CN103451222A - 稳定沉默pkm2基因表达的结肠癌细胞株的制备方法和应用 - Google Patents
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Abstract
本发明提供了一种稳定沉默PKM2基因表达的结肠癌细胞株的制备方法，包括人PKM2基因的shRNA慢病毒表达载体的构建，慢病毒的包装、收获，DLD1结肠癌细胞慢病毒感染，稳定细胞株嘌呤霉素筛选和Real-time PCR、Western blot鉴定。实验结果证明，shRNA寡核苷酸序列可成功插入pLKO.1-puro表达载体中，且对PKM2基因表达的抑制效果显著、持续且稳定。制得的细胞株为研究PKM2在大肠癌细胞能量代谢、增殖、迁移和炎症等恶性特征中的调控作用提供实验材料。
Description
技术领域
本发明涉及基因工程技术领域，具体是一种稳定沉默PKM2基因表达的结肠癌细胞株的制备方法，以及得到的细胞株在研究PKM2调控大肠癌细胞能量代谢、增殖、迁移和炎症等恶性特征中的应用。
背景技术
葡萄糖代谢异常是肿瘤细胞的一个重要特征。肿瘤细胞即使在有氧存在的情况下，也主要通过糖酵解而不是氧化磷酸化进行代谢，消耗大量葡萄糖并产生乳酸，即“Warburg效应”。越来越多的研究认为，糖酵解过程中产生的许多中间产物能够被肿瘤细胞所用，以合成蛋白质、核酸及脂类等生物大分子，为肿瘤细胞的生长和增殖提供必需的物质基础。
丙酮酸激酶（pyruvate kinase,PK）是糖酵解途径中一个重要的限速酶，催化磷酸烯醇式丙酮酸向丙酮酸转化。在哺乳动物细胞中，存在PKM1、PKM2、PKL和PKR四种同工酶，其中PKL和PKR由相同的基因PKL编码，通过不同的组织特异性启动子而分别表达于肝脏和红细胞中；PKM1和PKM2由PKM基因编码，是前体mRNA经不同选择性剪切的产物，PKM1包含外显子9，主要表达于骨骼肌和脑组织；PKM2包含外显子10，主要表达于胚胎组织以及增殖的细胞，尤其是肿瘤细胞中。
研究表明，肿瘤细胞特异性高表达PKM2在促进肿瘤细胞“Warburg效应”中发挥重要作用。PKM2可以通过调控糖酵解、磷酸戊糖和丝氨酸合成途径为肿瘤细胞提供大量增殖所必须的中间产物。此外，PKM2还具有重要的信号转导功能，它参与调控β-catenin、HIF-1α、NF-κB等重要信号促进肿瘤细胞对微环境的适应。然而，PKM2是否对肿瘤其它恶性特征发挥作用目前还不清楚。本发明通过慢病毒转染获得PKM2稳定低表达细胞株，为进一步探讨PKM2在肿瘤细胞能量代谢及其它恶性特征中的调控作用提供了有力工具。
发明内容
本发明的目的是提供一种稳定沉默PKM2基因表达的DLD1稳定细胞株，为研究PKM2在大肠癌细胞能量代谢、增殖、迁移和炎症等恶性特征中的调控作用提供实验材料。
本发明提供一种稳定沉默PKM2基因表达的DLD1稳定细胞株的制备方法，包括如下步骤：
（1）shRNA寡核苷酸序列的设计：根据PKM2基因的mRNA全序列，经Blast同源性比对证实特异性后应用RNA Structure4.4软件对靶mRNA序列的二级结构进行评估得到靶核苷酸序列，设计并合成靶向PKM2基因的shRNA寡核苷酸序列：SEQ ID NO：1，SEQ ID NO：2；
（2）shRNA慢病毒表达载体的构建和鉴定：将合成的shRNA寡核苷酸：SEQ ID NO：1、SEQ ID NO：2等量混合，退火形成双链的shRNA，同时双酶切pLKO.1-puro载体，电泳，切胶回收载体，用T4DNA连接酶将双链shRNA连接到载体pLKO.1-puro上，转化大肠杆菌感受态，克隆扩增获得pLKO.1-puro-shPKM2重组质粒，重组质粒经双酶切、DNA测序鉴定；
（3）DLD1稳定细胞株的构建与筛选：培养HEK-293T细胞，并接种到100cm的培养皿中，待细胞长到70%时，pLKO.1-puro-shPKM2重组质粒与慢病毒包装质粒pMD2.G、psPAX2以质量比4：3：1共转染HEK-293T细胞；培养48h、72h后分别收集含病毒的上清培养基，合并后用于感染生长状态良好的DLD1细胞，用浓度为6μg/mL的puromycin筛选出单细胞克隆，并扩大培养，采用Real-time PCR和Western blot技术分别从mRNA和蛋白水平验证PKM2基因的抑制效果。
实验结果证明本发明提供的shRNA寡核苷酸序列可成功插入pLKO.1-puro表达载体中，且对PKM2基因表达的抑制效果显著、持续且稳定。相比对照细胞，本发明得到的稳定沉默PKM2基因表达的结肠癌细胞株中谷氨酰胺代谢相关酶及受体的基因表达量均上升，说明沉默PKM2基因的表达引起了细胞谷氨酰胺的代偿作用。
附图说明
图1为重组质粒pLKO.1-puro-shPKM2双酶切鉴定结果示意图。
图2为嘌呤霉素筛选细胞后荧光定量PCR检测结果示意图。
图3为嘌呤霉素筛选细胞后Western Blot检测结果示意图。
图4为稳定细胞株谷氨酰胺代谢相关酶及受体的基因表达的检测结果示意图。
图5为MTT法检测稳定细胞株谷氨酰胺的代偿作用的结果示意图。
具体实施方式
下面结合附图与具体实施例对本发明做进一步说明。
实施例1
（1）靶向PKM2基因的shRNA寡核苷酸序列的设计
在GenBank查找到PKM2基因的mRNA全序列（NM-001206796），经Blast同源性比对证实特异性后应用RNA Structure4.4软件对靶mRNA序列的二级结构进行评估，最后筛选得到一条21nt靶核苷酸序列：SEQ ID NO：5，CGGGTGAACTTTGCCATGAAT，靶点位置为946-966。
根据靶核苷酸序列设计合成一条shRNA的DNA模板单链，命名为shRNA1，另外再设计一对对照序列shRNAc，具体序列见表1。设计的shRNA寡核苷酸链由上海英骏生物技术有限公司合成。
表1 靶向PKM2基因的shRNA寡核苷酸序列
（2）shRNA慢病毒表达载体的构建
将合成的shRNA寡核苷酸单链SEQ ID NO：1、SEQ ID NO：2等量混合，退火形成双链的shRNA，提取pLKO.1-puro质粒，用限制性内切酶Age I、EcoR I双酶切，电泳，切胶回收载体，再用T4DNA连接酶将双链shRNA1、shRNAc连接到载体pLKO.1-puro上，形成重组质粒pLKO.1-shRNA1、pLKO.1-shRNAc；将连接产物转化感受态细胞DH5α，分别涂布于含Amp抗性的LB培养基的平板上，37℃恒温箱培养过夜。分别挑取3个单菌落于20μL培养液中，各取2μL菌液作为模板，进行菌液PCR鉴定，将PCR产物正确的菌液于LB培养基（含100μg/mL氨苄青霉素）中扩大培养，提取质粒后进行Nco I、EcoR I双酶切鉴定，结果如图1所示，阳性重组菌送至上海英骏生物技术有限公司测序。取测序正确的阳性重组菌液20μL接种到15mL LB培养基中，37℃，200rpm培养16h，用Endo-Free Plasmid MaxiKit（Omega公司）抽提重组质粒，测其纯度和浓度。
（3）慢病毒包被
将慢病毒包装质粒psPAX2和pMD2.G的穿刺菌接种到LB培养基中扩大培养，并用Endo-Free Plasmid Maxi Kit抽提质粒psPAX2和pMD2.G，检测纯度和浓度。
培养HEK-293T细胞，取生长状态良好的细胞接种到100cm的培养皿中，待细胞长到70%时，利用磷酸钙法将提取的去内毒素重组质粒和慢病毒包装质粒psPAX2、pMD2.G按质量比为4:3:1共转染HEK-293T细胞，培养48h、72h后分别收集含病毒的上清培养基于无菌离心管中合并，用除菌的浓缩管浓缩病毒液用于转导结肠癌细胞DLD1。
（4）慢病毒转导结肠癌细胞DLD1
培养结肠癌细胞DLD1，取生长状态良好的细胞接种到60cm的培养皿中，待细胞长到50%时，加入含终浓度为8μg/mL的polybrene的新鲜培养基，然后加入病毒液。培养24h后，弃去含病毒的培养液，加入新鲜的培养基，用终浓度为6μg/mL的嘌呤霉素筛选细胞，挑出单克隆，并将生长状态良好的单克隆扩大培养。
（5）荧光定量PCR检测PKM2基因的表达量
根据PKM2和GAPDH基因mRNA序列，利用引物设计软件Primer5.0和Oligo7.0设计引物，如表2所示。
表2 荧光定量PCR引物序列
分别接种DLD1细胞及各沉默细胞至24孔板，待细胞密度为80%～90%时，提取各组细胞的总RNA，利用PrimeScrip RT reagent Kit将mRNA逆转录成cDNA，逆转录条件为：37℃，15min，85℃，5s，4℃，∞。反转录结束后，加入90μL的RNase Free dH2O稀释cDNA，-20℃保存备用。取各组细胞的cDNA2μL作为模板，以GAPDH为内参，荧光定量PCR检测PKM2基因的相对表达量，设置反应条件：94℃30s，一个循环；94℃5s；60℃34s，40个循环。结果如图2所示，从PKM2的mRNA相对表达量来看，shRNA1的沉默效果可高达82%。
（6）Western blot检测PKM2蛋白的表达量
取DLD1细胞及沉默细胞各一瓶，弃培养基，用预冷的PBS洗三次，用细胞刮收集细胞并离心，依细胞量加入适量细胞裂解液，于冰上放置30min后，4℃，13000rpm，离心20min，取上清，采用BCA法测蛋白浓度。
统一取60μg蛋白加入5×SDS-PAGE Loading Buffer，100℃变性5min，进行10%SDS-PAGE凝胶电泳，将蛋白电转至PVDF膜上，5%脱脂奶粉封闭2h；分别加入PKM2抗体、GAPDH抗体，4℃孵育过夜，TBST洗膜3次，每次10min，再加入二抗，室温孵育2h，TBST洗膜3次，加入Western Blot化学发光试剂后进行成像分析。结果如图3所示，shRNA1的PKM2的蛋白表达量显著下降。
实施例2荧光定量PCR检测谷氨酰胺代谢相关酶及受体的基因的表达情况
采用实施案例1（5）中的各cDNA作为模板，以GAPDH为内参，荧光定量PCR检测谷氨酰胺代谢相关酶的基因的相对表达量，设置反应条件：94℃30s，一个循环；94℃5s；60℃34s，40个循环。结果如图4所示，相比shRNAc细胞，shRNA1细胞中谷氨酰胺代谢相关酶及受体的基因表达量上升，说明沉默PKM2基因的表达引起了细胞谷氨酰胺的代偿作用。
实施例3MTT法（噻唑蓝比色法）检测稳定细胞株的谷氨酰胺代偿作用
分别接种生长状态良好的shRNAc细胞及shRNA1细胞8×103个/孔至96孔板中，于37℃培养箱孵育24h后，加入终浓度为0.5mM的Don（谷氨酰胺酶抑制剂），设五个复孔，并用不加药的培养基作为对照。待药物作用48h后，每孔加5mg/mL MTT溶液20μL，继续孵育4h后终止培养。小心吸弃孔内培养上清液，每孔加150μL DMSO，振荡10min，使紫色结晶物充分溶解。在酶联免疫检测仪上于570nm波长，测定各孔光吸收值(A值)，结果如图5所示，shRNA1细胞的存活率要低于对照细胞，这说明PKM2对肿瘤细胞的存活有一定的调控作用；且在Don处理后，shRNA1细胞的存活率相比对照细胞下降了22%，进一步说明shRNA1细胞引起了谷氨酰胺的代偿作用。
Claims (3)
1.一种稳定沉默PKM2基因表达的结肠癌细胞株的制备方法，其特征在于，包括以下步骤：
（1）根据PKM2基因的mRNA全序列，经Blast同源性比对证实特异性后应用RNAStructure4.4软件对靶mRNA序列的二级结构进行评估得到靶核苷酸序列，设计并合成靶向PKM2基因的shRNA寡核苷酸序列：SEQ ID NO：1，SEQ ID NO：2；
（2）将合成的shRNA寡核苷酸序列：SEQ ID NO：1、SEQ ID NO：2等量混合，退火形成双链的shRNA，同时双酶切pLKO.1-puro载体，电泳，切胶回收载体，用T4DNA连接酶将双链shRNA连接到载体pLKO.1-puro上，转化大肠杆菌感受态，克隆扩增获得pLKO.1-puro-shPKM2重组质粒，重组质粒经双酶切、DNA测序鉴定；
（3）培养HEK-293T细胞，并接种到培养皿中，待细胞长到70%时，pLKO.1-puro-shPKM2重组质粒与慢病毒包装质粒pMD2.G、psPAX2以质量比4：3：1共转染HEK-293T细胞；培养48h、72h后分别收集含病毒的上清培养基，合并后用于感染生长状态良好的DLD1细胞，用浓度为6μg/mL的puromycin筛选出单细胞克隆，并扩大培养即可。
2.如权利要求1所述的方法得到的稳定沉默PKM2基因表达的结肠癌细胞株。
3.如权利要求2所述的稳定沉默PKM2基因表达的结肠癌细胞株在研究PKM2调控大肠癌细胞能量代谢、增殖、迁移和炎症恶性特征中的应用。
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| CN109913498A (zh) * | 2017-12-12 | 2019-06-21 | 中国科学院大连化学物理研究所 | 低表达gfpt1的du145稳定细胞株及构建与应用 |
| CN109913498B (zh) * | 2017-12-12 | 2022-08-02 | 中国科学院大连化学物理研究所 | 低表达gfpt1的du145稳定细胞株及构建与应用 |
| CN112175997A (zh) * | 2020-09-08 | 2021-01-05 | 上海健康医学院 | 靶向外泌体pkm2改善非小细胞肺癌顺铂耐药性的应用 |
| CN114107293A (zh) * | 2021-10-26 | 2022-03-01 | 江苏大学 | 特异性抑制KSR1基因表达的shRNA及其应用 |
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| CN117431241B (zh) * | 2023-04-14 | 2024-03-22 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | 靶向LMP1的shRNA、载体及其应用 |
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