CN114908094B - 一类反义寡核苷酸分子及其在制备治疗恶性肿瘤药物中的应用 - Google Patents
一类反义寡核苷酸分子及其在制备治疗恶性肿瘤药物中的应用 Download PDFInfo
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Abstract
本发明公开了一类反义寡核苷酸分子及其在制备治疗恶性肿瘤药物中的应用。本发明发现特定核苷酸序列的反义寡核苷酸分子能够特异性地抑制STAT3蛋白的活性,降低肿瘤细胞中STAT3mRNA水平和蛋白质表达,显著抑制肿瘤细胞增殖并诱导肿瘤细胞发生凋亡,进而在在研发和制备恶性肿瘤治疗药物上具有广泛的用途。
Description
技术领域
本发明涉及医药技术领域,具体涉及一类反义寡核苷酸分子及其在制备治疗恶性肿瘤药物中的应用。
背景技术
恶性肿瘤由于分化不成熟并且生长不受控,严重危害当代人类的健康。随着人口增长和全球老龄化,恶性肿瘤成为许多国家过早死亡和预期寿命降低的主要原因。目前临床上肿瘤的治疗策略是手术治疗、化疗、放疗、免疫治疗等,但是治疗阶段仍会存在许多问题,比如肿瘤化疗耐药、转移、药物响应率低和毒副作用等问题。目前,肿瘤的治疗正朝着精准靶向治疗的方向前进,核酸药物治疗就是其中一种靶向治疗手段。
信号转导及转录激活因子3(Signal transducer and activator oftranscription 3,STAT3)在正常细胞中受到各种负性调控因子严格调控,从而STAT3的活性被维持在较低状态。然而,在信号分子的刺激下,STAT3会发生活化,进而可以调节细胞增殖、转移和免疫抑制相关基因的表达,参与一系列重要生理过程。当STAT3与磷酸化酪氨酸残基结合后,其主要通过705位点的酪氨酸和727位点的丝氨酸残基发生磷酸化效应,随后发生二聚化,两分子STAT3形成STAT3-STAT3二聚体转入细胞核内。STAT3二聚体能够结合到特定靶基因的启动子序列,从而发挥信号传导和调节靶基因的表达的功能。在大部分实体瘤和血液肿瘤中,STAT3会处于不受调控的异常激活状态,比如肝癌、乳腺癌、胃癌、肺癌、前列腺癌、头颈癌、胰腺癌、白血病等。过度活化的STAT3可上调其下游Survivin、Bcl-xL、Bcl-1、Mcl-1等相关抗凋亡蛋白的表达,增加血管内皮生长因子的表达,上调基质金属蛋白酶1型、基质金属蛋白酶2型、基质金属蛋白酶9型蛋白水平以及转录因子Snail、Twist等的表达,能够导致癌症增殖、生存、血管生成、转移和化疗耐药。因此,STAT3被认为是肿瘤治疗的有力靶点。
目前靶向STAT3的药物开发策略主要包括靶向STAT3上游激活分子和直接靶向STAT3,其中直接靶向STAT3的主要是通过作用于重要功能区域,如介导STAT3-STAT3二聚化的SH2结构域或与启动子序列结合的DBD结构域。然而,SH2结构域在STAT家族中十分保守,使得药物的设计开发难以实现高选择性。其次,STAT3和靶基因上启动子的结合位点通常很大且不一定依赖于二聚化,利用小分子阻断其相互作用也非常困难。此外,STAT3在肿瘤中容易发生突变,在临床癌症患者中就检测到几种不同类型的STAT3突变,特别是SH2结构域中的突变,这可能会导致靶向特定结构域的STAT3抑制剂治疗作用减弱,产生脱靶效应。目前迫切开发选择性好、药效高、副作用低的STAT3分子靶向药物。
反义寡核苷酸是最早应用的一类核酸药物,通常由16-20个核苷酸组成,能够与靶RNA链以碱基互补配对原则结合,然后反义寡核苷酸会通过不同的机制去调节mRNA的水平和翻译,主要分为诱导mRNA切割降解和立体阻断,这主要取决于反义寡核苷酸的设计和化学修饰。反义寡核苷酸作为肿瘤治疗药物最大的优点是能高特异性地抑制靶点mRNA的表达水平,在靶向难以成药性靶点上具有明显优势。反义寡核苷酸可以应用于肿瘤发生发展过程中基因异常表达的环节,可以针对于过度活化的STAT3基因,从而成为治疗恶性肿瘤的策略。
综上所述,靶向STAT3是有效的肿瘤治疗策略,靶向STAT3药物具有良好前景,阻断STAT3的核酸类药物对STAT3过活化的恶性肿瘤治疗具有非常重要的研究意义和临床应用价值。
发明内容
本发明的目的在于克服现有靶向STAT3的药物的缺乏,提供一类反义寡核苷酸分子。本发明发现特定核苷酸序列的反义寡核苷酸分子能够特异性地抑制STAT3蛋白的活性,降低肿瘤细胞中STAT3mRNA水平和蛋白质表达,显著抑制肿瘤细胞增殖并诱导肿瘤细胞发生凋亡,进而在在研发和制备恶性肿瘤治疗药物上具有广泛的用途。
本发明的另一目的在于提供一种表达载体。
本发明的另一目的在于提供一种宿主细胞。
本发明的另一目的在于提供上述反义寡核苷酸分子或其药学上可以接受的盐在制备治疗恶性肿瘤药物中的应用。
本发明的另一目的在于提供上述反义寡核苷酸分子或其药学上可以接受的盐或溶剂合物在制备治疗恶性肿瘤药物中的应用。
本发明的另一目的在于提供上述反义寡核苷酸分子或其药学上可以接受的盐在制备降低STAT3mRNA的水平和/或抑制STAT3蛋白质表达的药物中的应用。
为了实现本发明的上述目的,本发明提供了如下技术方案:
一类反义寡核苷酸分子,其序列为如下核苷酸序列中的一条或几条:
STAT3 ASO-1:5’-TGACGCCTCCTTCTTTGCTG-3’(如SEQ ID NO:1所示);
STAT3 ASO-2:5’-CTCTGCCAGTGTAGTCAGCT-3’(如SEQ ID NO:2所示);
STAT3 ASO-3:5’-GCGGGGGGACATCGGCAGGT-3’(如SEQ ID NO:3所示);
STAT3 ASO-4:5’-TTTCCGAATGCCTCCTCCTT-3’(如SEQ ID NO:4所示);
STAT3 ASO-5:5’-GTGATTCTTTGCTGGCCGC-3’(如SEQ ID NO:5所示);
STAT3 ASO-6:5’-ATTCCCACATCTCTGCTCC-3’(如SEQ ID NO:6所示);
STAT3 ASO-7:5’-TTTATAGTTGAAATCAAAG-3’(如SEQ ID NO:7所示)。
本发明提供的反义寡核苷酸分子可分为两类,其中STAT3 ASO-1~STAT3ASO-4均由20个核苷酸组成,STAT3 ASO-5~STAT3 ASO-7均由19个核苷酸组成.
研究发现,该系列反义寡核苷酸分子能够特异性地抑制STAT3蛋白的活性,降低肿瘤细胞中STAT3mRNA水平和蛋白质表达,显著抑制肿瘤细胞增殖并诱导肿瘤细胞发生凋亡,在研发和制备恶性肿瘤治疗药物上具有广泛的用途。
对反义寡核苷酸分子进行修饰可进一步提升其性能。
优选地,所述反义寡核苷酸分子的每个核苷酸间通过硫代磷酸酯键连接。该修饰可提升反义寡核苷酸分子的稳定性。硫代磷酸酯键连接是指核苷之间的连接,用硫原子取代磷酸二酯键中非桥接的氧原子之一来修饰二酯键。
优选地,所述反义寡核苷酸分子包含3或5个连接核苷组成的5’-翼和3’-翼,中间有13或10个2’-脱氧核苷组成的缺口。
优选地,所述反义寡核苷酸分子中5’-翼和3’-翼的3或5个连接核苷包含修饰的糖,所述修饰的糖包含2’-O-CH3基团。
具体地各序列的修饰方式如下:
STAT3 ASO-1:5’-TGACGCCTCCTTCTTTGCTG-3’(5’-翼的5个连接核苷酸包含修饰的糖,3’-翼的5个连接核苷酸包含修饰的糖,如下划线处的连接核苷酸,下同)。
STAT3 ASO-2:5’-CTCTGCCAGTGTAGTCAGCT-3’(5’-翼的5个连接核苷酸包含修饰的糖,3’-翼的5个连接核苷酸包含修饰的糖)。
STAT3 ASO-3:5’-GCGGGGGGACATCGGCAGGT-3’(5’-翼的5个连接核苷酸包含修饰的糖,3’-翼的5个连接核苷酸包含修饰的糖)。
STAT3 ASO-4:5’-TTTCCGAATGCCTCCTCCTT-3’(5’-翼的5个连接核苷酸包含修饰的糖,3’-翼的5个连接核苷酸包含修饰的糖)。
STAT3 ASO-5:5’-GTGATTCTTTGCTGGCCGC-3’(5’-翼的3个连接核苷酸包含修饰的糖,3’-翼的3个连接核苷酸包含修饰的糖)。
STAT3 ASO-6:5’-ATTCCCACATCTCTGCTCC-3’(5’-翼的3个连接核苷酸包含修饰的糖,3’-翼的3个连接核苷酸包含修饰的糖)。
STAT3 ASO-7:5’-TTTATAGTTGAAATCAAAG-3’(5’-翼的3个连接核苷酸包含修饰的糖,3’-翼的3个连接核苷酸包含修饰的糖)。
无下划线的是2’-脱氧核苷,具有下划线的核苷是含有修饰的糖,核苷之间通过硫代磷酸酯键连接。
术语“2’-O-CH3”(也称2’-OMe、2’-甲氧基)是指呋喃糖基环的2’位的甲氧基修饰(羟基修饰为甲氧基)。
甲氧基的修饰可增加稳定性、亲和力及降低免疫激活。
一种表达载体,含有上述反义寡核苷酸分子。
一种宿主细胞,含有上述反义寡核苷酸分子,或含有上述表达载体。
上述反义寡核苷酸分子或其药学上可以接受的盐在制备治疗恶性肿瘤药物中的应用也在本发明的保护范围内。
经研究发现,本发明的反义寡核苷酸分子能够显著抑制恶性肿瘤细胞增殖和诱导肿瘤细胞凋亡,进而具有制备治疗恶性肿瘤药物的用途。
术语“药学上可以接受的”是指某载体、稀释剂或赋形剂,和/或所形成的盐通常在化学上或物理上与构成某药物剂型的其它成分相兼容,并在生理上与受体相兼容。
术语“可接受的盐”是指上述化合物或其立体异构体,与无机和/或有机酸和碱形成的酸式和/或碱式盐,也包括两性离子盐(内盐),还包括季铵盐,例如烷基铵盐。这些盐可以是在化合物的最后分离和纯化中直接得到。也可以是通过将上述化合物,或其立体异构体,与一定数量的酸或碱适当(例如等当量)进行混合而得到。这些盐可能在溶液中形成沉淀而以过滤方法收集,或在溶剂蒸发后回收而得到,或在水介质中反应后冷冻干燥制得。
具体地,药学上可以接受的盐包括但不限于:硫酸盐、柠檬酸盐、乙酸盐、草酸盐、氯化物、溴化物、碘化物、硝酸盐、硫酸氢盐、磷酸盐、酸式磷酸盐、异烟酸盐、乳酸盐、水杨酸盐、酸式柠檬酸盐、酒石酸盐、油酸盐、鞣酸盐、泛酸盐、酒石酸氢盐、抗坏血酸盐、琥珀酸盐、马来酸盐、龙胆酸盐、富马酸盐、葡糖酸盐、葡糖醛酸盐、糖酸盐、甲酸盐、苯甲酸盐、谷氨酸盐,甲烷磺酸盐(甲磺酸盐)、乙烷磺酸盐、苯磺酸盐、对甲苯磺酸盐、和双羟萘酸盐;或者铵盐(例如伯胺盐、仲胺盐、叔胺盐、季铵盐)、金属盐(例如钠盐、钾盐、钙盐、镁盐、锰盐、铁盐、锌盐、铜盐、锂盐、铝盐)。
优选地,所述恶性肿瘤为肺癌、胰腺癌、乳腺癌、胃癌、卵巢癌、前列腺癌、肠癌、头颈癌、白血病或淋巴癌中的一种或几种。
优选地,所述反义寡核苷酸分子或其药学上可以接受的盐在制备抑制肿瘤的生长和/或增殖的药物中的应用。
上述反义寡核苷酸分子或其药学上可以接受的盐在制备诱导肿瘤细胞凋亡的药物中的应用也在本发明的保护范围内。
上述反义寡核苷酸分子或其药学上可以接受的盐在制备降低STAT3mRNA的水平和/或抑制STAT3蛋白质表达的药物中的应用也在本发明的保护范围内。
优选地,所述药物还包括药学上可接受的载体。
优选地,所述药物的剂型为注射剂、胶囊剂、片剂、丸剂或颗粒剂。
相对于现有技术,本发明具有如下的优点及效果:
本发明发现特定核苷酸序列的反义寡核苷酸分子能够特异性地抑制STAT3蛋白的活性,降低肿瘤细胞中STAT3mRNA水平和蛋白质表达,显著抑制肿瘤细胞增殖并诱导肿瘤细胞发生凋亡,进而在在研发和制备恶性肿瘤治疗药物上具有广泛的用途。
附图说明
图1为STAT3 ASO对肺癌PC-9细胞和肝癌HepG2细胞中STAT3mRNA水平的影响结果图,图1A为低浓度STAT3 ASO对肺癌PC-9细胞中STAT3mRNA水平的影响,图1B为高浓度STAT3ASO对肺癌PC-9细胞中STAT3mRNA水平的影响,图1C为低浓度STAT3 ASO对肝癌HepG2细胞中STAT3mRNA水平的影响,图1D为高浓度STAT3 ASO对肝癌HepG2细胞中STAT3mRNA水平的影响。
图2为STAT3 ASO对肝癌HepG2细胞中STAT3蛋白表达水平的影响结果图。
图3为STAT3 ASO-2和STAT3 ASO-3对肝癌HepG2和MHCC-97H细胞、肺癌PC-9和A549细胞、乳腺癌MDA-MB-468和MDA-MB-231细胞的细胞活性影响结果图,左图为不同浓度STAT3ASO-2对以上肿瘤细胞的细胞活性影响,右图是不同浓度STAT3 ASO-3对以上肿瘤细胞的细胞活性影响。
图4为STAT3 ASO-2和STAT3 ASO-3对肝癌HepG2细胞的克隆形成影响结果图,左图是克隆形成的影响,右图是对应的克隆集落数目统计图。
图5为STAT3 ASO-2和STAT3 ASO-3对肝癌HepG2细胞的增殖能力影响结果图,左图是细胞EdU染色结果,右图是对应的增殖细胞比例统计图。
图6为STAT3 ASO-2和STAT3 ASO-3对肝癌HepG2细胞凋亡影响结果图,左图是流式细胞分析结果,右图是流式分析统计图。
具体实施方式
以下结合实施例和附图进一步解释本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,本发明所用试剂和材料均为市购。
本发明各实施例中提及的序列信息如下:
1.STAT3转录本变异体1序列(如SEQ ID NO:8):
GTCGCAGCCG AGGGAACAAG CCCCAACCGG ATCCTGGACA GGCACCCCGG CTTGGCGCTGTCTCTCCCCC TCGGCTCGGA GAGGCCCTTC GGCCTGAGGG AGCCTCGCCG CCCGTCCCCG GCACACGCGCAGCCCCGGCC TCTCGGCCTC TGCCGGAGAA ACAGTTGGGA CCCCTGATTT TAGCAGGATG GCCCAATGGAATCAGCTACA GCAGCTTGAC ACACGGTACC TGGAGCAGCT CCATCAGCTC TACAGTGACA GCTTCCCAATGGAGCTGCGG CAGTTTCTGG CCCCTTGGAT TGAGAGTCAA GATTGGGCAT ATGCGGCCAG CAAAGAATCACATGCCACTT TGGTGTTTCA TAATCTCCTG GGAGAGATTG ACCAGCAGTA TAGCCGCTTC CTGCAAGAGTCGAATGTTCT CTATCAGCAC AATCTACGAA GAATCAAGCA GTTTCTTCAG AGCAGGTATC TTGAGAAGCCAATGGAGATT GCCCGGATTG TGGCCCGGTG CCTGTGGGAA GAATCACGCC TTCTACAGAC TGCAGCCACTGCGGCCCAGC AAGGGGGCCA GGCCAACCAC CCCACAGCAG CCGTGGTGAC GGAGAAGCAG CAGATGCTGGAGCAGCACCT TCAGGATGTC CGGAAGAGAG TGCAGGATCT AGAACAGAAA ATGAAAGTGG TAGAGAATCTCCAGGATGAC TTTGATTTCA ACTATAAAAC CCTCAAGAGT CAAGGAGACA TGCAAGATCT GAATGGAAACAACCAGTCAG TGACCAGGCA GAAGATGCAG CAGCTGGAAC AGATGCTCAC TGCGCTGGAC CAGATGCGGAGAAGCATCGT GAGTGAGCTG GCGGGGCTTT TGTCAGCGAT GGAGTACGTG CAGAAAACTC TCACGGACGAGGAGCTGGCT GACTGGAAGA GGCGGCAACA GATTGCCTGC ATTGGAGGCC CGCCCAACAT CTGCCTAGATCGGCTAGAAA ACTGGATAAC GTCATTAGCA GAATCTCAAC TTCAGACCCG TCAACAAATT AAGAAACTGGAGGAGTTGCA GCAAAAAGTT TCCTACAAAG GGGACCCCAT TGTACAGCAC CGGCCGATGC TGGAGGAGAGAATCGTGGAG CTGTTTAGAA ACTTAATGAA AAGTGCCTTT GTGGTGGAGC GGCAGCCCTG CATGCCCATGCATCCTGACC GGCCCCTCGT CATCAAGACC GGCGTCCAGT TCACTACTAA AGTCAGGTTG CTGGTCAAATTCCCTGAGTT GAATTATCAG CTTAAAATTA AAGTGTGCAT TGACAAAGAC TCTGGGGACG TTGCAGCTCTCAGAGGATCC CGGAAATTTA ACATTCTGGG CACAAACACA AAAGTGATGA ACATGGAAGA ATCCAACAACGGCAGCCTCT CTGCAGAATT CAAACACTTG ACCCTGAGGG AGCAGAGATG TGGGAATGGG GGCCGAGCCAATTGTGATGC TTCCCTGATT GTGACTGAGG AGCTGCACCT GATCACCTTT GAGACCGAGG TGTATCACCAAGGCCTCAAG ATTGACCTAG AGACCCACTC CTTGCCAGTT GTGGTGATCT CCAACATCTG TCAGATGCCAAATGCCTGGG CGTCCATCCT GTGGTACAAC ATGCTGACCA ACAATCCCAA GAATGTAAAC TTTTTTACCAAGCCCCCAAT TGGAACCTGG GATCAAGTGG CCGAGGTCCT GAGCTGGCAG TTCTCCTCCA CCACCAAGCGAGGACTGAGC ATCGAGCAGC TGACTACACT GGCAGAGAAA CTCTTGGGAC CTGGTGTGAA TTATTCAGGGTGTCAGATCA CATGGGCTAA ATTTTGCAAA GAAAACATGG CTGGCAAGGG CTTCTCCTTC TGGGTCTGGCTGGACAATAT CATTGACCTT GTGAAAAAGT ACATCCTGGC CCTTTGGAAC GAAGGGTACA TCATGGGCTTTATCAGTAAG GAGCGGGAGC GGGCCATCTT GAGCACTAAG CCTCCAGGCA CCTTCCTGCT AAGATTCAGTGAAAGCAGCAAAGAAGGAGG CGTCACTTTC ACTTGGGTGG AGAAGGACAT CAGCGGTAAG ACCCAGATCCAGTCCGTGGA ACCATACACA AAGCAGCAGC TGAACAACAT GTCATTTGCT GAAATCATCA TGGGCTATAAGATCATGGAT GCTACCAATA TCCTGGTGTC TCCACTGGTC TATCTCTATC CTGACATTCC CAAGGAGGAGGCATTCGGAA AGTATTGTCG GCCAGAGAGC CAGGAGCATC CTGAAGCTGA CCCAGGTAGC GCTGCCCCATACCTGAAGAC CAAGTTTATC TGTGTGACAC CAACGACCTGCAGCAATACC ATTGACCTGC CGATGTCCCCCCGCACTTTA GATTCATTGATGCAGTTTGG AAATAATGGT GAAGGTGCTG AACCCTCAGC AGGAGGGCAGTTTGAGTCCC TCACCTTTGA CATGGAGTTG ACCTCGGAGT GCGCTACCTC CCCCATGTGA GGAGCTGAGAACGGAAGCTG CAGAAAGATA CGACTGAGGC GCCTACCTGC ATTCTGCCAC CCCTCACACA GCCAAACCCCAGATCATCTG AAACTACTAA CTTTGTGGTT CCAGATTTTT TTTAATCTCC TACTTCTGCT ATCTTTGAGCAATCTGGGCA CTTTTAAAAA TAGAGAAATG AGTGAATGTG GGTGATCTGC TTTTATCTAA ATGCAAATAAGGATGTGTTC TCTGAGACCC ATGATCAGGG GATGTGGCGG GGGGTGGCTA GAGGGAGAAA AAGGAAATGTCTTGTGTTGT TTTGTTCCCC TGCCCTCCTT TCTCAGCAGC TTTTTGTTAT TGTTGTTGTT GTTCTTAGACAAGTGCCTCC TGGTGCCTGC GGCATCCTTC TGCCTGTTTC TGTAAGCAAA TGCCACAGGC CACCTATAGCTACATACTCC TGGCATTGCA CTTTTTAACC TTGCTGACAT CCAAATAGAA GATAGGACTA TCTAAGCCCTAGGTTTCTTT TTAAATTAAG AAATAATAAC AATTAAAGGG CAAAAAACAC TGTATCAGCA TAGCCTTTCTGTATTTAAGA AACTTAAGCA GCCGGGCATG GTGGCTCACG CCTGTAATCC CAGCACTTTG GGAGGCCGAGGCGGATCATA AGGTCAGGAG ATCAAGACCA TCCTGGCTAA CACGGTGAAA CCCCGTCTCT ACTAAAAGTACAAAAAATTA GCTGGGTGTG GTGGTGGGCG CCTGTAGTCC CAGCTACTCG GGAGGCTGAG GCAGGAGAATCGCTTGAACC TGAGAGGCGG AGGTTGCAGT GAGCCAAAAT TGCACCACTG CACACTGCAC TCCATCCTGGGCGACAGTCT GAGACTCTGT CTCAAAAAAA AAAAAAAAAA AAAGAAACTT CAGTTAACAG CCTCCTTGGTGCTTTAAGCA TTCAGCTTC CTTCAGGCTGG TAATTTATAT AATCCCTGAA ACGGGCTTCA GGTCAAACCCTTAAGACATC TGAAGCTGCA ACCTGGCCTT TGGTGTTGAA ATAGGAAGGTTTAAGGAGAA TCTAAGCATTTTAGACTTTT TTTTATAAAT AGACTTATTT TCCTTTGTAA TGTATTGGCC TTTTAGTGAG TAAGGCTGGGCAGAGGGTGC TTACAACCTT GACTCCCTTT CTCCCTGGAC TTGATCTGCT GTTTCAGAGG CTAGGTTGTTTCTGTGGGTG CCTTATCAGG GCTGGGATAC TTCTGATTCT GGCTTCCTTC CTGCCCCACC CTCCCGACCCCAGTCCCCCT GATCCTGCTA GAGGCATGTC TCCTTGCGTG TCTAAAGGTC CCTCATCCTG TTTGTTTTAGGAATCCTGGT CTCAGGACCT CATGGAAGAA GAGGGGGAGA GAGTTACAGG TTGGACATGA TGCACACTATGGGGCCCCAG CGACGTGTCT GGTTGAGCTC AGGGAATATG GTTCTTAGCC AGTTTCTTGG TGATATCCAGTGGCACTTGT AATGGCGTCT TCATTCAGTT CATGCAGGGC AAAGGCTTAC TGATAAACTT GAGTCTGCCCTCGTATGAGG GTGTATACCT GGCCTCCCTC TGAGGCTGGT GACTCCTCCC TGCTGGGGCC CCACAGGTGAGGCAGAACAG CTAGAGGGCC TCCCCGCCTG CCCGCCTTGG CTGGCTAGCT CGCCTCTCCT GTGCGTATGGGAACACCTAG CACGTGCTGG ATGGGCTGCC TCTGACTCAG AGGCATGGCC GGATTTGGCA ACTCAAAACCACCTTGCCTC AGCTGATCAG AGTTTCTGTG GAATTCTGTT TGTTAAATCA AATTAGCTGG TCTCTGAATTAAGGGGGAGA CGACCTTCTC TAAGATGAAC AGGGTTCGCC CCAGTCCTCC TGCCTGGAGA CAGTTGATGTGTCATGCAGA GCTCTTACTT CTCCAGCAAC ACTCTTCAGT ACATAATAAG CTTAACTGAT AAACAGAATATTTAGAAAGG TGAGACTTGG GCTTACCATT GGGTTTAAAT CATAGGGACC TAGGGCGAGG GTTCAGGGCTTCTCTGGAGC AGATATTGTC AAGTTCATGG CCTTAGGTAG CATGTATCTG GTCTTAACTC TGATTGTAGCAAAAGTTCTG AGAGGAGCTG AGCCCTGTTG TGGCCCATTA AAGAACAGGG TCCTCAGGCC CTGCCCGCTTCCTGTCCACT GCCCCCTCCC CATCCCCAGC CCAGCCGAGG GAATCCCGTG GGTTGCTTAC CTACCTATAAGGTGGTTTAT AAGCTGCTGT CCTGGCCACT GCATTCAAAT TCCAATGTGT ACTTCATAGT GTAAAAATTTATATTATTGT GAGGTTTTTT GTCTTTTTTT TTTTTTTTTT TTTTTGGTAT ATTGCTGTAT CTACTTTAACTTCCAGAAAT AAACGTTATA TAGGAACCGTC
2.STAT3 ASO-1:5’-TGACGCCTCCTTCTTTGCTG-3’(5’-翼的5个连接核苷酸包含修饰的糖,3’-翼的5个连接核苷酸包含修饰的糖,如下划线处的连接核苷酸,下同)。
3.STAT3 ASO-2:5’-CTCTGCCAGTGTAGTCAGCT-3’(5’-翼的5个连接核苷酸包含修饰的糖,3’-翼的5个连接核苷酸包含修饰的糖)。
4.STAT3 ASO-3:5’-GCGGGGGGACATCGGCAGGT-3’(5’-翼的5个连接核苷酸包含修饰的糖,3’-翼的5个连接核苷酸包含修饰的糖)。
5.STAT3 ASO-4:5’-TTTCCGAATGCCTCCTCCTT-3’(5’-翼的5个连接核苷酸包含修饰的糖,3’-翼的5个连接核苷酸包含修饰的糖)。
6.STAT3 ASO-5:5’-GTGATTCTTTGCTGGCCGC-3’(5’-翼的3个连接核苷酸包含修饰的糖,3’-翼的3个连接核苷酸包含修饰的糖)。
7.STAT3 ASO-6:5’-ATTCCCACATCTCTGCTCC-3’(5’-翼的3个连接核苷酸包含修饰的糖,3’-翼的3个连接核苷酸包含修饰的糖)。
8.STAT3 ASO-7:5’-TTTATAGTTGAAATCAAAG-3’(5’-翼的3个连接核苷酸包含修饰的糖,3’-翼的3个连接核苷酸包含修饰的糖)。
无下划线的是2’-脱氧核苷,具有下划线的核苷是含有修饰的糖,核苷之间通过硫代磷酸酯键连接。
下面结合实施例,进一步阐述本发明。
实施例1 STAT3 ASO抑制肺癌和肝癌细胞中人STAT3mRNA水平
(1)设计了靶向STAT3核酸的反义寡核苷酸并且在体外测试其对STAT3mRNA表达的影响。
各序列及其修饰方式如前述。
具体合成过程如下:利用单链核酸合成仪采用固相亚磷酰胺三酯法合成并修饰序列,通过重复脱DMT、活化耦连、加帽和氧化四个步骤合成序列。然后利用单链核酸纯化仪纯化单链寡核苷酸,纯度测定时OD值为2。
(2)PC-9细胞和HepG2细胞在100mm细胞培养皿中用含有10%血清的RIPM1640培养基(PC-9细胞)或DMEM高糖培养基(HepG2细胞)培养。待细胞生长到至汇集度达到70-80%,消化细胞,以每孔350000个细胞把细胞分别接种到6孔板中,培养过夜。
(3)配置以下溶液:
溶液A:每份3μL Lipofectamine 2000+97μL opti MEM,按所需处理的孔数配若干份。
溶液B:终浓度为200nM/400nM该体积的寡核苷酸母液+opti MEM,共100μL,按所需处理的孔数配若干份。
(4)细胞转染:吸去细胞培养基,六孔板中每孔加入800μL无血清培养基。将以上溶液A和B分别室温静置5min后,溶液A和溶液B混合均匀,室温静置15min,之后按每孔200μL混合溶液加入到六孔板中。将六孔板置于37℃的CO 2培养箱培养6h,之后吸去细胞中的培养基,换成10%血清的RIPM1640培养基或DMEM高糖培养基培养。
(5)在培养48h后,从细胞分离RNA且通过定量实时PCR测量STAT3mRNA水平。使用人STAT3引物探针(上游:5’-CTGCCCCATACCTGAAGACC-3’,下游:5’-TCCTCACATGGGGGAGGTAG-3’,分别如SEQ ID NO:9和SEQ ID NO:10所示)测量mRNA水平,结果如图1所示。从图1可知,肺癌和肝癌细胞经过STAT3 ASO处理后细胞中STAT3mRNA水平下降,其中STAT3 ASO-2和STAT3 ASO-3降低mRNA水平的作用效果较好。在肺癌PC-9细胞中,200nM的STAT3 ASO以及400nM的STAT3 ASO使mRNA相对水平降低至50%以下。在肝癌HepG2细胞中,400nM的STAT3ASO降低mRNA水平的效果强于以200nM浓度转染时的效果,400nM的STAT3 ASO能使mRNA相对水平降低至50%以下。
实施例2 STAT3 ASO抑制肝癌细胞中人STAT3蛋白表达
(1)细胞铺板:按照实施例1中的培养方法,以每孔350000个细胞把细胞分别接种到6孔板中,培养过夜。
(2)细胞转染:按照实施例1中的方法,400nM浓度的STAT3 ASO孵育HepG2细胞,以阿斯利康公司处于临床试验阶段唯一靶向STAT3的反义核酸药物AZD9150作为阳性对照。
(3)在培养72h后,从细胞分离总蛋白并通过蛋白质免疫印迹法测量STAT3蛋白水平,结果如图2所示。从图2可知,肝癌细胞经过STAT3 ASO处理后细胞中STAT3蛋白表达减少,提示STAT3 ASO抑制肿瘤细胞中STAT3的蛋白表达。其中,STAT3 ASO-2和STAT3 ASO-3对STAT3蛋白表达的抑制作用优于阳性药物AZD9150。
实施例3 STAT3 ASO-2和STAT3 ASO-3抑制肝癌、肺癌、乳腺癌等癌症细胞的增殖能力
根据STAT3 ASO对STAT3 mRNA水平以及蛋白质表达的影响结果来看,STAT3 ASO-2和STAT3 ASO-3的抑制效果较好,所以接下来以STAT3 ASO-2和STAT3 ASO-3作为研究对象。
(1)细胞铺板:按照实施例1中的培养方法,以每孔5000个/孔接种到96孔板上,每孔加入含有10%血清的RIPM1640培养基(肺癌细胞PC-9、肺癌细胞A549和乳腺癌细胞MDA-MB-468)或DMEM高糖培养基(肝癌细胞HepG2、肝癌细胞MHCC-97H、乳腺癌细胞MDA-MB-231)至100μL,每种细胞重复接种三个孔。每种细胞组另设三个不含药物组,还有三个不含细胞核药物组作为空白对照。96孔板放入培养箱中,培养过夜。
(2)细胞转染:按照实施例1中的方法,以25nM、50nM、100nM、200nM、300nM和400nM浓度的STAT3 ASO-2或STAT3 ASO-3孵育PC-9、A549、MDA-MB-468、HepG2、MHCC-97H、MDA-MB-231细胞。
(3)CCK-8细胞增殖检测:在培养72h后,取出96孔板,每孔中加入10μLCCK8溶液,然后在37℃培养箱中避光孵育。用酶标仪测定450nm波长处的吸光值,OD值在0.8-1.5之间,实验重复3次。计算细胞活性(%)=[A(药物)-Ab(空白)]/[A(对照)-Ab(空白)]×100%,结果如图3所示。图3为不同浓度STAT3 ASO-2和STAT3 ASO-3对肿瘤细胞PC-9、A549、MDA-MB-468、HepG2、MHCC-97H和MDA-MB-231的细胞增殖抑制曲线,可知STAT3 ASO-2和STAT ASO-3显著抑制肺癌细胞PC-9和A549、乳腺癌细胞MDA-MB-468和MDA-MB-231、以及肝癌细胞HepG2和MHCC-97H的生长增殖。
实施例4 STAT3 ASO-2和STAT3 ASO-3抑制肝癌细胞的克隆形成
(1)细胞铺板:按照实施例1中的培养方法,以每孔2000个细胞把细胞分别接种到6孔板中,培养过夜。
(2)细胞转染:按照实施例1中的方法,以400nM浓度的STAT3 ASO-2或STAT3 ASO-3孵育HepG2细胞。
(3)固定与染色:继续培养至出现可见的克隆。弃去培养基并润洗后,加入4%多聚甲醇固定15min,然后用清水轻轻冲洗。再加入结晶紫避光染色30min,用清水冲洗。
(4)倒置干燥,拍照记录,结果如图4所示。从图4可知,STAT3 ASO-2和STAT3 ASO-3能够显著抑制肝癌HepG2细胞的克隆形成,且效果优于阳性药物AZD9150。这说明STAT3ASO-2和STAT3 ASO-3良好的抗癌活性及抑制肿瘤细胞的增殖生存的能力。
实施例5 STAT3 ASO-2和STAT3 ASO-3抑制肝癌细胞的增殖能力
(1)细胞铺板:按照实施例1中的培养方法,以每孔350000个细胞把细胞分别接种到6孔板中,培养过夜。
(2)细胞转染:按照实施例1中的方法,以400nM浓度的STAT3 ASO-2或STAT3 ASO-3孵育HepG2细胞。
(3)EdU处理:在培养72h后,加入合适浓度的EdU工作液,继续培养2h。
(4)固定与染色:弃去培养基,用PBS润洗后,每孔加入4%多聚甲醛固定15min。然后用3%BSA洗涤液,洗涤5min,重复三次。加入1mL 0.3%Triton X-100通透液处理15min,再用3%BSA洗涤液洗涤。加入250μL配制Click反应液(以1份样品为例:Click ReactionBuffer:430μL,CuSO4:20μL,Azide 555:1μL,Click Additive Solution:50μL)避光孵育30min,再用3%BSA洗涤液洗涤。最后加入500μL Hoechst 33342避光孵育30min,再用3%BSA洗涤液洗涤。
(5)成像:用荧光显微镜拍摄细胞,结果如图5所示。EdU染色是细胞生长增殖的重要标志,因此从图5可知,STAT3 ASO-2和STAT3 ASO-3可明显抑制肝癌HepG2细胞的生长增殖。
实施例6 STAT3 ASO-2和STAT3 ASO-3诱导肝癌细胞发生细胞凋亡
(1)样品处理;按照实施例1中的培养方法,以每孔350000个细胞把细胞分别接种到6孔板中,培养过夜。以400nM浓度的STAT3 ASO-2或STAT3 ASO-3孵育HepG2细胞,培养72h后消化细胞,收集细胞到离心管中。离心后,向离心管中加入400μL结合液,置于冰上。
(2)细胞染色:加入2.5μL的AnnexinV-FITC染色液,冰上避光15min。随后再加入5μLPI染色液,冰上避光5min。
(3)用流式细胞仪进行检测,结果如图6所示。从图6可知,STAT3 ASO-2和STAT3ASO-3处理后肝癌细胞明显发生凋亡,说明STAT3 ASO-2和STAT3 ASO-3能够通过诱导细胞凋亡从而发挥抗肿瘤功能。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 中山大学
<120> 一类反义寡核苷酸分子及其在制备治疗恶性肿瘤药物中的应用
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<170> SIPOSequenceListing 1.0
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<213> 反义寡核苷酸分子(STAT3 ASO-1)
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<213> 反义寡核苷酸分子(STAT3 ASO-4)
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<211> 19
<212> DNA
<213> 反义寡核苷酸分子(STAT3 ASO-5)
<400> 5
gtgattcttt gctggccgc 19
<210> 6
<211> 19
<212> DNA
<213> 反义寡核苷酸分子(STAT3 ASO-6)
<400> 6
attcccacat ctctgctcc 19
<210> 7
<211> 19
<212> DNA
<213> 反义寡核苷酸分子(STAT3 ASO-7)
<400> 7
tttatagttg aaatcaaag 19
<210> 8
<211> 4921
<212> DNA
<213> STAT3转录本变异体1(1)
<400> 8
gtcgcagccg agggaacaag ccccaaccgg atcctggaca ggcaccccgg cttggcgctg 60
tctctccccc tcggctcgga gaggcccttc ggcctgaggg agcctcgccg cccgtccccg 120
gcacacgcgc agccccggcc tctcggcctc tgccggagaa acagttggga cccctgattt 180
tagcaggatg gcccaatgga atcagctaca gcagcttgac acacggtacc tggagcagct 240
ccatcagctc tacagtgaca gcttcccaat ggagctgcgg cagtttctgg ccccttggat 300
tgagagtcaa gattgggcat atgcggccag caaagaatca catgccactt tggtgtttca 360
taatctcctg ggagagattg accagcagta tagccgcttc ctgcaagagt cgaatgttct 420
ctatcagcac aatctacgaa gaatcaagca gtttcttcag agcaggtatc ttgagaagcc 480
aatggagatt gcccggattg tggcccggtg cctgtgggaa gaatcacgcc ttctacagac 540
tgcagccact gcggcccagc aagggggcca ggccaaccac cccacagcag ccgtggtgac 600
ggagaagcag cagatgctgg agcagcacct tcaggatgtc cggaagagag tgcaggatct 660
agaacagaaa atgaaagtgg tagagaatct ccaggatgac tttgatttca actataaaac 720
cctcaagagt caaggagaca tgcaagatct gaatggaaac aaccagtcag tgaccaggca 780
gaagatgcag cagctggaac agatgctcac tgcgctggac cagatgcgga gaagcatcgt 840
gagtgagctg gcggggcttt tgtcagcgat ggagtacgtg cagaaaactc tcacggacga 900
ggagctggct gactggaaga ggcggcaaca gattgcctgc attggaggcc cgcccaacat 960
ctgcctagat cggctagaaa actggataac gtcattagca gaatctcaac ttcagacccg 1020
tcaacaaatt aagaaactgg aggagttgca gcaaaaagtt tcctacaaag gggaccccat 1080
tgtacagcac cggccgatgc tggaggagag aatcgtggag ctgtttagaa acttaatgaa 1140
aagtgccttt gtggtggagc ggcagccctg catgcccatg catcctgacc ggcccctcgt 1200
catcaagacc ggcgtccagt tcactactaa agtcaggttg ctggtcaaat tccctgagtt 1260
gaattatcag cttaaaatta aagtgtgcat tgacaaagac tctggggacg ttgcagctct 1320
cagaggatcc cggaaattta acattctggg cacaaacaca aaagtgatga acatggaaga 1380
atccaacaac ggcagcctct ctgcagaatt caaacacttg accctgaggg agcagagatg 1440
tgggaatggg ggccgagcca attgtgatgc ttccctgatt gtgactgagg agctgcacct 1500
gatcaccttt gagaccgagg tgtatcacca aggcctcaag attgacctag agacccactc 1560
cttgccagtt gtggtgatct ccaacatctg tcagatgcca aatgcctggg cgtccatcct 1620
gtggtacaac atgctgacca acaatcccaa gaatgtaaac ttttttacca agcccccaat 1680
tggaacctgg gatcaagtgg ccgaggtcct gagctggcag ttctcctcca ccaccaagcg 1740
aggactgagc atcgagcagc tgactacact ggcagagaaa ctcttgggac ctggtgtgaa 1800
ttattcaggg tgtcagatca catgggctaa attttgcaaa gaaaacatgg ctggcaaggg 1860
cttctccttc tgggtctggc tggacaatat cattgacctt gtgaaaaagt acatcctggc 1920
cctttggaac gaagggtaca tcatgggctt tatcagtaag gagcgggagc gggccatctt 1980
gagcactaag cctccaggca ccttcctgct aagattcagt gaaagcagca aagaaggagg 2040
cgtcactttc acttgggtgg agaaggacat cagcggtaag acccagatcc agtccgtgga 2100
accatacaca aagcagcagc tgaacaacat gtcatttgct gaaatcatca tgggctataa 2160
gatcatggat gctaccaata tcctggtgtc tccactggtc tatctctatc ctgacattcc 2220
caaggaggag gcattcggaa agtattgtcg gccagagagc caggagcatc ctgaagctga 2280
cccaggtagc gctgccccat acctgaagac caagtttatc tgtgtgacac caacgacctg 2340
cagcaatacc attgacctgc cgatgtcccc ccgcacttta gattcattga tgcagtttgg 2400
aaataatggt gaaggtgctg aaccctcagc aggagggcag tttgagtccc tcacctttga 2460
catggagttg acctcggagt gcgctacctc ccccatgtga ggagctgaga acggaagctg 2520
cagaaagata cgactgaggc gcctacctgc attctgccac ccctcacaca gccaaacccc 2580
agatcatctg aaactactaa ctttgtggtt ccagattttt tttaatctcc tacttctgct 2640
atctttgagc aatctgggca cttttaaaaa tagagaaatg agtgaatgtg ggtgatctgc 2700
ttttatctaa atgcaaataa ggatgtgttc tctgagaccc atgatcaggg gatgtggcgg 2760
ggggtggcta gagggagaaa aaggaaatgt cttgtgttgt tttgttcccc tgccctcctt 2820
tctcagcagc tttttgttat tgttgttgtt gttcttagac aagtgcctcc tggtgcctgc 2880
ggcatccttc tgcctgtttc tgtaagcaaa tgccacaggc cacctatagc tacatactcc 2940
tggcattgca ctttttaacc ttgctgacat ccaaatagaa gataggacta tctaagccct 3000
aggtttcttt ttaaattaag aaataataac aattaaaggg caaaaaacac tgtatcagca 3060
tagcctttct gtatttaaga aacttaagca gccgggcatg gtggctcacg cctgtaatcc 3120
cagcactttg ggaggccgag gcggatcata aggtcaggag atcaagacca tcctggctaa 3180
cacggtgaaa ccccgtctct actaaaagta caaaaaatta gctgggtgtg gtggtgggcg 3240
cctgtagtcc cagctactcg ggaggctgag gcaggagaat cgcttgaacc tgagaggcgg 3300
aggttgcagt gagccaaaat tgcaccactg cacactgcac tccatcctgg gcgacagtct 3360
gagactctgt ctcaaaaaaa aaaaaaaaaa aaagaaactt cagttaacag cctccttggt 3420
gctttaagca ttcagcttcc ttcaggctgg taatttatat aatccctgaa acgggcttca 3480
ggtcaaaccc ttaagacatc tgaagctgca acctggcctt tggtgttgaa ataggaaggt 3540
ttaaggagaa tctaagcatt ttagactttt ttttataaat agacttattt tcctttgtaa 3600
tgtattggcc ttttagtgag taaggctggg cagagggtgc ttacaacctt gactcccttt 3660
ctccctggac ttgatctgct gtttcagagg ctaggttgtt tctgtgggtg ccttatcagg 3720
gctgggatac ttctgattct ggcttccttc ctgccccacc ctcccgaccc cagtccccct 3780
gatcctgcta gaggcatgtc tccttgcgtg tctaaaggtc cctcatcctg tttgttttag 3840
gaatcctggt ctcaggacct catggaagaa gagggggaga gagttacagg ttggacatga 3900
tgcacactat ggggccccag cgacgtgtct ggttgagctc agggaatatg gttcttagcc 3960
agtttcttgg tgatatccag tggcacttgt aatggcgtct tcattcagtt catgcagggc 4020
aaaggcttac tgataaactt gagtctgccc tcgtatgagg gtgtatacct ggcctccctc 4080
tgaggctggt gactcctccc tgctggggcc ccacaggtga ggcagaacag ctagagggcc 4140
tccccgcctg cccgccttgg ctggctagct cgcctctcct gtgcgtatgg gaacacctag 4200
cacgtgctgg atgggctgcc tctgactcag aggcatggcc ggatttggca actcaaaacc 4260
accttgcctc agctgatcag agtttctgtg gaattctgtt tgttaaatca aattagctgg 4320
tctctgaatt aagggggaga cgaccttctc taagatgaac agggttcgcc ccagtcctcc 4380
tgcctggaga cagttgatgt gtcatgcaga gctcttactt ctccagcaac actcttcagt 4440
acataataag cttaactgat aaacagaata tttagaaagg tgagacttgg gcttaccatt 4500
gggtttaaat catagggacc tagggcgagg gttcagggct tctctggagc agatattgtc 4560
aagttcatgg ccttaggtag catgtatctg gtcttaactc tgattgtagc aaaagttctg 4620
agaggagctg agccctgttg tggcccatta aagaacaggg tcctcaggcc ctgcccgctt 4680
cctgtccact gccccctccc catccccagc ccagccgagg gaatcccgtg ggttgcttac 4740
ctacctataa ggtggtttat aagctgctgt cctggccact gcattcaaat tccaatgtgt 4800
acttcatagt gtaaaaattt atattattgt gaggtttttt gtcttttttt tttttttttt 4860
tttttggtat attgctgtat ctactttaac ttccagaaat aaacgttata taggaaccgt 4920
c 4921
<210> 9
<211> 20
<212> DNA
<213> 人工合成引物(STAT3)(9)
<400> 9
ctgccccata cctgaagacc 20
<210> 10
<211> 20
<212> DNA
<213> 人工合成引物(STAT3)(10)
<400> 10
tcctcacatg ggggaggtag 20
Claims (3)
1.一种反义寡核苷酸分子或其药学上可以接受的盐在制备治疗恶性肿瘤药物中的应用,其特征在于,所述反义寡核苷酸分子的核苷酸序列如下所示:
STAT3 ASO-2:5’-CTCTGCCAGTGTAGTCAGCT-3’;
所述恶性肿瘤为肺癌、乳腺癌或肝癌中的一种或几种。
2.根据权利要求1所述应用,其特征在于,所述药物包括药学上可接受的载体。
3.根据权利要求1所述应用,其特征在于,所述药物的剂型为注射剂、胶囊剂、片剂、丸剂或颗粒剂。
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