的应用。
6.根据权利要求5所述的应用,其特征在于,所述肿瘤为结直肠癌或非小细胞肺癌。
7.一种重组载体,其含有权利要求1至4任一项所述的DKC1特异小干扰RNA。
8.一种宿主细胞,其含有根据权利要求7所述的重组载体。
9.一种试剂盒,其含有权利要求1至4任一项所述的DKC1特异小干扰RNA。
10.一种肿瘤治疗方法,其使用权利要求1至4任一项所述的DKC1特异小干扰RNA来治疗肿瘤。
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