CN114410632A - 特异性抑制PTEN基因的shRNA及应用 - Google Patents
特异性抑制PTEN基因的shRNA及应用 Download PDFInfo
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- CN114410632A CN114410632A CN202210106401.XA CN202210106401A CN114410632A CN 114410632 A CN114410632 A CN 114410632A CN 202210106401 A CN202210106401 A CN 202210106401A CN 114410632 A CN114410632 A CN 114410632A
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Abstract
本发明提供了一种特异性抑制PTEN基因的shRNA及应用，属于基因工程和生物医药领域；在本发明中，提供了特异性抑制PTEN基因表达的shRNA序列shPTEN‑1、shPTEN‑2和shPTEN‑3；所述shRNA序列能够使卵巢癌细胞中PTEN基因PTEN mRNA下调，在非治疗与诊断目的的特异性沉默卵巢癌细胞中PTEN基因和制备促进卵巢癌细胞凋亡的基因药物中有着很好的应用。
Description
技术领域
本发明属于基因工程和生物医药领域，具体涉及一种特异性抑制PTEN基因的shRNA及应用。
背景技术
卵巢癌是妇科肿瘤中常见的恶性肿瘤，发病率仅次于子宫颈癌和子宫体癌。由于卵巢癌的早期症状不明显，且目前早期诊断技术还未成熟，多数被诊断出患有卵巢癌的患者都处于中晚期状态，导致治疗及预后效果极差。卵巢癌因其不同的病理分型而需要应用不同的治疗方案，标准的治疗方案包括手术切除结合基于铂、紫杉醇的化学疗法等进行综合治疗，但常规治疗方案效果并不尽如人意，术后的复发率高达70％，且复发后肿瘤大多对常用化疗药物产生耐药性，寻求一种高效、术后复发率低且不易引起肿瘤耐药性的治疗方法与手段已成为卵巢癌治疗领域亟待解决的热点与难题之一。大量研究表明导致卵巢癌高发病率和高死亡率的主要原因是该恶性肿瘤超强的增殖特征，因此寻找一种能够有效抑制卵巢癌细胞增殖的方法，是降低卵巢癌患者术后复发率，提高患者生存率的关键。
RNA干扰技术（RNA interference, RNAi）是高保守的基因防御机制，即依赖于双链RNA特异性短序列实现转录后基因沉默的技术。目前实验室常用的RNAi技术主要有siRNA寡核苷酸载体和shRNA慢病毒质粒表达载体。siRNA是短双链RNA，大小19-29 nt左右，3´端有两个游离碱基，可激活RNA干扰，通过与目标mRNA互补序列结合，特异性降解mRNA。但是siRNA在细胞中的表达是瞬时的，发挥作用的时间较短。相较于siRNA，shRNA可通过病毒介导的转导保持稳定，能够减少脱靶效应。shRNA包括两个短的反向重复序列，中间由茎环结构进行分割，组成一个类似发夹的结构。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RNA诱导的沉默复合物RISC，此复合物中含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，从而阻断了mRNA的遗传信息传递。
PTEN是迄今为止发现的第一个具有磷酸酶活性的抑癌基因，其编码的蛋白具有脂质磷酸酶和蛋白磷酸酶双重特异性磷酸酶活性。PTEN的功能与其亚细胞定位有关。在细胞质中，PTEN可与胞质靶点相结合调节细胞增殖、细胞周期进展、细胞凋亡、细胞粘附、迁移和侵袭，并通过对PI3K/Akt信号通路的拮抗作用发挥抑癌功能；而在细胞核中，PTEN则在染色体稳定性、DNA修复、细胞周期阻滞和细胞稳定性等方面发挥重要作用。此外，PTEN还参与DNA损伤修复，尤其是双链损伤修复（double-strand break repair，DSBR）。虽然PTEN在之前研究中都被看作抑癌基因，但近年来的研究发现，PTEN水平的降低或缺失可导致同源重组（homologous recombination，HR）缺陷。
PTEN缺失常见于胶质母细胞瘤、甲状腺癌、乳腺癌、子宫内膜癌、卵巢癌、前列腺癌、结直肠癌和黑色素瘤中，可显著影响癌症的发展和严重程度。大量研究表明，PTEN作为主要检查点和癌症调节剂，因此PTEN的突变和缺失与癌症的发生密切相关。由于PTEN是PI3K信号通路的拮抗因子，PTEN蛋白的表达或活性的重构可以减少PI3K介导的致癌信号，这在PTEN缺失的癌症中具有潜在的治疗价值。尽管PTEN在许多类型的癌症中扮演着重要的肿瘤抑制因子的角色，但在特定的情况下，PTEN抑制剂可能作为一种抗癌治疗新方法。
发明内容
针对现有技术中存在不足，本发明提供了一种特异性抑制PTEN基因的shRNA及应用。在本发明中，通过观察沉默PTEN基因对卵巢癌细胞的影响来判断沉默PTEN基因的shRNA序列的特异性，验证了可沉默PTEN基因的shRNA序列对卵巢癌细胞生长的影响。
本发明中首先提供了特异性抑制PTEN基因表达的shRNA，所述shRNA为shPTEN-1、shPTEN-2或shPTEN-3；
其中，shPTEN-1的序列为：
F:CCGGGTCTGCCAGCTAAAGGTGAAGATATACTCGAGTATATCTTCACCTTTAGCTGGCAGACTTTTTG（SEQ.ID.NO.1）；
R:AATTCAAAAAGTCTGCCAGCTAAAGGTGAAGATATACTCGAGTATATCTTCACCTTTAGCTGGCAGAC（SEQ.ID.NO.2）；
所述，shPTEN-2的序列为：
F:CCGGGCCGTTACCTGTGTGTGGTGATATCACTCGAGTGATATCACCACACACAGGTAACGGCTTTTTG（SEQ.ID.NO.3）；
R:AATTCAAAAAGCCGTTACCTGTGTGTGGTGATATCACTCGAGTGATATCACCACACACAGGTAACGGC（SEQ.ID.NO.4）；
所述，shPTEN-3的序列为：
F:CCGGGAGCGTGCAGATAATGACAAGGAATACTCGAGTATTCCTTGTCATTATCTGCACGCTCTTTTTG（SEQ.ID.NO.5）；
R:AATTCAAAAAGAGCGTGCAGATAATGACAAGGAATACTCGAGTATTCCTTGTCATTATCTGCACGCTC（SEQ.ID.NO.6）。
本发明中还提供了一种shRNA慢病毒表达载体，所述shRNA慢病毒表达载体包含上述shRNA。
本发明中还提供了上述shRNA或shRNA慢病毒表达载体在非治疗与诊断目的的特异性沉默卵巢癌细胞中PTEN基因中的应用。
进一步的，所述应用为使卵巢癌细胞中PTEN基因PTEN mRNA下调。
本发明中还提供了上述shRNA或shRNA慢病毒表达载体在制备促进卵巢癌细胞凋亡的药物中的应用。
本发明中还提供了一种促进卵巢癌细胞凋亡的药物，所述药物包含上述shRNA或shRNA慢病毒表达载体。
与现有技术相比，本发明的有益效果在于：
本发明基于PTEN基因的mRNA序列设计了3对用于构建shRNA的引物。将设计好的3对引物分别退火，形成双链；同时将载体pLKO.1-TRC在BamI和EcoRI酶切位点处进行酶切。分别用3种双链引物与酶切后的载体连接。连接产物转化大肠杆菌DH5α，并筛选出构建成功的shRNA慢病毒表达载体pLKO.1-TRC-shPTEN。
本发明中，将构建成功的shPTEN质粒与pLP1、pLP2、pLPSVG一起构成慢病毒包装系统，经细胞转导，shPTEN质粒被导入靶细胞细胞核内，且被RNA聚合酶催化转录得到初始前体。在被Exportin5转运到细胞质之前，这些初始前体需要首先用Drosha酶及双链RNA结合伴侣DGCR8加工形成pre-shRNA。pre-shRNA后被Dicer和TRBP/PACT酶切，去除发卡结构，产生双链siRNA。这一有活性的siRNA之后被整合到RISC，siRNA通过碱基互补配对以序列特异性的方式结合到靶mRNA，利用Ago2裂解靶mRNA，达到沉默PTEN基因的目的。
本发明中，将构建成功的shRNA慢病毒表达载体pLKO.1-TRC-shPTEN，用EZ trans将其与辅助质粒pLP1、pLP2和pLPSVG共同转入293FT细胞中，制备并收集病毒，之后用含目标shPTEN序列的慢病毒感染卵巢癌细胞株ES2和SKOV3，用puromycin筛选出具有抗性的细胞用于实验，通过实验发现shPTEN-1、shPTEN-2和shPTEN-3对ES2和SKOV3细胞中PTEN基因的表达有显著的沉默作用。shPTEN-1、shPTEN-2和shPTEN-3都能促进SKOV3细胞的凋亡。本发明提供的shRNA序列在未来卵巢癌的靶向基因药物研发和治疗中具有巨大的潜在价值。
附图说明
图1为shRNA慢病毒表达载体pLKO.1-TRC-shPTEN的电泳图；图中，M为DNA marker，1、3、5分别为pLKO.1-shPTEN-1、pLKO.1-shPTEN-2、pLKO.1-shPTEN-3，2、4、6分别为用Bam1和EcoR1两酶切后的3种pLKO.1-shPTEN质粒。
图2为shRNA慢病毒表达载体pLKO.1-TRC-shPTEN对SKOV3（a）及ES2（b）细胞中PTEN基因表达量的影响图；图中，“1”为对照组，用pLKO.1-TRC空载体转导；其他为实验组，“2”转导pLKO.1-shPTEN-1，“3”转导pLKO.1-shPTEN-2，“4”转导pLKO.1-shPTEN-3。**表示P＜0.01，***表示P＜0.001，n=3。
图3为重组质粒pLKO.1-TRC-shPTEN对SKOV3（a）及ES2（b）细胞中PTEN蛋白表达水平的抑制情况图；图中，“1”为对照组，用pLKO.1-TRC空载体转导；其他为实验组，“2”转导pLKO.1-shPTEN-1，“3”转导pLKO.1-shPTEN-2，“4”转导pLKO.1-shPTEN-3。“5”为PTEN蛋白，“6”为Actin蛋白。
图4为shPTEN促进SKOV3细胞凋亡的情况图（a）及统计图（b）；图中，“1”为对照组，用pLKO.1-TRC空载体转导；其他为实验组，“2”转导pLKO.1-shPTEN-1，“3”转导pLKO.1-shPTEN-2，“4”转导pLKO.1-shPTEN-3。***表示P＜0.001，n=3。
具体实施方式
下面结合附图以及具体实施例对本发明作进一步的说明，但本发明的保护范围并不限于此。
下列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：沉默PTEN基因表达的慢病毒质粒的构建
首先基于PTEN基因的mRNA序列（NCBI Reference Sequence: NM_000314.8）设计了3对shRNA引物，命名为shRNA-PTEN（3对shRNA分别记为shPTEN-1、shPTEN-2和shPTEN-3），其寡核苷酸链序列如表1所示。将这3对引物退火形成3条双链寡核苷酸短片段，连接到Bam1和EcoR1酶切的慢病毒载体Plko.1-TRC（购自Open Biosystems）上，得到三条连接产物。
表1. shRNA序列表
将上述得到的三条连接产物，即shRNA慢病毒表达载体pLKO.1-TRC-shPTEN，通过热激法转化到DH5α大肠杆菌感受态细胞（购自Vazyme）中，置于含氨苄抗性的LB固体培养基（含酵母提取物、蛋白胨、氯化钠、琼脂粉、氨苄西林）中，37 ℃培养16 h，挑取单个菌落于含氨苄抗性LB液体培养基（氨苄终浓度为50 μg/ml）中摇菌，37 ℃、220 rpm、16 h，然后收集菌体提取shRNA慢病毒表达载体pLKO.1-TRC-shPTEN，并对提取的shRNA慢病毒表达载体进行酶切、琼脂糖凝胶电泳验证。
图1即电泳验证图，图中M为DNA marker，1、3、5分别为pLKO.1-shPTEN-1、pLKO.1-shPTEN-2、pLKO.1-shPTEN-3，2、4、6分别为用Bam1和EcoR1两酶切后的3种pLKO.1-shPTEN质粒。从图中可以看出， pLKO.1-TRC-shPTEN重组质粒是由pLKO.1-TRC质粒连接了shPTEN而组成，酶切后的pLKO.1-TRC-shPTEN与完整重组质粒pLKO.1-TRC-shPTEN相比，在6000 bp以下有3条被切下的小片段，即可证明重组质粒pLKO.1-TRC-shPTEN构建成功。结果显示构建成功的慢病毒质粒将用于后续实验。
实施例2：细胞慢病毒的制备及慢病毒转染细胞
（1）试剂的准备：
A液的制备：2.25 μg pLP1（购自Thermo Fisher）+ 1.05 μg pLP2（购自ThermoFisher）+ 1.47 μg pLPSVG（购自Thermo Fisher）+1.83 μg目的质粒；在此分为对照组和实验组1、2、3；其中对照组的目的质粒是Plko.1-TRC（购自Open Biosystems），实验组的目的质粒分别是Plko.1-TRC-shPTEN-1，-2，-3。将A液加到250 μL无血清高糖DMEM（购自英潍捷基），轻轻混匀，室温孵育5 min，备用。
B液的制备：取18 μL EZ Trans细胞转染试剂（购自李记生物）加入到250 μL无血清高糖DMEM中，轻柔混匀，室温孵育5 min，备用。
（2）细胞慢病毒的制备：
将B液全部加入到配好的A液中，室温孵育20 min，形成带正电的转染复合物。在培养皿中消化293FT细胞（购自中国科学研究院），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，形成293FT细胞混悬液。这里我们用6 cm培养皿，含4 ml 293FT细胞混悬液。
将全部转染复合物轻轻滴入4 ml 293FT细胞混悬液中，轻轻摇晃，使混匀并铺平底部。将培养皿放置在培养箱中孵育12 h后，给293FT细胞换成新鲜培养基，继续培养。培养两天后，用灭菌注射器吸取细胞上清液，再经0.45 μm微孔滤膜过滤掉细胞上清杂质即可收集到病毒。
（3）慢病毒转染细胞：
用步骤（2）收集到的病毒分别转染ES2及SKOV3细胞（购自中国科学研究院），具体操作步骤如下：
第一天，种板：
数细胞，调整细胞密度为6 cm培养皿底面积的十分之一；
第二天，病毒感染：
拿出前一天种的细胞，弃上清，将病毒液与1640 + fcs培养基（购自英潍捷基）按体积比1：1混合加入，并加入终浓度为8 μg/mL的聚凝胺（polybrene），放入培养箱，孵育12h；孵育结束后，弃上清，给细胞换成1640 + fcs培养基，继续放入培养箱培养两天；培养两天后，弃上清，用1000 mg/mL 嘌呤霉素（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： PTEN RNA在ES2、SKOV3细胞中含量的检测：
收集实施例2中获得的具有抗性的ES2和SKOV3细胞，即病毒感染成功的细胞，用Vazyme提RNA试剂盒，提取病毒感染成功细胞中的RNA，并逆转录成cDNA（用Vazyme反转录试剂盒），利用定量PCR法（Vazyme染料法定量PCR检测试剂盒，ΔΔCT法计算表达量），检测相对PTEN RNA表达量。
图2为重组质粒pLKO.1-TRC-shPTEN对SKOV3（a）及ES2（b）细胞中PTEN基因表达量的影响图；图中，“1”为对照组，用pLKO.1-TRC空载体转导；其他为实验组，“2”转导pLKO.1-shPTEN-1，“3”转导pLKO.1-shPTEN-2，“4”转导pLKO.1-shPTEN-3。**表示P＜0.01，***表示P＜0.001，n=3。如图所示，成功转导pLKO.1-TRC-shPTEN病毒的SKOV3和ES2细胞中，PTEN RNA相比与对照组的含量更低，说明pLKO.1-TRC-shPTEN敲低了SKOV3和ES2细胞中的PTEN RNA。
实施例4： PTEN蛋白在ES2、SKOV3细胞中的表达水平的检测：
收集实施例2中获得的具有抗性的ES2和SKOV3细胞，即病毒感染成功的细胞，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1 h之后，一抗孵育过夜。一抗包括：PTEN抗体（购自Cell signaling）和Actin抗体（购自Sigma）。洗膜后，二抗孵育1 h。二抗包括：鼠二抗（购自Jackson Immuno Research）和兔二抗（购自JacksonImmuno Research）。洗膜后，将膜置于暗匣中拿到暗室曝光，曝光时将胶片固定在膜上，在发光液和显影液的作用下蛋白的条带会印迹在胶片上。
图3为重组质粒pLKO.1-TRC-shPTEN对SKOV3（a）及ES2（b）细胞中PTEN蛋白表达水平的抑制情况图；图中，“1”为对照组，用pLKO.1-TRC空载体转导；其他为实验组，“2”转导pLKO.1-shPTEN-1，“3”转导pLKO.1-shPTEN-2，“4”转导pLKO.1-shPTEN-3。“5”为PTEN蛋白，“6”为Actin蛋白。在Actin含量一致的情况下，2、3、4组的PTEN含量都比1组PTEN含量低，这说明了shPTEN-1 ~ -3可下调PTEN蛋白。
实施例5： shPTEN对SKOV3细胞细胞凋亡影响的检测：
（1）细胞消化：
在病毒转导且药筛结束后的细胞培养皿中，收集上清培养基中的凋亡细胞。然后将贴壁的SKVO3用PBS洗后，胰酶消化3 min，用含血清1640培养基终止消化，离心收集细胞沉淀。混合上清中的凋亡细胞及消化下来的贴壁细胞，用预冷 PBS 离心洗涤，收集全部细胞沉淀。
（2）流式分析：
用500 μL 1×Binding Buffer（购自Vazyme）重悬收集到的细胞沉淀，每管加入5μL Annexin V-FITC（购自Vazyme）和10 μL PI（购自Vazyme），轻柔涡旋混匀后，室温避光孵育15 min。进行流式分析：上机后圈出要分析的细胞群，调出FITC和PI两种荧光染料的检测通道，用流式软件中的十字门区分早期凋亡、晚期凋亡、坏死和活细胞，并进行统计。
图4为shPTEN促进SKOV3细胞凋亡的情况图（a）及统计图（b）；图中，“1”为对照组，用pLKO.1-TRC空载体转导；其他为实验组，“2”转导pLKO.1-shPTEN-1，“3”转导pLKO.1-shPTEN-2，“4”转导pLKO.1-shPTEN-3。***表示P＜0.001，n=3。从图中可以看出，对照组中，SKVO3早期凋亡占比约7.09%，晚期凋亡占比约0.84%。shPTEN-1早期凋亡占比约11.3%，晚期凋亡占比约20.0%。shPTEN-2早期凋亡占比约10.1%，晚期凋亡占比约20.6%。shPTEN-3早期凋亡占比约18.7%，晚期凋亡占比约18.4%。与对照组相比，shPTEN-1 ~ -3均可促进SKOV3发生凋亡。
综上所述，shPTEN可以显著促进SKOV3细胞凋亡，沉默PTEN基因对卵巢癌细胞SKOV3的凋亡有明显的促进作用。本发明设计的shRNA可以用于制备特异性沉默卵巢癌细胞的药物。
所述实施例为本发明的优选的实施方式，但本发明并不限于上述实施方式，在不背离本发明的实质内容的情况下，本领域技术人员能够做出的任何显而易见的改进、替换或变型均属于本发明的保护范围。
序列表
<110> 江苏大学
<120> 特异性抑制PTEN基因的shRNA及应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 68
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ccgggtctgc cagctaaagg tgaagatata ctcgagtata tcttcacctt tagctggcag 60
actttttg 68
<210> 2
<211> 68
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
aattcaaaaa gtctgccagc taaaggtgaa gatatactcg agtatatctt cacctttagc 60
tggcagac 68
<210> 3
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<212> DNA
<213> 人工序列(Artificial Sequence)
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ccgggccgtt acctgtgtgt ggtgatatca ctcgagtgat atcaccacac acaggtaacg 60
gctttttg 68
<210> 4
<211> 68
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
aattcaaaaa gccgttacct gtgtgtggtg atatcactcg agtgatatca ccacacacag 60
gtaacggc 68
<210> 5
<211> 68
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ccgggagcgt gcagataatg acaaggaata ctcgagtatt ccttgtcatt atctgcacgc 60
tctttttg 68
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<212> DNA
<213> 人工序列(Artificial Sequence)
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aattcaaaaa gagcgtgcag ataatgacaa ggaatactcg agtattcctt gtcattatct 60
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Claims (6)
1.一种特异性抑制PTEN基因表达的shRNA，其特征在于，所述shRNA为shPTEN-1、shPTEN-2或shPTEN-3；
其中，shPTEN-1的序列为：
F:CCGGGTCTGCCAGCTAAAGGTGAAGATATACTCGAGTATATCTTCACCTTTAGCTGGCAGACTTTTTG（SEQ ID NO.1）；
R:AATTCAAAAAGTCTGCCAGCTAAAGGTGAAGATATACTCGAGTATATCTTCACCTTTAGCTGGCAGAC（SEQ ID NO.2）；
所述，shPTEN-2的序列为：
F:CCGGGCCGTTACCTGTGTGTGGTGATATCACTCGAGTGATATCACCACACACAGGTAACGGCTTTTTG（SEQ ID NO.3）；
R:AATTCAAAAAGCCGTTACCTGTGTGTGGTGATATCACTCGAGTGATATCACCACACACAGGTAACGGC（SEQ ID NO.4）；
所述，shPTEN-3的序列为：
F:CCGGGAGCGTGCAGATAATGACAAGGAATACTCGAGTATTCCTTGTCATTATCTGCACGCTCTTTTTG（SEQ ID NO.5）；
R:AATTCAAAAAGAGCGTGCAGATAATGACAAGGAATACTCGAGTATTCCTTGTCATTATCTGCACGCTC（SEQ ID NO.6）。
2.一种shRNA慢病毒表达载体，其特征在于，所述shRNA慢病毒表达载体包含权利要求1所述shRNA。
3.权利要求1所述shRNA或权利要求2所述shRNA慢病毒表达载体在非治疗与诊断目的的特异性沉默卵巢癌细胞中PTEN基因中的应用。
4. 根据权利要求3所述的应用，其特征在于，所述应用为使卵巢癌细胞中PTEN基因PTEN mRNA下调。
5.权利要求1所述shRNA或权利要求2所述shRNA慢病毒表达载体在制备促进卵巢癌细胞凋亡的药物中的应用。
6.一种促进卵巢癌细胞凋亡的药物，其特征在于，所述药物包含权利要求1所述shRNA或权利要求2所述shRNA慢病毒表达载体。
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