CN107875402A - 一种靶向基因UBE2J2的修饰siRNA的用途 - Google Patents

一种靶向基因UBE2J2的修饰siRNA的用途 Download PDF

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CN107875402A
CN107875402A CN201711175486.2A CN201711175486A CN107875402A CN 107875402 A CN107875402 A CN 107875402A CN 201711175486 A CN201711175486 A CN 201711175486A CN 107875402 A CN107875402 A CN 107875402A
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sirna
ube2j2
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angiogenesis
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CN107875402B (zh
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蒋宇扬
陈少鹏
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Shenzhen Graduate School Tsinghua University
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Abstract

本发明属于基因治疗领域，具体涉及作为抗血管生成剂和/或在制备抗血管生成药物中的应用。该修饰siRNA由正义链和反义链组成；所述正义链的序列如SEQ ID NO：1所示，所述反义链的序列如SEQ ID NO：2所示，其中，序列中U均为2’‑Fluoro‑dU修饰。经过体外实验与体内动物模型测试证明，本发明的UBE2J2 siRNA能显著抑制血管生成，可作为抗血管生成与辅助抗肿瘤治疗的新靶标，具有潜在的临床应用前景。

Description

一种靶向基因UBE2J2的修饰siRNA的用途
技术领域
本发明属于基因治疗领域，更具体地，涉及一种靶向基因UBE2J2的修饰siRNA的用途。
背景技术
肿瘤是目前人类仍未攻克的难题之一。恶性肿瘤发展到一定体积，需要生成新的血管为肿瘤细胞提供足够的营养与氧气，血液循环系统同时带走肿瘤细胞快速繁殖代谢所产生的代谢废物。肿瘤细胞一方面分泌各种细胞生长因子诱发新生血管生成，反过来新生的血管内皮细胞也会分泌各种生长因子改造肿瘤细胞周围环境，使周围环境更加适合肿瘤细胞与新生血管细胞生长。
自从30年前folkman第一次提出抑制血管生成从而抑制肿瘤生长以来，抗血管生成的研究从不间断，但进展非常缓慢，目前应用于临床的抗血管生成药物寥寥可数，且副作用不小。
因此，亟需找到一种新的有效的抗血管生成治疗方法。
发明内容
本发明的目的是提供一种靶向肿瘤相关的基因UBE2J2(SEQ ID NO：5所示)的修饰siRNA的应用。通过设计合成UBE2J2的siRNA并加以化学修饰基因(UBE2J2 StableTMsiRNA)，用血管成环实验模型与Matrigel plugs小鼠体内血管生成动物模型进行验证，确定UBE2J2 StableTM siRNA具有体内外抑制血管生成的活性。
本发明提供一种靶向基因UBE2J2的修饰siRNA作为抗血管生成剂和/或在制备抗血管生成药物中的应用，该修饰siRNA由正义链和反义链组成；所述正义链的序列如SEQ IDNO：1所示，所述反义链的序列如SEQ ID NO：2所示，
5’-CCAGAGAAUUUCCUUUCAATT-3’(SEQ ID NO：1)
5’-UUGAAAGGAAAUUCUCUGGTT-3’(SEQ ID NO：2)
其中，序列中U均为2’-Fluoro-dU修饰。该Fluoro-dU修饰进一步提高了siRNA的稳定性。
根据本发明，优选地，所述抗血管生成的途径为抑制血管内皮细胞的增殖。所述血管内皮细胞包括但不限于HUVEC人脐静脉内皮细胞。
经过体外实验与体内动物模型测试证明，本发明的UBE2J2siRNA能显著抑制血管生成，可作为抗血管生成与辅助抗肿瘤治疗的新靶标，具有潜在的临床应用前景。
本发明的其它特征和优点将在随后具体实施方式部分予以详细说明。
附图说明
通过结合附图对本发明示例性实施方式进行更详细的描述。
图1-2示出了血管成环实验分析UBE2J2 StableTM siRNA体外抑制血管生成的作用。
图3-4示出了Matrigel plugs小鼠实验验证UBE2J2 StableTM siRNA具有体内抑制血管生成的活性。
具体实施方式
下面将更详细地描述本发明的优选实施方式。虽然以下描述了本发明的优选实施方式，然而应该理解，可以以各种形式实现本发明而不应被这里阐述的实施方式所限制。
下述实施例中所述实验方法，如无特殊说明，均为常规方法；所述试剂和生物材料，如无特殊说明，均可从商业途径获得。
以下实施例中采用Shcramble StableTM siRNA作为对照组，其序列为：
5’-UUCUCCGAACGUGUCACGUTT-3’(SEQ ID NO：3)
5’-ACGUGACACGUUCGGAGAATT-3’(SEQ ID NO：4)。
实施例1
体外血管成环实验验证UBE2J2 StableTM siRNA能抑制新生血管生成。
六孔板用不含生长因子的matrigel胶预处理。10μg UBE2J2 StableTMsiRNA或Shcramble StableTM siRNA加上2μl lipid2000溶于50ul PBS，转染到人脐静脉内皮细胞HUVEC(2×104)。细胞接种至matrigel预处理的六孔板，37度培养箱过夜培养。第二天置于倒置显微镜4倍物镜拍照观察。随机选取5个不同区域，用Image-Pro-Plus 6.0软件进行分析成环情况。
如图1-2所示，血管成环实验结果显示，UBE2J2 StableTM siRNA对血管成环的抑制率达到78％，具有显著的体外抑制效果。
实施例2
体内验证UBE2J2 StableTM siRNA具有抑制体内血管生成的活性。
10只6周龄Balb/c无胸腺免疫小鼠分成两组，SPF级饲料无菌室喂养。500μl不含生长因子的matrigel，加入bFGF(100ng/ml)与肝素(50U/500μl)，同时分别加入UBE2J2StableTM siRNA或Shcramble StableTM siRNA(10μg)与lipid2000(2μl)的混合液，打入小鼠右前肢腋下。1周后解剖小鼠，取出matrigel胶块，拍照，福尔马林固定后做石蜡切片，并做马森三色染色。染色的切片用倒置显微镜4倍及10倍物镜观察拍照，图片用Image-Pro-Plus 6.0进行处理，随机选取5个视野进行统计血细胞数量。
如图3-4所示，Matrigel plugs小鼠模型结果显示，UBE2J2 StableTMsiRNA显著抑制小鼠体内新生血管的生长，抑制率为81％，具有良好的抗血管生成活性。
以上已经描述了本发明的各实施例，上述说明是示例性的，并非穷尽性的，并且也不限于所披露的各实施例。在不偏离所说明的各实施例的范围和精神的情况下，对于本技术领域的普通技术人员来说许多修改和变更都是显而易见的。
序列表
<110> 清华大学深圳研究生院
<120> 一种靶向基因UBE2J2的修饰siRNA的用途
<130> 1700558
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 1
ccagagaauu uccuuucaat t 21
<210> 2
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 2
uugaaaggaa auucucuggt t 21
<210> 3
<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 3
ucuccgaacg ugucacgutt 20
<210> 4
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 4
acgugacacg uucggagaat t 21
<210> 5
<211> 624
<212> DNA/RNA
<213> Homo sapiens
<400> 5
atgacccctt atgaaggtgg ctattatcat ggaaaactaa tttttcccag agaatttcct 60
ttcaaacctc ccagtatcta tatgatcact cccaacggga ggtttaagtg caacaccagg 120
ctgtgtcttt ctatcacgga tttccacccg gacacgtgga acccggcctg gtctgtctcc 180
accatcctga ctgggctcct gagcttcatg gtggagaagg gccccaccct gggcagtata 240
gagacgtcgg acttcacgaa aagacaactg gcagtgcaga gtttagcatt taatttgaaa 300
gataaagtct tttgtgaatt atttcctgaa gtcgtggagg agattaaaca aaaacagaaa 360
gcacaagacg aactcagtag cagaccccag actctcccct tgccagacgt ggttccagac 420
ggggagacgc acctcgtcca gaacgggatt cagctgctca acgggcatgc gccgggggcc 480
gtcccaaacc tcgcagggct ccagcaggcc aaccggcacc acggactcct gggtggcgcc 540
ctggcgaact tgtttgtgat agttgggttt gcagcctttg cttacacggt caagtacgtg 600
ctgaggagca tcgcgcagga gtga 624

Claims (3)

1.一种靶向基因UBE2J2的修饰siRNA作为抗血管生成剂和/或在制备抗血管生成药物中的应用，其特征在于，该修饰siRNA由正义链和反义链组成；所述正义链的序列如SEQ IDNO：1所示，所述反义链的序列如SEQ ID NO：2所示，
5’-CCAGAGAAUUUCCUUUCAATT-3’(SEQ ID NO：1)
5’-UUGAAAGGAAAUUCUCUGGTT-3’(SEQ ID NO：2)
其中，序列中U均为2’-Fluoro-dU修饰。
2.根据权利要求1所述的应用，其中，所述抗血管生成的途径为抑制血管内皮细胞的增殖。
3.根据权利要求2所述的应用，其中，所述血管内皮细胞为HUVEC人脐静脉内皮细胞。
CN201711175486.2A 2017-11-22 2017-11-22 一种靶向基因UBE2J2的修饰siRNA的用途 Active CN107875402B (zh)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101353655A (zh) * 2008-07-24 2009-01-28 中国科学院广州生物医药与健康研究院 抑制血管内皮生长因子受体基因表达的siRNA及其应用
CN107142260A (zh) * 2016-08-11 2017-09-08 武汉泽智生物医药有限公司 沉默内皮细胞生长因子受体2表达的siRNA及其用途

Patent Citations (2)

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CN101353655A (zh) * 2008-07-24 2009-01-28 中国科学院广州生物医药与健康研究院 抑制血管内皮生长因子受体基因表达的siRNA及其应用
CN107142260A (zh) * 2016-08-11 2017-09-08 武汉泽智生物医药有限公司 沉默内皮细胞生长因子受体2表达的siRNA及其用途

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SHAOPENG CHEN，等: "UBE2J2 promotes hepatocellular carcinoma cell epithelial-mesenchymal transition and invasion in vitro", 《ONCOTARGET》 *
SUN T ET AL.: "Expression and functional significance of Twist1 in hepatocellular carcinoma: its role in vasculogenic mimicry", 《HEPATOLOGY》 *
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