CN112359039A - 靶向沉默BRD4基因表达的shRNA序列及其用途 - Google Patents
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Abstract
本发明属于生物医药领域，具体涉及一种靶向沉默BRD4基因表达的shRNA序列及其用途。本发明基于BRD4基因的mRNA序列设计并合成shRNA分子，并构建shRNA的寡核苷酸序列，shRNA序列通过慢病毒载体包装成慢病毒后感染人小细胞肺癌细胞，能够与BRD4基因的mRNA结合，高效地干扰其转录和翻译，降低BRD4基因在小细胞肺癌细胞中的表达，抑制小细胞肺癌细胞的增殖。本发明提供的shRNA序列在将为以BRD4抑制剂为核心的小细胞肺癌治疗新策略提供理论基础，有望应用于制备高效、特异性强、副作用小的抗癌基因药物，尤其针对小细胞肺癌基因药物的研发，具有巨大的社会和经济效益。
Description
技术领域
本发明属于生物医药领域，具体涉及一种靶向沉默BRD4基因表达的shRNA序列及其用途。
背景技术
肺癌是目前最为常见的恶性肿瘤，其死亡率居恶性肿瘤之首。肺癌可分为非小细胞肺癌（NSCLC）和小细胞肺癌（SCLC），后者约占所有肺癌病例的12-14％。SCLC最显著的特征之一是某些重要基因突变频率高，如肿瘤蛋白53基因（TP53）的突变率为75%–90%，视网膜母细胞瘤1（RB1）基因的突变率接近100%。目前在临床上，SCLC最常用的治疗方法包括手术、化疗和放疗等。SCLC初期对化疗和放疗比较敏感，但由于SCLC倍增时间短，恶性程度高，非常容易发生耐药和复发，绝大多数患者的诊断后生存期短，预后较差。目前，与其它肿瘤相比，SCLC的治疗方式仍比较局限单一，对病人的生存期改善效果也不明显。因此，迫切地需要找到更多安全有效的药物靶向，以提高SCLC患者的生存期。
溴结构域蛋白4（BRD4）位于染色体19p13区，是溴结构域和超末端结构域家族重要的功能蛋白。在SCLC中，BRD4可直接结合于无刚毛鳞甲复合体样1（ASCL1）基因的增强子上，进而促进SCLC的发展；ASCL1的表达水平与SCLC细胞对BRD4的抑制剂JQ1的敏感性有关。BRD4抑制剂ABBV-075可通过诱导Caspase3/7活性、促进前凋亡蛋白BIM的表达、促进Bcl2-BIM复合物的形成等方式，引发细胞凋亡。
RNA干扰技术(RNAi)是一种依赖于双链RNA特异性短序列实现转录后的基因沉默的技术。目前实验室常用的RNAi技术主要有siRNA寡核苷酸载体与shRNA慢病毒质粒表达载体。短发夹核糖核酸（shRNA）可以通过病毒介导的转染保持稳定，能够减少脱靶效应。
shRNA首先被插入到慢病毒载体上形成重组慢病毒质粒，慢病毒质粒与其他辅助质粒借助于293FT细胞形成慢病毒，最终用慢病毒转染细胞发挥shRNA的沉默作用。在内切核酸酶Dicer作用下，shRNA被裂解成21-25nt的核苷酸链，由正义链和反义链组成。反义链与特定的酶结合形成由RNA诱导的沉默复合物RISC（RISC复合物中含siRNA、核酸外切酶、核酸内切酶以及解旋酶等元件），核苷酸双链会被活化后的RISC解聚成为两条单链，随后反义链识别并结合与其同源的靶mRNA，在反义链的引导下活化型RISC会切割靶mRNA的特定位置，同时切割的mRNA会被RISC复合物中的酶特异性降解，从而阻断mRNA的遗传信息传递。但如何利用shRNA技术，研究出特异性针对小细胞肺癌细胞的shRNA，对于小细胞肺癌的预防或治疗具有重要的意义。
发明内容
有鉴于此，本发明的目的在于提供靶向沉默BRD4基因的shRNA序列及其应用。本发明根据沉默BRD4基因对小细胞肺癌增殖方面的影响，判断沉默BRD4基因的shRNA序列的特异性，验证可沉默BRD4基因的shRNA序列对小细胞肺癌细胞的抑制作用。
为实现上述发明目的，本发明采取的技术方案为：
本发明提供了一种靶向沉默BRD4基因表达的shRNA序列，所述的shRNA记为shBRD4-1和shBRD4-2，所述shBRD4-1的序列如SEQ ID NO.1所示，shBRD4-2的序列如SEQ ID NO.2所示。
本发明还提供了用于制备上述shBRD4-1的寡核苷酸序列，其正义链shBRD4-1-F如SEQ ID NO.3所示，即：5´-CCGGTgccaacgcagccagcaccaacCTCGAGgttggtgctggctgcgttggcATTTTT-3´，反义链shBRD4-1-R如SEQ ID NO.4所示，即：5´-AAAAATgccaacgcagccagcaccaacCTCGAGgttggtgctggctgcgttggcACCGG-3´；
本发明还提供了用于制备上述shBRD4-2的寡核苷酸序列，其正义链shBRD4-2-F如SEQID NO.5所示，即：shBRD4-2-F：5´-CCGGTagcagctcaagtgctgcagcgCTCGAGcgctgcagcacttgagctgctATTTTT-3´，反义链shBRD4-2-R如SEQ ID NO.6所示，即：5´-AAAAATagcagctcaagtgctgcagcgCTCGAGcgctgcagcacttgagctgctACCGG-3´。
在一些实施方案中，本发明还提供了一种包含上述的靶向沉默BRD4基因表达的shRNA序列的慢病毒表达载体，所述载体为pLKO.1-TRC-shBRD4-1或pLKO.1-TRC-shBRD4-2。所述慢病毒表达载体用于表达如权利要求1所述的shRNA序列，所述慢病毒表达载体中包含用于沉默目的基因BRD4的靶位点序列shBRD4-1或shBRD4-2。
进一步地，所述慢病毒表达载体采用引物退火合成shBRD4-1或shBRD4-2序列；将合成的shBRD4-1或shBRD4-2序列克隆到慢病毒表达载体pLKO.1-TRC中获得，所述shBRD4-1的引物序列如SEQ ID NO.3~4所示，所述shBRD4-2的引物序列如SEQ ID NO.5~6所示。
在一些实施方案中，本发明还提供了上述沉默BRD4基因表达的shRNA序列在制备抑制BRD4基因表达的药物中的用途，所述应用为制备治疗/预防小细胞肺癌的药物。
本发明还提供了一种治疗小细胞肺癌的药物，所述药物包含上述的靶向沉默BRD4基因表达的shRNA序列。
与现有技术相比，本发明的有益效果是：
本发明基于BRD4基因的mRNA序列设计并合成shRNA分子，并构建shRNA的寡核苷酸序列，这两对shRNA序列通过慢病毒载体包装成慢病毒后感染人小细胞肺癌细胞，能够与BRD4基因的mRNA结合，高效地干扰其转录和翻译，降低BRD4基因在小细胞肺癌细胞中的表达，抑制小细胞肺癌细胞的增殖。本发明设计的shRNA序列限制抑制小肺癌细胞H446的生长速率，对小细胞肺癌的治疗具有十分重要的意义。为以BRD4抑制剂为核心的小细胞肺癌治疗新策略提供理论基础，有望应用于制备高效、特异性强、副作用小的抗癌基因药物，尤其针对小细胞肺癌基因药物的研发，具有巨大的社会和经济效益。
附图说明
图1是Western blot检测shBRD4对BRD4表达的沉默；
图2是shBRD4对人小细胞肺癌细胞H446增殖的影响图。
具体实施方式
以下结合实施例对本发明作进一步说明，实施例是用于说明本发明，而不是用于限制本发明的范围。本领域技术人员可以确定本发明的基本特征，并且在不偏离本发明精神和范围的情况下，可以对本发明做出各种修改和改变，以使其使用各种用途和条件。
以下实施例中，所用材料及试剂部分来源如下：
慢病毒包装载体pLP1、pLP2、pLPSVG、慢病毒敲减质粒载体Plko.1-TRC均购买于美国Addgene公司。本发明所用人小细胞肺癌细胞H446购自于美国菌种保藏中心（ATCC）。
实施例中未注明具体条件者，皆按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。除特殊注明外，本发明所采用的均为该领域现有技术。
实施例1：慢病毒载体的构建
根据GenBank数据库(http://www.ncbi.nlm.nih.gov/genbank)获得人源BRD4基因(NM_058243)的mRNA序列，根据shRNA设计原理，设计了针对人源BRD4基因的shRNA，其靶点序列分别如SEQ ID NO.1和SEQ ID NO.2所示，用于沉默人源BRD4基因。SEQ ID NO.1即：5´-gccaacgcagccagcaccaac-3´，其对应编码区为NM_001330384.1（核苷酸位点400-420）； SEQID NO.2即：5´-agcagctcaagtgctgcagcg-3´，其对应编码区为XM_011527854.2（核苷酸位点1126-1146）。根据上述两段靶点序列分别设计合成两对互补的寡核苷酸序列shBRD4-1-1和shBRD4-1-2。其中，shBRD4-1-1的正义链shBRD4-1-F如SEQ ID NO.3所示，即：5´-CCGGTgccaacgcagccagcaccaacCTCGAGgttggtgctggctgcgttggcATTTTT-3´；反义链shBRD4-1-R如SEQID NO.4所示，即：5´-AAAAATgccaacgcagccagcaccaacCTCGAGgttggtgctggctgcgttggcACCGG-3´；
shBRD4-1-2的正义链shBRD4-2-F如SEQ ID NO.5所示，即：5´-CCGGTagcagctcaagtgctgcagcgCTCGAGcgctgcagcacttgagctgctATTTTT-3´，反义链shBRD4-2-R如SEQ ID NO.6所示，即：5´-AAAAATagcagctcaagtgctgcagcgCTCGAGcgctgcagcacttgagctgctACCGG-3´。本发明中所述序列均由生工生物工程(上海)股份有限公司合成。
将上述4条双链寡核苷酸短片段分别连接到双酶切的慢病毒载体Plko.1-TRC上。将连接产物通过热激法转化到DH5α大肠杆菌感受态细胞中，置于含氨苄抗性的LB固体培养基上37℃培养16h，挑取单个菌落于含氨苄抗性LB液体培养基中摇菌，37℃、220rpm、16h，收集菌体提取质粒，酶切验证质粒，将酶切的阳性质粒送于华大基因测序验证。测序显示构建成功的慢病毒质粒用于后续实验。
通过上述方法，分别构建得到含有目的质粒的慢病毒载体pLKO.1-TRC-shBRD4-1和pLKO.1-TRC-shBRD4-2。
实施例2：细胞慢病毒的制备及慢病毒转染细胞
参照Lipofectamine 2000试剂说明书包装慢病毒。分别以实施例1所制备的两种慢病毒载体转染细胞作为实验组，同时以pLKO.1-TRC慢病毒空载体转染细胞作为对照组。lipofectmin2000将目的质粒与辅助质粒pLP1、pLP2和pLPSVG转入293FT细胞中，制备并收集病毒，之后用含目标shBRD4序列的慢病毒感染人小细胞肺癌细胞H446，用嘌呤霉素（puromycin）筛选出具有抗性的细胞用于实验。
将0.75 μg pLP1、0.35 μg pLP2、0.49 μg pLPSVG、0.61 μg慢病毒载体pLKO.1-TRC-shBRD4-1分别加入到0.5 mL的低血清OPTI-MEM培养基中，轻轻混匀，室温孵育5 min，得到液A，待用。
取9μL脂质体Lipofectmin2000加到0.5mL的OPTI-MEM培养基中，轻轻混匀，室温放置5min得到液B，待用。
将液A与液B液按1:1的比例混匀得到混合液，室温孵育20 min；消化293FT细胞，并在显微镜下计数，调整细胞密度为培养皿底面积的三分之一。将293FT的细胞混悬液与混合液按1:1的比例轻柔充分混匀，并转移到培养皿中，轻轻摇晃，使混匀铺平底部。将培养皿放置在培养箱中孵育12h后，给细胞换液，继续培养。培养两天后，用5mL的灭菌注射器吸取细胞上清液，再经0.45μm微孔滤膜过滤掉细胞上清杂质，收集病毒。慢病毒载体pLKO.1-TRC-shBRD4-2和慢病毒空载体pLKO.1-TRC包装慢病毒的方法与上述慢病毒载体pLKO.1-TRC-shBRD4-1包装慢病毒的方法相同。
用收集到的病毒分别转染H446细胞：第一天，种板，数细胞，调整细胞密度为6cm培养皿底面积的三分之一；第二天，病毒感染，拿出6cm培养皿，弃上清，病毒与H446细胞培养基1：1混合加入，并加入8mg/mL聚凝胺（polybrene）使作用浓度为8ug/mL，放入培养箱，孵育8h；孵育8h后，弃上清，给细胞换液，继续放入培养箱培养两天；培养两天后，弃上清，用1000mg/mL puromycin按1：1000稀释，筛选2-3天；重新种板：将筛选两天后的细胞消化下来重新种回培养皿中，获得具有抗性的活细胞，用于后续实验。
实施例3：Western blot检测BRD4基因在H446细胞中的蛋白表达水平
收集病毒感染的H446细胞，提取总蛋白，测定蛋白浓度，将蛋白变性之后跑胶转膜，5%脱脂牛奶封闭膜1h之后，使用BRD4抗体和β-actin抗体过夜孵育，然后用连有辣根过氧化物酶的山羊抗鼠的抗体室温孵育1h，抗体孵育过后，用TBS-T溶液室温洗膜3次，每次4min，最后将膜置于经过暗处理的暗匣中拿到暗室中曝光，曝光时将片固定在膜上，在发光液和显影液的作用下蛋白的条带会印迹在胶片上。收集puromycin筛选后实验组和对照组存活的H446细胞，分别提取细胞总蛋白，Western blotting检测不同组细胞中目的蛋白BRD4的表达情况。图1是shBRD4对BRD4基因的表达水平Western blotting图；图中，β-actin为内参蛋白，Vector为用pLKO.1-TRC空载体感染的对照组，shBRD4-1和shBRD4-2为分别用Plko.1-TRC-shBRD4-1和Plko.1-TRC-shBRD4-2感染的实验组。由图1可见，与对照组Vector相比，shBRD4-1和shBRD4-2均能显著沉默H446细胞中BRD4的表达，对H446细胞中BRD4的表达有显著的沉默作用。
实施例4：MTT实验检测shBRD4对H446细胞增殖的影响
（1）处理细胞：收集病毒感染的 H446 细胞，用胰酶消化，200 g 离心 5 min，培养基重悬，使用细胞计数板进行细胞计数，使用 96 孔板，根据细胞生长特性，每孔 100 μL 培养基， 含 4×103 个细胞，种三个平行孔。
（2） MTT 检测：种完板，每隔 24 h，在待检测孔加 5 mg/mL 的 MTT 溶液 10 μL，于培养箱中孵育 1.5 h，显微镜下观察有蓝紫色甲瓒结晶，H446 细胞直接吸弃上清液，每孔加 100 μL DMSO 溶解结晶，轻轻拍匀，使用酶标仪在 550 nm 波长处测定每孔吸光值，计算细胞的相对存活率。
图2是shBRD4对H446细胞增殖的影响图，vector组为对照组，shBRD4-1和shBRD4-2组是实验组。如图2所述，shBRD4-1和shBRD4-2组均能显著抑制小细胞肺癌细胞H446的增殖，可见，本发明设计的shRNA可以用于制备治疗小细胞肺癌的药物。
以上显示和描述了本发明的基本原理、主要特征以及本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
序列表
<110> 江苏大学
<120> 靶向沉默BRD4基因表达的shRNA序列及其用途
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Claims (8)
1.一种靶向沉默BRD4基因表达的shRNA序列，其特征在于，记为shBRD4-1和 shBRD4-2；所述shBRD4-1的靶位点序列如SEQ ID NO.1所示，所述shBRD4-2的靶位点序列如SEQ IDNO.2所示。
2.根据权利要求1所述的shRNA序列，其特征在于，制备所述shBRD4-1的寡核苷酸序列的正义链序列如SEQ ID NO.3所示，反义链序列如SEQ ID NO.4所示。
3.根据权利要求1所述的shRNA序列，其特征在于，制备所述shBRD4-2的寡核苷酸序列的正义链序列如SEQ ID NO.5所示，反义链序列如SEQ ID NO.6所示。
4.一种慢病毒表达载体，其特征在于，所述慢病毒表达载体用于表达如权利要求1所述的shRNA序列，所述慢病毒表达载体中包含用于沉默目的基因BRD4的靶位点序列shBRD4-1或shBRD4-2。
5.根据权利要求4所述的慢病毒表达载体的构建方法，其特征在于，所述慢病毒表达载体采用引物退火合成shBRD4-1或shBRD4-2序列；将合成的shBRD4-1或shBRD4-2序列克隆到慢病毒表达载体pLKO.1-TRC中获得，所述shBRD4-1的引物序列如SEQ ID NO.3~4所示，所述shBRD4-2的引物序列如SEQ ID NO.5~6所示。
6.根据权利要求1所述的shRNA序列在制备抑制BRD4基因表达的药物方面的应用。
7.根据权利要求1所述的shRNA序列在制备治疗/预防小细胞肺癌相关的药物方面的应用。
8.一种治疗小细胞肺癌的药物，其特征在于，包含如权利要求1所述的靶向沉默BRD4基因表达的shRNA序列。
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| CN114045264A (zh) * | 2021-11-16 | 2022-02-15 | 珠海中科先进技术研究院有限公司 | 一种获取驱动元件的方法 |
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