CN113750218B - 多肽zc3h12a及其突变体在制备抗肝癌药物中的应用 - Google Patents

多肽zc3h12a及其突变体在制备抗肝癌药物中的应用 Download PDF

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CN113750218B
CN113750218B CN202110916954.7A CN202110916954A CN113750218B CN 113750218 B CN113750218 B CN 113750218B CN 202110916954 A CN202110916954 A CN 202110916954A CN 113750218 B CN113750218 B CN 113750218B
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CN113750218A (zh
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吴雨萌
沈爱国
岳燕萍
杨磊
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Nantong Tumor Hospital
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Abstract

本发明公开了多肽ZC3H12A及其突变体在制备抗肝癌药物中的应用，属于生物技术领域。本发明利用生物工程技术，将多肽ZC3H12A及突变体分别重组到GV347真核诱导表达载体，经酶切和序列分析证明重组成功后包装成诱导表达病毒，将此重组质粒转通过病毒染肝癌细胞Huh7中构建稳转细胞系，免疫印记检测蛋白的表达。免疫共沉淀实验结果证明，多肽ZC3H12A及其突变体ZC3H12AC157A与MYC mRNA具有直接相互作用，同时可以降解MYC mRNA。CCK8实验证明在肿瘤细胞中过表达ZC3H12A和ZC3H12AC157A可以降低MYC mRNA水平从而抑制肿瘤细胞的增殖。

Description

多肽ZC3H12A及其突变体在制备抗肝癌药物中的应用
技术领域
本发明属于生物技术领域，更具体地说，涉及多肽ZC3H12A及其突变体在制备抗肝癌药物中的应用。
背景技术
肝癌是世界全世界范围内常见的消化系统恶性肿瘤。根据2018年公布的数据，全球肝癌的年新发病例居于恶性肿瘤第6位，是第二大恶性肿瘤致死原因。靶向药物的研发一直是肝癌治疗的热点研究方向，随着靶向药物的开发及应用显著提高了肿瘤患者的生存时间。尽管如此，目前靶向药物仍旧满足不了临床的需求。
c-Myc(MYC)蛋白失调控是人类肿瘤发生的主要驱动因素，作为一种重要的转录因子，MYC与MAX的形成异源二聚体结合到靶基因启动子内的Ebox基序(CACGTG)结合，通过募集转录共激活子调控靶基因转录的启动、暂停释放和延伸，从而影响人类基因组中约15％的基因。在正常情况下MYC的蛋白和mRNA水平受到严格控制，但在大多数人类癌症中MYC变得失控和过度表达。MYC的过量表达可通过逆转录病毒启动子插入、染色体易位/扩增、MYC基因内超级增强子的激活和/或上游信号通路的突变来增强MYC的稳定性。在转基因小鼠模型中的研究表明，即使是短暂的MYC失活也会引起肝脏肿瘤的退化，这表明调控致癌的MYC可以用于治疗癌症患者。
以MYC为靶点的靶向药物研发一直是临床抗肿瘤药物研发的热点，由于转录因子一些固有的特性使直接靶向MYC蛋白设计药物非常困难，目前的研究策略集中在靶向MYC转录调控和靶向MYC翻译后调控层面，相关药物目前多属于临床前阶段。但目前尚无直接靶向降解MYC mRNA的药物。
ZC3H12A是一种锌指蛋白，最初被发现于经MCP-1处理的人外周血单核细胞中，具有转录因子、RNA酶、去泛素化酶等作用。
发明内容
针对现有技术存在的上述问题，本发明所要解决的技术问题在于提供多肽ZC3H12A及其突变体在制备抗肝癌药物中的应用。本发明所要解决的另一技术问题在于提供含有多肽ZC3H12A及其突变体的抗肝癌药物。
为了解决上述技术问题，本发明所采用的技术方案如下：
多肽ZC3H12A在制备抗肝癌药物中的应用。
所述的应用是构建含有编码所述多肽ZC3H12A的核酸的表达载体，将所述表达载体由病毒载体进行包装，作为降低肝癌细胞增殖能力的药物。
进一步地，所述的表达载体为GV347真核Tet on表达载体。
进一步地，所述的病毒载体为慢病毒载体。
一种抗肝癌药物，是一种包装有表达载体的病毒载体，所述表达载体含有编码所述多肽ZC3H12A的核酸。
多肽ZC3H12AC157A在制备抗肝癌药物中的应用，所述多肽ZC3H12AC157A是多肽ZC3H12A的突变体。
所述的应用是构建含有编码所述多肽ZC3H12AC157A的核酸的表达载体，将所述表达载体由病毒载体进行包装，作为降低肝癌细胞增殖能力的药物。
进一步地，所述的表达载体为GV347真核Tet on表达载体。
进一步地，所述的病毒载体为慢病毒载体。
一种抗肝癌药物，是一种包装有表达载体的病毒载体，所述表达载体含有编码所述多肽ZC3H12AC157A的核酸。
相比于现有技术，本发明的有益效果为：
与现有技术相比，本发明利用生物工程技术，将一段599个氨基酸的多肽ZC3H12A及突变体分别重组到13.1kb的GV347真核Tet on表达载体，该载体转入细胞后使用四环素进行诱导表达，通过四环素浓度调控诱导蛋白的表达水平，载体经酶切和序列分析证明重组成功后包装成诱导表达病毒，将此重组质粒转通过病毒染肝癌细胞Huh7中构建稳转细胞系，免疫印记检测蛋白的表达。通过免疫共沉淀实验和CCK8实验表明，多肽ZC3H12A及其突变体ZC3H12AC157A能特异性的结合MYC mRNA，通过降解MYC mRNA抑制MYC下游信号通路的激活，起到抑制肝癌细胞增殖和抗肿瘤的作用，因此本发明将在肝癌靶向药物研发上具有重要的应用价值。
附图说明
图1是肝细胞癌组织中ZC3H12A和MYC mRNA RIP(RNA结合蛋白免疫沉淀)实验结果；
图2是多肽ZC3H12A及突变体的编码基因载体质粒图；
图3是多肽ZC3H12A及突变体重组真核表达质双酶切电泳鉴定结果图；
图4是ZC3H12A及突变体Huh7稳转细胞系鉴，不同浓度DOX(单位：ng/mL)诱导下ZC3H12A和GAPHD蛋白表达结果；
图5是肝癌细胞Huh7中过表达ZC3H12A及突变体与MYC mRNA RIP实验结果图；
图6是PCR实验检测ZC3H12A及ZC3H12AC157A降解MYC mRNA图；
图7是CCK8检测ZC3H12A及ZC3H12AC157A后对肝肿瘤细胞增殖的影响图；
图8是克隆形成实验检测ZC3H12A及ZC3H12AC157A对肝肿瘤细胞增殖的影响图。
具体实施方式
下面结合具体实施例对本发明进一步进行描述。实施列中未注明具体条件的实验方法，通常按照常规条件或按照制造商建议条件进行。
实施例1：肝细胞癌组织中ZC3H12A与MYC mRNA免疫共沉淀
材料：RNA结合蛋白免疫沉淀(RIP)试剂盒由Merck公司生产，ZC3H12A抗体由abcam公司生产，IgG由Thermo Fisher Scientific公司生产，逆转录试剂、定量聚合酶链式反应(qPRC)试剂由诺唯赞公司生产，MYC引物由生工公司合成，其序列为：上游5′-CCTGGTGCTCCATGAGGAGAC-3′；下游5′-CAGACTCTGACCTTTTGCCAGG-3′。
1.RIP实验
按照试剂盒说明书提取组织和细胞样品RNA，新鲜肝细胞癌组织切成小块后预冷的0.01M PBS洗涤3次后匀浆获得单细胞悬浮液，收集细胞后使用RIP裂解缓冲液裂解冰上裂解5分钟，随后贮藏于-80℃冰箱备用(对于培养的细胞，取3瓶T-75瓶细胞用10mL预冷的PBS洗涤2次后从每个烧瓶或平板上刮下细胞，转移到一个离心管中。在4℃下，离心收集细胞，用等体积完全RIP裂解缓冲液重悬细胞后吹打均匀，于冰上继续裂解5分钟后贮藏于-80℃冰箱备用)；吹打重悬磁珠至完全分散状态，吸取50μL磁珠转移至离心管中。向每个离心管加入0.5mL RIP洗涤缓冲液短暂涡旋，将离心管放在磁力架中弃去上清，重复洗涤两次后用100μL RIP洗涤缓冲液重悬。在离心管中加入约5μg ZC3H12A抗体或IgG，室温旋转孵育30分钟，0.5mL RIP洗涤缓冲液洗涤孵育好的磁珠两次。每个离心管加入900μLRIP免疫沉淀缓冲液。快速解冻RIP裂解产物，4℃，14000rpm，离心10分钟。取100μL上清液加至磁珠-抗体复合物中，免疫沉淀反应的最终体积将为1.0mL。取RIP裂解产物的上清液10μL，标记为“input”备用。将所有离心管放置在旋转器上，4℃下，孵育3h后短暂离心免疫沉淀反应离心，放在磁力架中，弃去上清液。取出离心管，每管中加入0.5mLRIP洗涤缓冲液，涡旋后把离心管放在磁力架中，弃去上清液重复此洗涤步骤6次。向每管中加入150μL蛋白酶K缓冲液重悬磁珠，在input样品管中加入107μL RIP洗涤缓冲液、15μL 10％SDS和18μL蛋白酶K，定容至总体积150μL。使所有离心管置于55℃下振荡孵育30分钟。离心管放在磁力架上，转移上清至新管并加入250μL RIP洗涤缓冲液。在每个离心管中加入400μL苯酚∶氯仿∶异戊醇，涡旋15秒，室温，14000rpm，离心10分钟，小心转移350μL水相至新管中，加入400μL氯仿后涡旋15秒，室温，14000rpm，离心10分钟。小心转移350μL水相至新管中，每管加50μL盐溶液I、15μL盐溶液II、5μL沉淀增强剂、850μL无水乙醇，混匀后-80℃下保存过夜沉淀RNA。4℃下14000rpm离心30分钟，小心弃去上清液用80％乙醇洗涤沉淀一次。4℃下14000rpm离心15分钟。小心弃去上清液，使沉淀自然风干重悬RNA沉淀于不含核糖核酸酶的10μL水中，将离心管置于冰上备用。
2.RNA逆转录
提取的RNA使用紫外分光光度计进行浓度测定，已下按照说明书操作，取1μg RNA加入RNase-free离心管随后加入RNase-free ddH2O至16μL，向管中再加入4μL 5×HiScriptIII qRT SuperMix，混匀后放于PCR仪器中，并以37℃，15min，85℃，5sec运行，以获取cDNA。
3.PCR检测MYC
cDNA使用紫外分光光度计进行浓度测定，已下按照说明书操作，在qPCR管中加入10μL AceQ Universal SYBR qPCR Master Mix、MYC上下游引物各0.4μL、1μg cDNA加入ddH2O至20μL。涡旋离心后放入PCR仪，按95℃5min；95℃10sec，60℃30sec，40循环；95℃10sec，60℃60sec，95℃15sec条件进行PCR扩增。
结果如图1所示：在肝癌组织中存在ZC3H12A和MYC mRNA的相互作用。
实施列2：ZC3H12A及ZC3H12A突变体诱导表达载体构建稳转株验证及诱导表达ZC3H12A及ZC3H12A突变体与MYC mRNA免疫共沉淀。
ZC3H12A及ZC3H12A突变体诱导表达载体构建及慢病毒包装委托吉凯基因公司进行完成，将病毒感染Huh7细胞构建稳转细胞系后使用四环素诱导蛋白表达，使用RIP试剂盒检测表达蛋白与MYC mRNA相互作用。
材料：ZC3H12A及其突变体ZC3H12A诱导表达病毒、RIP试剂盒、IgG由ThermoFisher Scientific公司生产、逆转录试剂、定量聚合酶链式反应(qPRC)试剂MYC引物、Huh7细胞，DMEM、FBS由gibico公司生产，四环素、Glutamax、Sodium Pyruvate Solution由Thermo Fisher Scientific公司生产，感染增强剂HitransGA由吉凯基因公司生产，免疫印迹试剂盒由上海雅酶生物科技有限公司生产。
1.诱导表达慢病毒的构建和鉴定
ZC3H12A及ZC3H12A突变体诱导表达病毒载体构建和鉴定委托吉凯基因公司完成，载体的质粒图谱如图2所示。
构建步骤如下：所使用载体均为GV347载体，其元件顺序为：TetIIP-MCS-EGFP-3FLAG-Ubi-TetR-IRES-Puromycin，克隆位点为AgeI/AgeI，使用AgeI酶切后琼脂糖凝胶电泳回收酶切载体，表达载体经酶切鉴定正确，结果如图3。分别使用对应的引物PCR扩增获取ZC3H12A全长或突变体目的基因片段，引物对应关系如下表所示：
ID Seq(5′-3′)
ZC3H12A-p1 AACCGTCAGATCGCACCGGCGCCACCATGAGTGGCCCCTGTGGAGAG
ZC3H12A-p2 CACCATGGTGGCGACCGGCTCACTGGGGTGCTGGGACTTGTAG
ZC3H12AΔ305-325-P1 AACCGTCAGATCGCACCGGCGCCACCATGAGTGGCCCCTGTGGAGAG
ZC3H12AΔ305-325-P2 CACCATGGTGGCGACCGGCTCACTGGGGTGCTGGGACTTGTAG
ZC3H12AC157A-P1 AACCGTCAGATCGCACCGGCGCCACCATGAGTGGCCCCTGTGGAGAG
ZC3H12AC157A-P2 GCACCATGGTGGCGACCGGCTCACTGGGGTGCTGGGACTTGTAG
ZC3H12AD225-226A-P1 AACCGTCAGATCGCACCGGCGCCACCATGAGTGGCCCCTGTGGAGAG
ZC3H12AD225-226A-P2 CACCATGGTGGCGACCGGCTCACTGGGGTGCTGGGACTTGTAG
将PCR产物交换入线性化表达载体转入大肠杆菌，挑去单克隆后经过测序验证。ZC3H12A及其突变体的序列如序列表中所示：SEQ ID NO.1：ZC3H12A；SEQ ID NO.2：ZC3H12AΔ305-325；SEQ ID NO.3：ZC3H12AD225/226A；SEQ ID NO.4：ZC3H12AC157A。
2.ZC3H12A及ZC3H12A突变体诱导表达Huh7细胞稳转株构建及验证
使用DMEM作为基培加入10％FBS、1％Glutamax、1％Sodium Pyruvate配制Huh7完全培养基，按2×104个/mL细胞悬液500μL接种24孔板中，37℃培养24h，至细胞汇合度为20-30％后更换为DMEM。分别加入空载病毒、ZC3H12A及ZC3H12A突变体病毒4μL每孔，HitransGA2μL每孔，37℃、5％CO2及饱和湿度的细胞培养箱中培养12-16h，更换为完全培养基，继续培养72h。分别加入浓度50、100、200、400、800ng/mL四环素继续培养24h诱导蛋白表达，使用免疫印迹方法检测ZC3H12A蛋白的表达，如图4，不同浓度四环素诱导下ZC3H12A及突变体蛋白表达水平的变化表明诱导表达稳转株构建成功。
3.过表达ZC3H12A及ZC3H12A突变体与MYC mRNA RIP实验
对过表达ZC3H12A及ZC3H12A突变体的细胞按照实施例1进行RIP实验。提取的RNA逆转录后qPCR检测MYC，结果如图5。只有ZC3H12A及ZC3H12AC157A突变体可以与MYC mRNA相互作用。
实施列3：ZC3H12A及ZC3H12AC157A突变体降解MYC mRNA
材料：ZC3H12A及ZC3H12A突变体稳转Huh7细胞、稳转空载Huh7细胞系、四环素，放线菌酮D，mRNA逆转录试剂盒、PCR扩增试剂、MYC引物，RNA提取试剂盒由诺唯赞公司生产。
方法：
ZC3H12A及ZC3H12A突变体稳转Huh7细胞及对照细胞系2×105接种于6孔板中，待细胞培养融合接近70％-80％时加入四环素诱导蛋白表达，24h后再加入放线菌素D处理细胞，1h后按照RNA提取试剂盒说明书操作收集细胞总RNA，使用qPCR检测MYC mRNA水平。结果如图6，只有ZC3H12A及ZC3H12AC157A突变体可以降解MYC mRNA。
实施列4：ZC3H12A及ZC3H12AC157A突变体抗肝癌功能检测
材料：ZC3H12A及ZC3H12AC157A突变体稳转Huh7细胞系、稳转空载Huh7细胞系、DMEM、完全培养基、四环素、细胞增殖及毒性检测试剂盒(CCK-8)上海弈杉公司提供，4％多聚甲醛、GIMSA染色液
方法：
1.CCK8实验证明ZC3H12A及ZC3H12AC157A突变体抑制肝癌细胞的增殖
在96孔板中接种ZC3H12A及ZC3H12AC157A突变体稳转Huh7细胞系和正常Huh7细胞系5000个每孔，细胞生长至70％-80％时加入四环素诱导细胞表达ZC3H12A及ZC3H12AC157A突变体蛋白，24h后每孔加入100μL含10μL CCK-8溶液的细胞完全培养基，放回培养箱中继续培养24h、48h、72h、96h。检测时拿出将酶标仪检测波长调整至450nm后检测96孔板吸光度。如图7所示，与对照组相比，ZC3H12A及ZC3H12AC157A突变体稳转Huh7细胞细胞增殖能力明显下降。
2.克隆形成实验证明ZC3H12A及ZC3H12A突变体抑制肝癌细胞的增殖
取对数生长期的稳转细胞，分别用0.25％胰蛋白酶消化并吹打成单个细胞，并把细胞悬浮在完全培养基中备用。细胞分别以每皿500个接种含10mL37℃预温培养基的皿中，并轻轻转动，使细胞分散均匀。置37℃5％CO2及饱和湿度的细胞培养箱中培养24h后更换含有200ng/mL四环素的完全培养基，继续培养。经常观察，当培养皿中出现肉眼可见的克隆时，终止培养。弃去上清液，用PBS小心浸洗2次。加4％多聚甲醛固定细胞5mL固定15分钟。然后去固定液，加适量GIMSA应用染色液染10～30分钟，然后用流水缓慢洗去染色液，空气干燥。将平皿倒置并在显微镜下拍照，结果如图8所示，与对照组相比过表达ZC3H12A及ZC3H12AC157A突变体的Huh7细胞克隆数目明显减少，ZC3H12AC157A突变体抑制Huh7细胞克隆能力最为明显。
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Claims (4)

1.多肽ZC3H12AC157A在制备抗肝癌药物中的应用；所述多肽ZC3H12AC157A的序列如SEQ IDNO .4所示。
2.根据权利要求1所述的应用，其特征在于，构建含有编码所述多肽ZC3H12AC157A的核酸的表达载体，将所述表达载体由病毒载体进行包装，作为降低肝癌细胞增殖能力的药物。
3.根据权利要求2所述的应用，其特征在于，所述的表达载体为GV347真核Tet on表达载体。
4.根据权利要求2所述的应用，其特征在于，所述的病毒载体为慢病毒载体。
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