CN114181937B - 一种沉默人LINC01614表达的shRNA分子及其应用 - Google Patents

一种沉默人LINC01614表达的shRNA分子及其应用 Download PDF

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CN114181937B
CN114181937B CN202111494647.0A CN202111494647A CN114181937B CN 114181937 B CN114181937 B CN 114181937B CN 202111494647 A CN202111494647 A CN 202111494647A CN 114181937 B CN114181937 B CN 114181937B
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linc01614
shrna
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陶方方
张志潜
刘文洪
徐烨
刘青玲
李俊峰
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Zhejiang Chinese Medicine University ZCMU
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Abstract

一种沉默人LINC01614表达的shRNA分子及其应用，属于生物医学技术领域。本发明一方面提供了一种沉默人LINC01614表达的shRNA分子的靶标基因，另一方面提供了一种沉默人LINC01614表达的shRNA分子及其应用。本发明得到的shRNA分子能够干扰LINC01614表达，从而降低肿瘤细胞的迁移和侵袭能力，抑制EMT分子表达，并于体内动物模型中抑制肿瘤形成和肺转移。本发明为乳腺癌的靶向治疗提供新的方案。

Description

一种沉默人LINC01614表达的shRNA分子及其应用
技术领域
本发明属于生物医学技术领域，具体涉及一种沉默人LINC01614表达的shRNA分子及其应用。
背景技术
乳腺癌是一种多见的恶性肿瘤，因其高发病率而位居女性癌症首位。近年来我国女性乳腺癌发病率呈逐年上升趋势且逐渐年轻化，抗肿瘤治疗方法的发现和发展虽一定程度上提高了肿瘤患者的生存率和生活质量，然而接受肿瘤药物治疗的患者通常会产生明显的耐药性，导致疗效降低、肿瘤复发、转移，最终导致治疗失败。乳腺癌的侵袭转移是一个多因素、多步骤的复杂过程，受到多种基因的表达调控，其具体的分子机制目前尚不完全清楚。
LncRNA是一类长度大于200nt的不具备编码蛋白质能力的功能性RNA分子，参与调控细胞内多种生物学过程。现已有上千种LncRNA参与调控哺乳动物基因表达，在多种恶性肿瘤中均有LncRNA异常表达的报道，其对人类疾病进程的影响越来越突出。LINC01614作为一种新的肿瘤诊断和预后指标，有团队研究发现LINC01614的高表达与细胞粘附信号通路中多个基因呈正相关，并与上皮-间质转化(Epithelial-Mesenchymal Transition,EMT)相关，影响多种癌症的转移和预后。在公共数据库中可查询到其在多种癌症中均高表达，包括乳腺癌、非小细胞肺癌、胃癌、胶质瘤等，提示其与肿瘤的发生发展密切相关，但迄今为止有关LINC01614的文献报道并不多见。
RNA干扰(RNA interference，RNAi)是机体内广泛存在的一种双链RNA介导的序列特异性基因沉默现象，因其可特异性剔除或关闭特定基因的表达而被广泛应用于探索基因功能和传染性疾病及恶性肿瘤的治疗领域。其中，短发卡RNA(short hairpin RNA，shRNA)是一段具有茎环结构的外源性RNA序列，能够在细胞内加工成siRNA，siRNA进而与蛋白质结合形成RNA诱导沉默复合物(RNA-inducedsilencing complex，RISC)结合到同源的mRNA上并诱导其降解。shRNA的作用具有严格的靶向性，特异性靶位点的选择具有位置效应，不同的靶位点对同一基因的干扰效率有着很大的差异。
本发明构建了特异性沉默LINC01614的shRNA，所述shRNA转入肿瘤细胞后，有效抑制了肿瘤细胞的转移和侵袭，并抑制了EMT相关分子的表达，为进一步深入探索LINC01614在乳腺癌转移侵袭方面的分子机制提供应用基础。
发明内容
针对现有技术存在的问题，本发明的目的在于设计提供一种沉默人LINC01614表达的shRNA分子及其应用的技术方案。
为实现上述目的，本发明采用如下技术方案：
本发明一方面提供了一种沉默人LINC01614表达的shRNA分子的靶标基因，所述沉默人LINC01614表达的shRNA分子靶标基因如SEQ ID N0.1和SEQ ID N0.2所示。
本发明另一方面提供了一种沉默人LINC01614表达的shRNA分子，包括shRNA-1和/或shRNA-2，所述shRNA-1的正义链核苷酸序列如SEQ ID N0.3所示，所述shRNA-1的反义链核苷酸序列如SEQ ID N0.4所示，所述shRNA-2的正义链核苷酸序列如SEQ ID N0.5所示，所述shRNA-2的反义链核苷酸序列如SEQ ID N0.6所示。
本发明另一方面提供了一种沉默人LINC01614表达的shRNA分子在制备用于抑制细胞LINC01614表达药物中的应用。
本发明另一方面提供了一种沉默人LINC01614表达的shRNA分子在制备用于抑制人乳腺癌细胞MDA-MB-231和Hs578T转移和侵袭药物中的应用。
本发明另一方面提供了一种沉默人LINC01614表达的shRNA分子在制备用于抑制乳腺癌转移和侵袭药物中的应用。
本发明具有以下有益效果：
其一，通过特异性降低LINC01614的表达，抑制了乳腺癌的肿瘤形成、转移和侵袭能力，在控制乳腺癌转移中发挥治疗作用。
其二，将shRNA构建在慢病毒载体上，通过shRNA-慢病毒感染两种常见乳腺癌细胞来进行基因沉默，提高了基因沉默的效率。
本发明获得的shRNA分子未见以往公开报道。以上shRNA分子能够干扰LINC01614表达，从而降低肿瘤细胞的迁移和侵袭能力，抑制EMT分子表达，并于体内动物模型中抑制肿瘤形成和肺转移。为乳腺癌的靶向治疗提供新的方案。
附图说明
图1慢病毒介导的shRNA对人乳腺癌细胞MDA-MB-231和Hs578T细胞中LINC01614基因沉默的验证效果。*表示与sh-Control相比，*P<0.05，**P<0.01，***P<0.001。
图2转染慢病毒后两种癌细胞克隆形成能力的检测。
图3转染慢病毒后两种癌细胞迁移和侵袭能力的检测。
图4转染慢病毒后两种癌细胞中EMT相关分子E-cadherin与N-cadherin的蛋白表达检测。
图5转染慢病毒沉默LINC01614后的MDA-MB-231进行裸鼠皮下成瘤造模，肿瘤体积及重量检测。
图6转染慢病毒沉默LINC01614后的MDA-MB-231尾静脉注射裸鼠建立乳腺癌肺转移模型，肺组织中转移灶情况检测。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
统计学分析
数据用SPSS 22.0软件进行统计学分析。计量资料以均值±标准差(x±s)表示，组间比较单因素方差分析(One-way ANOVA)和t检验，P<0.05表明差异有显著性。
实施例1：沉默人LINC01614表达的特异性干扰序列shRNA的获得
根据网站：
www.sigmaaldrich.cn/CN/zh/semi-configurators/shrna？activeLink＝product Search设计shRNA序列，获得的靶基因序列：
shRNA-1：TTCCTTAAAGTAGCAATCTTAGC(如SEQ ID N0.1所示)；
shRNA-2：GACAAGTTCAGTGGAAACTTTCT(如SEQ ID N0.2所示)。
shRNA-1的正义链核苷酸序列为：
5’-CCGGTTCCTTAAAGTAGCAATCTTAGCCTCGAGGCTAAGATTGCTACTTTAAGGAATTTTTG-3’(如SEQ ID N0.3所示)；
shRNA-1的反义链核苷酸序列为：
5’-AATTCAAAAATTCCTTAAAGTAGCAATCTTAGCCTCGAGGCTAAGATTGCTACTTTAAGGAA-3’(如SEQ ID N0.4所示)。
shRNA-2的正义链核苷酸序列为：
5’-CCGGGACAAGTTCAGTGGAAACTTTCTCTCGAGAGAAAGTTTCCACTGAACTTGTCTTTTTG-3’(如SEQ ID N0.5所示)；
shRNA-2的反义链核苷酸序列为：
5’-AATTCAAAAAGACAAGTTCAGTGGAAACTTTCTCTCGAGAGAAAGTTTCCACTGAACTTGTC-3’(如SEQ ID N0.6所示)；
sh-LINC01614-1：
CCGGTTCCTTAAAGTAGCAATCTTAGCCTCGAGGCTAAGATTGCTACTTTAAGGAATTTTTG；
sh-LINC01614-2：
CCGGGACAAGTTCAGTGGAAACTTTCTCTCGAGAGAAAGTTTCCACTGAACTTGTCTTTTTG。
sh-Control：TTCTCCGAACGTGTCACGT(如SEQ ID N0.7所示)。
实施例2：LINC01614的shRNA-慢病毒表达载体的构建
将hU6-MCS-CBh-gcGFP-IRES-puromycin载体，采用EcoR I/Age I进行双酶切线状化，并切胶回收；根据shRNA设计特异性引物完成引物退火；将shRNA和酶切回收的线状载体同源重组形成环状，转入制备好的细菌感受态细胞，挑选单克隆菌落进行测序鉴定，比对正确的克隆即为构建成功的shRNA沉默载体。
表1：shRNA设计特异性引物
具体步骤如下：
(1)载体线状化酶切回收，酶切体系如表2所示：
表2酶切体系
37℃(最适温度)反应1h后，对载体酶切产物进行1％琼脂糖凝胶电泳，回收目的片段。
(2)目的片段与载体的重组，通过T4 DNA ligase将双酶切线性化的载体和退火双链DNA连接，16℃连接1-3h，或连接过夜。反应体系如表3：
表3链接体系
*根据载体大小做相应调整
(3)转化及阳性克隆的PCR鉴定与测序
将5μl连接产物加入到50μl大肠杆菌感受态细胞中，冰浴30min，42℃热激80s，冰浴3min；加入450μL无抗生素的LB液体培养基，150rpm于37℃摇床震荡培养45min；取菌液均匀涂抹在含有Amp的LB固体培养基上，于37℃培养箱中过夜培养。次日，挑取单克隆，一个离心管中放一个单克隆，管内加8ml含有氨苄青霉素的液体LB培养基，37℃，220rpm，振荡培养14h。培养至菌液混浊，收集菌液，并提质粒进行测序(上海吉凯基因有限公司)，进行测序比对后，鉴定阳性克隆即为构建成功的LINC01614-shRNA慢病毒表达载体。
(4)慢病毒包装及滴度测定
将提取的慢病毒表达载体质粒分别与包装质粒共转染293T细胞(转染试剂由上海吉凯公司提供)，于48h收集病毒上清，观察细胞形态和GFP表达，将提取的病毒纯化和浓缩，采用梯度稀释法测定病毒滴度，将制备完成的病毒浓缩液分装于-80℃保存。
通过该实施例得到sh-LINC01614-1、sh-LINC01614-2、sh-Control三种慢病毒。
实施例3：LINC01614-shRNA慢病毒转染人乳腺癌细胞
将MDA-MB-231和Hs578T细胞制备以2.5×105个/孔的密度接种于6孔板，待细胞密度达到70％时，分别更换为sh-LINC01614-1、sh-LINC01614-2、sh-Control三种慢病毒溶解液(MOI＝10)与感染试剂至培养基，摇晃混匀孵育8-10h后更换为正常完全培养基继续培养，72h后荧光显微镜观察GFP绿色荧光表达量，约90％以上说明转染成功。
实施例4：两种细胞中LINC01614沉默效果检测
收集感染慢病毒72h后的各组细胞和同步培养的sh-Control组细胞，按照TotalRNA提取试剂盒(15596026，Invitrogen公司)说明书提取细胞总RNA，用反转录试剂盒(RR037A，Takara公司)反转录合成cDNA。进行Real-Time PCR检测LINC01614(扩增引物见表4)，程序为：95℃10min；(95℃15s，64℃30s，72℃20s)*40个循环；95℃15s；60℃，1min；95℃15s。Real-Time PCR采用2-△△Ct法进行相对定量分析。
表4引物序列
RT-qPCR检测发现sh-LINC01614-1组和sh-LINC01614-2组的LINC01614水平显著低于sh-Control组(P<0.001)，并且sh-LINC01614-2沉默效果更佳(见图1)。
实施例5：LINC01614低表达抑制乳腺癌细胞克隆形成
取对数生长期感染慢病毒后的各组细胞，胰酶消化后制成单细胞悬液，计数活细胞，按200个/孔接种于6孔板中，轻轻晃动使细胞均匀分散，于37℃，5％CO2培养箱内静置培养2-3周，当孔内出现肉眼可见的克隆时，终止培养。弃去上清液，用PBS小心洗2次。加1:3醋酸/甲醇5mL，固定15min，弃去固定液，加入结晶紫染液染色10-30min，用水缓慢洗去染色液，自然干燥，显微镜下计数大于50个细胞的克隆数，计算克隆形成率。
两种癌细胞经LINC01614-shRNA慢病毒转染后其克隆形成能力均显著降低(P<0.01)，见图2。
实施例6：LINC01614低表达抑制乳腺癌细胞迁移和侵袭
Transwell细胞迁移与侵袭实验：实验前将细胞饥饿12h，胰蛋白酶消化离心，用无血清培养基稀释重悬并进行细胞计数，使用具有8.0μm孔径的Transwell小室，取无血清细胞悬液(2×104个/100μL)接种在上室(侵袭实验在接种细胞前预先铺入100μl1:8稀释的Matrigel基质胶，37℃孵育2h待胶凝固)，下室加600μL含10％FBS的完全培养基；培养24h，取出Transwell小室，弃上室培养基，用PBS洗两次，棉签轻轻擦去上室表面细胞；下室加入4％多聚甲醛固定细胞30min，自然风干后用0.1％结晶紫溶液染色30min，随机计数5个视野细胞数量计算平均细胞数，实验独立重复3次。
两种癌细胞经LINC01614-shRNA慢病毒转染后其迁移和侵袭能力均显著降低(P<0.05)，见图3。
实施例7：LINC01614低表达抑制乳腺癌细胞上皮间质转化(EMT)
分别提取各组细胞的总蛋白，BSA法测定蛋白浓度。加入适量上样缓冲液于100℃煮沸变性10min；每孔20μg，80V恒压SDS-PAGE电泳，在4℃，300mA恒流条件下电转60min，将蛋白转移到PVDF膜上。5％脱脂牛奶室温封闭1.5h，分别加入一抗兔抗E-cadherin和N-cadherin(1:1000，CST公司)、兔抗β-actin单克隆抗体(1:5000，Abcam公司)4℃孵育过夜。分别加入HRP标记的羊抗兔IgG(1:5000，Abcam公司)，室温孵育1h，TBST漂洗10min×3次，ECL化学发光显影。
Western Blot检测发现，LINC01614低表达后EMT相关分子E-cadherin表达增高，N-cadherin表达降低，说明LINC01614的降低可抑制肿瘤细胞EMT的形成(见图4)。
实施例8：LINC01614低表达抑制乳腺癌肿瘤形成和肺转移
裸鼠乳腺癌皮下成瘤：将30只BALB/c-nu裸鼠随机分为三组，使用慢病毒感染MDA-MB-231细胞即sh-LINC01614-1、sh-LINC01614-2、sh-Control三组，将1×107个每组细胞混匀，分别接种于小鼠右侧腋下，并在SPF下饲养，待肉眼可见瘤体，每隔2天记录裸鼠体重，采用游标卡尺测量瘤体的长径(L)和短径(W)，计算肿瘤体积(V＝L×W2/2)。18天后，麻醉下处死裸鼠，剥离瘤体拍摄图像。
裸鼠肺乳腺癌转移模型构建：分组同上，将1×107个癌细胞通过尾静脉注射雌性BALB/c-nu裸鼠，接种细胞2周造模成功后，麻醉下处死裸鼠，剥离肺组织观察转移灶情况并拍摄图像。
经测量皮下瘤的体积与重量发现，LINC01614低表达可明显的抑制肿瘤的形成，与对照组相比sh-LINC01614-1组和sh-LINC01614-2组瘤体积明显减小(P<0.05)，见图5；此外，在裸鼠乳腺癌肺转移模型中发现，LINC01614低表达可显著降低肺部转移灶的形成(P<0.05)，见图6，表明其可抑制肿瘤细胞的远处转移。
以上已对本发明创造的较佳实施例进行了具体说明，但本发明创造并不限于所述实施例，熟悉本领域的技术人员在不违背本发明创造精神的前提下还可做出种种的等同的变型或替换，这些等同的变型或替换均包含在本申请权利要求所限定的范围内。
序列表
<110> 浙江中医药大学
<120> 一种沉默人LINC01614表达的shRNA分子及其应用
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Claims (4)

1.一种沉默人LINC01614表达的shRNA分子的靶标基因，其特征在于，所述沉默人LINC01614表达的shRNA分子靶标基因如SEQ ID N0.1和SEQ ID N0.2所示。
2.一种沉默人LINC01614表达的shRNA分子，其特征在于包括shRNA-1和/或shRNA-2，所述shRNA-1的正义链核苷酸序列如SEQ ID N0.3所示，所述shRNA-1的反义链核苷酸序列如SEQ ID N0.4所示，所述shRNA-2的正义链核苷酸序列如SEQ ID N0.5所示，所述shRNA-2的反义链核苷酸序列如SEQ ID N0.6所示。
3.如权利要求2所述的一种沉默人LINC01614表达的shRNA分子在制备用于抑制人乳腺癌细胞MDA-MB-231和Hs578T转移和侵袭药物中的应用。
4.如权利要求2所述的一种沉默人LINC01614表达的shRNA分子在制备用于抑制乳腺癌转移和侵袭药物中的应用。
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