CN105671195A - miR-520c核苷酸的用途、药物组合物和试剂盒 - Google Patents
miR-520c核苷酸的用途、药物组合物和试剂盒 Download PDFInfo
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Abstract
本发明公开了miR-520c核苷酸在制备用于诊断或预后实体瘤药物或试剂盒中的用途、药物组合物和试剂盒。miR-520c的核苷酸序列为下述SEQ？ID？NO:1所示miR-520c-3p的序列：5’-AAAGUGCUUCCUUUUAGAGGGU-3’；以及SEQ？ID？NO:2所示miR-520c-5p的序列：5’-CUCUAGAGGGAAGCACUUUCUC-3’。本发明的miR-520c核苷酸通过抑制肿瘤细胞增殖与细胞迁移的功能。MiR-520c核苷酸可以作为一种新型的药物作用靶点，对于由细胞增殖与迁移引起的鳞状细胞癌，乳头状腺癌，囊腺癌以及乳头状腺癌的预防及治疗在临床上具有重要意义。
Description
技术领域
本发明涉及内源性非编码小RNAs的新药用途技术领域，尤其是一种miR-520c核苷酸的用途、药物组合物和试剂盒。
背景技术
肿瘤是严重危害人类健康的主要疾病，发病率有逐年增高且呈年轻化的趋势大量临床研究显示，肿瘤的转移是造成临床死亡的最主要原因。侵袭转移是癌细胞从原发性部位转移到远端部位形成肿瘤的过程，它是恶性肿瘤的重要生物学特征，也是导致肿瘤复发和影响预后的重要因素，肿瘤发生侵袭和转移是一个多因素调控、多步骤、多阶段、连续复杂的生物学过程。对肿瘤转移的细胞生物学与分子调控机制不断地深入研究，如何检测癌细胞的侵袭转移以实现肿瘤的早期检测与评估，已成为抗癌转移研究的重要共识。
MicroRNA(miRNA)是真核生物中一类长度约为22个核苷酸的参与基因转录后水平调控的非编码小分子单链RNA,能通过与靶mRNA特异的碱基配对引起靶mRNA的降解或翻译抑制,从而对基因进行转录后的表达调控。目前普遍认为miRNA参与的基因调控是遗传程序中最基本的一步,调控细胞分化、生长、凋亡、代谢等功能。由于肿瘤本质上是一种多基因异常疾病，通过激活一种或多种原癌基因的表达及抑癌基因的突变缺失从而使肿瘤细胞逃避了正常生长调控机制，自主进行增殖和侵袭、出现恶性表型。研究表明多种miRNAs参与了肿瘤细胞重要的生物程序调控，直接或间接的起着促癌基因或抑癌基因的功能。在肿瘤的发生与发展中起了至关重要的作用。因此，将miRNA作为肿瘤疾病治疗的靶点，开发相关药物具有潜在的临床应用价值。单一的miRNA可以同时作用多个靶基因mRNA水平的表达，影响多个信号通路，从整体上达到治疗疾病的目的。由于miRNA在肿瘤不同发展阶段的表达水平不同，因此，可以通过检查miRNA的表达水平来预测诊断疾病。
目前研究发现许多miRNA通过调控细胞增殖与细胞迁移相关靶蛋白的表达，影响肿瘤细胞的恶性增殖与转移，这些miRNA有促进增殖的，也有抑制增殖的。寻找在肿瘤中特异性表达。参与肿瘤细胞迁移的miRNA，阐明其作用机制，对于开发以miRNA为策略的肿瘤疾病的诊断、治疗具有极其重要的意义的应用前景。
发明内容
为了克服现有的不足，本发明提供了一种miR-520c核苷酸的用途、药物组合物和试剂盒。
本发明解决其技术问题所采用的技术方案是：一种单链非编码miR-520c核苷酸的用途，miR-520c核苷酸在用于诊断或预后实体瘤药物或试剂盒中的用途，所述miR-520c核苷酸序列为SEQIDNO:1所示的miR-520c-3p的序列：5’-AAAGUGCUUCCUUUUAGAGGGU-3’；以及SEQIDNO:2所示miR-520c-5p的序列：5’-CUCUAGAGGGAAGCACUUUCUC-3’。
根据上述方案，进一步包括：所述实体瘤选自鳞状细胞癌，乳头状腺癌，囊腺癌，乳头状腺癌，恶性多形性腺瘤中的一种或多种。
一种用于诊断或预后实体瘤药物组合物，药物组合物由如下组成：miR-520c，药学上可接受的载体、病毒或辅料。
根据上述方案，进一步包括：所述miR-520c核苷酸序列为SEQIDNO:1所示的miR-520c-3p的序列：5’-AAAGUGCUUCCUUUUAGAGGGU-3’；以及SEQIDNO:2所示miR-520c-5p的序列：
5’-CUCUAGAGGGAAGCACUUUCUC-3’；
所述miR-520c含量为0.1-10g/ml。
根据上述方案，进一步包括：所述的载体选自胆固醇、纳米颗粒、腺病毒和脂质体中的一种或多种；所述病毒选自腺病毒、逆转录病毒、腺相关病毒、慢病毒等中的一种或多种;所述辅料选自PEG-PLA、PEG-DSPE、PEG-PCL、PLGA等中的一种或多种。
根据上述方案，进一步包括：所述的药物组合以口服或注射方式给药。
根据上述方案，进一步包括：所述注射方式为静脉注射或肌肉注射。
一种用于诊断或预后实体瘤试剂盒，所述试剂盒中含有miR-520c、生理盐水和缓冲液：
所述miR-520c核苷酸序列为SEQIDNO:1所示的miR-520c-3p的序列：5’-AAAGUGCUUCCUUUUAGAGGGU-3’；以及SEQIDNO:2所示miR-520c-5p的序列：5’-CUCUAGAGGGAAGCACUUUCUC-3’；
所述生理盐水为浓度0.9%氯化钠生理盐水；
所述缓冲液为Tris-HCl缓冲液、PIPES缓冲液、GOOD’S缓冲液中的一种或多种的组合
一种上述的药物组合物或上述的试剂盒在制备用于诊断或预后实体瘤中的用途。
根据上述方案，进一步包括：所述实体瘤选自鳞状细胞癌，乳头状腺癌，囊腺癌，乳头状腺癌，恶性多形性腺瘤中的一种或多种。
本发明的有益效果是，本发明通过实验发现miR-520c在鳞状细胞癌和乳头状腺癌中表达显著下调，并与肿瘤患者的分期，转移以及预后相关。通过构建miR-520c过表达和荷瘤小鼠中过表达miR-520c的表达发现，miR-520c过表达在细胞水平能抑制细胞增殖与迁移，荷瘤小鼠中过表达miR-520c与对照组相比肿瘤大小与肿瘤重量有了明显的降低，同时肿瘤相关基因的表达与对照组相比有了明显的降低。
具体的，本发明发现miR-520c在肿瘤细胞与正常对照相比有了明显的下降，对肿瘤细胞增殖、细胞迁移与对照组相比都有了明显的下降。荷瘤小鼠miR-520c过表达小鼠在肿瘤体积，肿瘤大小，小鼠体重方面与野生型相比都有了明显的降低。以上实验说明miR-520c能够抑制肿瘤细胞的增殖与肿瘤的生长转移，这意味了miR-520c可作为一种早期诊断和早期预防肿瘤的生物标志。
以上实验说明miR-520c通过抑制鳞状细胞癌和乳头状腺癌细胞增殖对肿瘤生长具有保护作用，它对许多肿瘤疾病具有潜在的预防和治疗价值，可称为一种治疗肿瘤的药物。具体的，可以合成miR-520c的核苷酸并与适当载体如胆固醇，纳米颗粒，腺病毒，脂质体等连接形成药物，通过口服，静脉或肌肉注射的方式，对鳞状细胞癌和乳头状腺癌等肿瘤疾病进行治疗。
附图说明
下面结合附图和实施例对本发明进一步说明。
图1为miR-520c在正常组织与肿瘤组织中的表达比较。
图2为miR-520c在不同肿瘤分期中的表达比较。
图3为miR-520c在肿瘤患者转移与未转移中的比较。
图4为miR-520c在肿瘤患者预后中表达比较。
图5为miR-520c过表达在肿瘤细胞增殖中与对照组相比抑制肿瘤细胞增殖。
图6为miR-520c过表达在肿瘤细胞迁移中与对照组相比抑制肿瘤细胞迁移。
图7为荷瘤小鼠中miR-520c过表达，与对照组在检测肿瘤体积大小中的比较。
图8为荷瘤小鼠中miR-520c过表达，与对照组在肿瘤重量大小中的比较。
具体实施方式
下面结合具体实施例来进一步描述本发明
除非特别指明，以下实施例中所用的小鼠购自常州卡文斯实验动物有限公司。
除非特别指明，以下实施例中所用的实验方法均为本领域中所用的常规方法。
除非特别指明，以下实施例中所用的试剂均为分析纯级试剂，且可以从正规渠道商购获得
实验例1miR-520c在肿瘤组织以及不同肿瘤分期中的表达情况检测
①选择2010年10月至2014年6月我院病理资料保存完整的肿瘤患者85例，年龄35-76，平均50.8岁，所有患者均经病理检查确诊，均无其他重要脏器合并症或肿瘤所有研究对象均知情同意。收集手术切除的新鲜正常组织，肿瘤组织以及肿瘤患者的病理切片，肿瘤组织采用Trizol（Invitrogen），病理切片使用mirVanamiRNAIsolationKit（Ambion，按照标准操作步骤）提取RNA，使用RIPA裂解液提取组织中的蛋白。RNA浓度与纯度和蛋白质浓度采用Epoch(BioTeck)分光光度计检测。
②通过Q-PCR检测miR-520c在肿瘤组织中的表达变化。提取的RNA用RevertAidTMFirstStrandcDNASynthesisKit(Fermentas)，按照操作说明逆转录为cDNA，采用ABI-Vii7Real-timeRT-PCR系统进行检测实验。扩增miR-520c的编码序列为
(TCTCAGGCTGTCGTCCTCTAGAGGGAAGCACTTTCTGTTGTCTGAAAGAAAAGAAAGTGCTTCCTTTTAGAGGGTTACCGTTTGAGASEQIDNO:3)及其上下游两端各将近50bp的序列，扩增的序列约218bp，具体为：
GGAGGATTGCCCGTTGATGAACAAGGCTAACCTGCTGATTCTTTGAAGCAAAGAACTGCAGATGGTCCTTTTAGGGATTTATGCTCTGGATTCCAGAAAACATGCAAACAGGGCAAATAAATGCATCTTTATTTTTGCGTCCATTTTAACCTGGTCAAGGAAGATTCCCAC(SEQIDNO:4)AAAAATCCACAGTGCCAGAGCAAGAAGATCTCAGGCTGTCGT CCTCTAGAGGGAAGCACTTTCTGTTGTCTGAAAGAAAAGAAAGTGCTTCCTTTTAGAGGGTTACCGTTTGAGAAAAGCAACATTGAAGTTGATGCTGATCTTGGTAATACATTTTCAGAGCATGCTTATCATCAGAGATGGATGATGGTGGGC(SEQIDNO:5)TTCTGTTTTTGTTTTGATTTTTTCTTTTTTTTTTTTTTGAGATGGAGTTTCGTTCTTGCTGCCCAAGCACTAGTATGTAGGAATTATTCTTTATGTGAGCATATAGTACATGGAACTTTG(SEQIDNO:6)其中加粗并带下划线标记为miR-520c的编码序列；其余的为它的上下游序列，加粗斜体为上下游引物序列，
上游引物为5’-CCTGGTCAAGGAAGATTCCCAC-3’(SEQIDNO:4)
下游引物为5’-GCCCACCATCATCCATCTCTGA-3’(SEQIDNO:5)
25μl体系含有12.5μlSYBRGreenPCR缓冲液，200nM引物，100ngcDNA。
检测到miR-520c的表达水平如图1至图4所示，miR-520c在肿瘤组织中表达水平与正常组织中相比miR-520c表达显著降低；同时随着肿瘤的发展，miR-520c表达水平降低。
实验例2miR-520c在肿瘤细胞增殖与细胞周期中的实验
③自液氮罐中取出冻存的肿瘤细胞，迅速置于37℃水浴锅中，轻轻摇动细胞，复苏细胞，取对数期生长的肿瘤细胞，应用LipofectamineTM2000(Invitrogen)按照每96孔板以每孔1×104个细胞/100μl培养基、5pmolRNA、0.25μlLipofectamineTM2000体系分别将其转染肿瘤细胞实验分组为:PBS(Gibco)对照组,miR-520c过表达以及miR-520c抑制组。置于5%CO2，37℃培养箱中培养，收集12H,24H,48H,72H的细胞,在培养结束前6小时，每孔加入3H-TdR20μl，终浓度为3.7х104Bq/ml，在FJ-2100液闪计数仪上测定每孔放射强度,计算增殖指数(SI).SI=(实验组-本底)/(对照组-本底)。
④含10％血清培养液的24孔板transwell小室，置于5%CO2，37℃培养箱中培养1H，细胞
浓度调至3.0x104/ml肿瘤细胞，悬浮于200μl无血清RPMll640培养液中，并种植在小室的上层，下室加入2.5%血清500μlRPMI1640。实验组加入miR-520c过表达以及miR-520c抑制剂，对照组加入同体积的PBS溶液。于37℃培养箱中培养。24H后，用PBS轻轻冲洗上室，用棉签去除膜上层未迁移的细胞。37℃风干20min。将小室膜固定、染色lH。PBS冲洗，切下小室膜，底面向上置于载玻片上，盖玻片覆盖固定，于显微镜下观察穿膜细胞数，随机取5个视野，计数5个视野穿膜细胞总数,实验重复至少3次。
实验结果如图5和图6所示，miR-520c过表达抑制肿瘤细胞增殖以及细胞迁移。
实施例3miR-520c在荷瘤小鼠肿瘤生长中作用的
⑤本实验中使用实施例2中的肿瘤细胞。取对数期肿瘤细胞，应用LipofectamineTM2000(Invitrogen)转染体系，建立miR-520c过表达与miR-520c抑制表达的肿瘤细胞系，选取4周龄的BALB/C裸鼠，将肿瘤细胞制成悬液，按照1х107细胞/小鼠注射到小鼠体内，隔天观察小鼠以及肿瘤生长情况。
⑥记录肿瘤体积大小，每周检测2次，以及记录小鼠生存期和肿瘤转移情况，并绘制生长曲线和/或生存曲线，到肿瘤体积达到规定大小时(最长直径不大于15mm)，处死小鼠。
小鼠体积大小测算方式：长×宽×宽/2
实验结果如图7与图8所示，miR-520c可以抑制荷瘤小鼠体内肿瘤的生长。
Claims (10)
1.一种单链非编码miR-520c核苷酸的用途，其特征在于，miR-520c核苷酸在用于诊断或预后实体瘤药物或试剂盒中的用途，所述miR-520c核苷酸序列为SEQIDNO:1所示的miR-520c-3p的序列：5’-AAAGUGCUUCCUUUUAGAGGGU-3’；以及SEQIDNO:2所示miR-520c-5p的序列：5’-CUCUAGAGGGAAGCACUUUCUC-3’。
2.根据权利要求1所述的单链非编码miR-520c核苷酸的用途，其特征在于，所述实体瘤选自鳞状细胞癌，乳头状腺癌，囊腺癌，乳头状腺癌，恶性多形性腺瘤中的一种或多种。
3.一种用于诊断或预后实体瘤药物组合物，其特征在于，药物组合物由如下组成：miR-520c，药学上可接受的载体、病毒或辅料。
4.根据权利要求3所述的药物组合物，其特征在于，所述miR-520c核苷酸序列为SEQIDNO:1所示的miR-520c-3p的序列：5’-AAAGUGCUUCCUUUUAGAGGGU-3’；以及SEQIDNO:2所示miR-520c-5p的序列：5’-CUCUAGAGGGAAGCACUUUCUC-3’；所述miR-520c含量为0.1-10g/ml。
5.根据权利要求3所述的药物组合物，其特征在于：所述的载体选自胆固醇、纳米颗粒、腺病毒和脂质体中的一种或多种；所述病毒选自腺病毒、逆转录病毒、腺相关病毒、慢病毒等中的一种或多种;所述辅料选自PEG-PLA、PEG-DSPE、PEG-PCL、PLGA等中的一种或多种。
6.权利要求3-5中的任一项所述的药物组合物，其特征在于，所述的药物组合以口服或注射方式给药。
7.权利要求6中的所述的药物组合物，其特征在于，所述注射方式为静脉注射或肌肉注射。
8.一种用于诊断或预后实体瘤试剂盒，其特征在于，所述试剂盒中含有miR-520c、生理盐水和缓冲液：所述miR-520c核苷酸序列为SEQIDNO:1所示的miR-520c-3p的序列：5’-AAAGUGCUUCCUUUUAGAGGGU-3’；以及SEQIDNO:2所示miR-520c-5p的序列：5’-CUCUAGAGGGAAGCACUUUCUC-3’；所述生理盐水为浓度0.9%氯化钠生理盐水；所述缓冲液为Tris-HCl缓冲液、PIPES缓冲液、GOOD’S缓冲液中的一种或多种的组合。
9.一种权利要求3所述的药物组合物或权利要求8所述的试剂盒在制备用于诊断或预后实体瘤中的用途。
10.根据权利要求9所述的用途，其特征在于，所述实体瘤选自鳞状细胞癌，乳头状腺癌，囊腺癌，乳头状腺癌，恶性多形性腺瘤中的一种或多种。
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