CN113262305B - Coro1a基因作为靶点在制备抗肿瘤药物中的应用 - Google Patents
Coro1a基因作为靶点在制备抗肿瘤药物中的应用 Download PDFInfo
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Abstract
本发明公开了Coro1a基因作为靶点在制备抗肿瘤药物中的应用，Coro1a蛋白作为靶点在制备抗肿瘤药物中的应用，以及抗肿瘤药物。本发明经研究发现，敲除Coro1a基因之后能通过抑制TEXs产生，增强机体的抗肿瘤免疫，从而对肿瘤产生明显的治疗效果；将Coro1a基因过表达后能增加外泌体分泌。
Description
技术领域
本发明涉及生物技术领域，特别是涉及Coro1a基因作为靶点在制备抗肿瘤药物中的应用。
背景技术
外泌体是各种活细胞分泌的直径介于30-150纳米具有脂质双分子层结构的小囊泡。外泌体通过内体途径产生。质膜向内凹陷形成早期内体，早期内体相互融合并向内出芽，形成含有大量腔内囊泡的晚期内体/多囊泡体。之后，多囊泡体与质膜融合，释放腔内囊泡至胞外形成外泌体。
大量的研究证据表明，肿瘤细胞来源的外泌体(Tumor cell-derived exosomes，TEXs)与肿瘤的转移密切相关。TEXs能“驯化”骨髓细胞，使骨髓细胞获得促血管生成的能力，促进形成肿瘤前转移小生境。富含整合素的TEXs能够通过整合素粘附到特定的肿瘤转移器官，从而介导肿瘤细胞往特定的器官转移。TEXs的RNAs可活化肺泡上皮细胞的TLR3信号，促进对中性粒细胞的招募，从而营造了肺前转移小生境。申请人前期的研究也表明，TEXs通过膜表面HSP72/105活化树突状细胞(Dendritic cells，DCs)的TLR2/4信号，促进其分泌IL-6，随后IL-6作用于肿瘤细胞，促进肿瘤细胞MMP9的表达，从而介导肿瘤转移的发生(Shen YY,Guo DF,Weng LX,Wang SJ,Ma ZY,Yang YS,et al.Tumor-derived exosomeseducate dendritic cells to promote tumor metastasis via HSP72/HSP105-TLR2/TLR4pathway.Oncoimmunology 2017,6(12).)。此外，TEXs在肿瘤免疫抑制微环境的形成过程也具有重要作用。TEXs含有较高水平的FasL，可以通过活化Fas信号，诱导细胞毒性T细胞的大量凋亡(Abusamra AJ,Zhong Z,Zheng X,Li M,Ichim TE,Chin JL,et al.Tumorexosomes expressing Fas ligand mediate CD8+T-cell apoptosis.Blood Cells MolDis.2005,35(2):169-173.)。TEXs可通过下调NK细胞活化性NKG2D受体抑制NK细胞的抗肿瘤活性(Clayton A,Mitchell JP,Court J,Linnane S,Mason MD,Tabi Z.Human tumor-derived exosomes down-modulate NKG2D expression.JImmunol.2008,180(11):7249-7258.)。TEXs携带膜PD-L1，能够通过PD-L1/PD-1抑制抗肿瘤T细胞反应(1、Chen G,HuangAC,Zhang W,Zhang G,Wu M,Xu W,et al.Exosomal PD-L1 contributes toimmunosuppression and is associated with anti-PD-1response.Nature 2018,560(7718):382-386.；2、Poggio M,Hu TY,Pai CC,Chu B,Belair CD,Chang A,etal.Suppression of exosomal PD-L1 induces systemic anti-tumor immunity andmemory.Cell2019,177(2):414-427.)。TEXs可通过其含有的IL-6和TGF-β1分别抑制树突细胞的分化及成熟；另外，TEXs还可通过诱导调节性T细胞(Regulatory T cells,Tregs)及髓系来源的抑制性细胞(Myeloid-derived suppressor cells,MDSCs)，抑制M1型并促进M2型巨噬细胞介导肿瘤免疫抑制微环境的形成(Liu YF,Gu Y,Cao XT.The exosomes in tumorimmunity.Oncoimmunology 2015,4(9).)。因此，抑制TEXs生成能抑制肿瘤转移并有效激发机体抗肿瘤免疫。
发明内容
本发明提供了一种调控TEXs生成的新靶点及其在肿瘤治疗中的应用。
本发明首先提供了Coro1a基因作为靶点在制备抗肿瘤药物中的应用。优选的，所述的应用，所述抗肿瘤药物用于降低Coro1a基因的表达。
本发明又提供了Coro1a蛋白作为靶点在制备抗肿瘤药物中的应用。优选的，所述的应用，所述抗肿瘤药物用于抑制Coro1a蛋白的活性。
本发明又提供了一种抗肿瘤药物，为以下一种：
(1)具有降低Coro1a基因表达作用的siRNA或相应的shRNA；
(2)具有降低Coro1a基因表达作用的小分子药物；
(3)具有抑制Coro1a蛋白活性的小分子药物或抗体。
优选的，所述的抗肿瘤药物，为具有降低Coro1a基因表达作用的siRNA，siRNA序列的正反链分别为：
5’-UGACAGCUCUAUCCGGUAUUUdTdT-3’；
5’-AAAUACCGGAUAGAGCUGUCAdTdT-3’，在做干扰时只需要选择其中一条序列使用即可。
本发明还提供了Coro1a基因在非疾病治疗为目的的调控外泌体分泌中的应用。优选的，所述的应用，通过敲除Coro1a基因或者降低Coro1a基因表达来抑制外泌体分泌，或者通过Coro1a基因的过表达来提高外泌体分泌。
本发明经研究发现，敲除Coro1a基因之后能通过抑制TEXs产生，增强机体的抗肿瘤免疫，从而对肿瘤产生明显的治疗效果；将Coro1a基因过表达后能增加外泌体分泌。
附图说明
图1为体外分别在MC38和B16-F10细胞中敲低和过表达Coro1a；在HEK293细胞过表达Coro1a，检测相应细胞外泌体的分泌水平，其中，A：免疫印迹鉴定MC38和B16-F10细胞中Coro1a的敲低效果；B：BCA法鉴定通过梯度离心及超速离心提取的相同数量对照干扰(NCsiRNA)或Coro1a干扰(Coro1a siRNA)的MC38和B16-F10细胞分泌外泌体的蛋白量；C：为免疫印迹鉴定MC38和B16-F10细胞中Coro1a的过表达效果；D：为BCA法鉴定通过梯度离心及超速离心提取的相同数量对照或Coro1a过表达的MC38和B16-F10细胞分泌外泌体的蛋白量。E：为免疫印迹鉴定HEK293细胞中Coro1a的过表达效果；F：为BCA法鉴定通过梯度离心及超速离心提取的相同数量对照或Coro1a过表达的HEK293细胞分泌外泌体的蛋白量。***表示两者在p<0.001具有显著性差异(下同)，ns表示两者没有显著性差异(下同)。
图2为敲除Coro1a的MC38和B16-F10细胞株的建立及对其分泌外体水平的检测，其中，A：免疫印迹鉴定MC38-Coro1a-/-和B16-F10-Coro1a-/-细胞中Coro1a的敲除效果；B：为BCA法鉴定通过梯度离心及超速离心提取的相同数量MC38和MC38-Coro1a-/-细胞分泌外泌体的蛋白量及B16-F10和B16-F10-Coro1a-/-细胞分泌外泌体的蛋白量。
图3为Coro1a敲除肿瘤细胞肿瘤生长情况及与外泌体的关系，其中，A：为皮下接种MC38和MC38-Coro1a-/-细胞或B16-F10和B16-F10-Coro1a-/-细胞并回输及不回输相应外泌体的肿瘤小鼠的肿瘤大小；B：为皮下接种MC38和MC38-Coro1a-/-细胞或B16-F10和B16-F10-Coro1a-/-细胞并回输及不回输相应外泌体的肿瘤小鼠的生存时间；C：为皮下接种MC38和MC38-Coro1a-/-细胞或B16-F10和B16-F10-Coro1a-/-细胞的肿瘤小鼠第5天血清外泌体的含量。**表示两者在p<0.01具有显著性差异(下同)。
图4为流式分析皮下接种MC38和MC38-Coro1a-/-细胞的肿瘤小鼠第20天肿瘤输出淋巴结中相应CD8+T细胞亚群的比例。
具体实施方式
实施例1
一、体外分别在MC38细胞(小鼠结肠癌细胞)和B16-F10细胞(小鼠黑色素瘤细胞)中敲低和过表达Coro1a(小鼠Coro1a基因的Gene ID：12721)，检测相应细胞外泌体的分泌水平，实验步骤如下：
(1)设计小鼠Coro1a siRNA，
mCoro1a siRNA：5’-UGACAGCUCUAUCCGGUAUUUdTdT-3’，
mCoro1a siRNA：5’-AAAUACCGGAUAGAGCUGUCAdTdT-3’。
上述两条siRNA为互补链，使用时两条均可以使用。
(2)体外培养MC38，B16-F10细胞系，12孔板铺板2×105个/孔，使用干扰试剂Transfection Reagent Mirus(TransIT-TKO，货号MIR 2150)，按照试剂说明书标准操作步骤进行操作：细胞提前按照2×105个/孔接种于12孔板细胞培养板中，37℃，5％CO2培养箱常规培养过夜。第二天待细胞完全贴壁，且密度约为70％左右即可进行干扰。0.5ODmCoro1asiRNA：5’-UGACAGCUCUAUCCGGUAUUUdTdT-3’，62.5μl DEPC水溶解并分装使用。取1.5ml EP管，按照每个孔100μl 
(Thermo Fisher，货号：31985062)，分别加入5μl的Transfection Reagent Mirus和3.6μl siRNA(工作浓度为60nM)，混匀，室温静置20min。
(3)设置阴性对照组加入NC siRNA，其序列为：
F：5’-UUCUCCGAACGUGUCACGUdTdT-3’
R：5’-ACGUGACACGUUCGGAGAAdTdT-3’，
操作同上。
(4)逐滴加入对应的细胞培养孔，轻摇混匀。
(5)37℃细胞培养箱，培养48h。
(6)收取细胞，进行免疫印迹实验检测Coro1a的表达情况，以评估干扰效果。具体操作为：使用5×SDS使上清液蛋白变性，混匀，样本煮沸5min，上样进行电泳，90V 1h20min；低温转膜，PVDF膜覆盖相应的条带位置，330mA 90min；脱脂牛奶溶于PBST缓冲液配成5％封闭液室温封闭膜2h；按照抗体的稀释比例，使用5％BSA溶液作为溶剂，使用anti-Coro1a抗体(Abcam，货号：ab228635)，进行4℃孵育，水平摇床过夜；回收抗体，PBST缓冲液冲洗膜四次，每次10min；同样使用5％BSA溶液作为溶剂，按照一定的比例以及对应的一抗种属稀释二抗，二抗室温反应，水平摇床1h；回收二抗，PBST缓冲液冲洗膜四次，每次10min；曝光，使用曝光液(新赛美，货号：P10300A、P10300B)，Tanon 4500成像仪进行曝光。
(7)收集细胞培养上清，将培养上清一分为二，一部分按照300g，10min、2000g，20min、10000g，30min的梯度转速离心，之后取上清液，100000g，4℃离心1h，弃上清，300μl/管重悬，使用BCA Protein Assay Kit(Thermo Fisher，货号：23225)对获得的外泌体浓度进行测定。
(8)B16-F10和MC38细胞提前按照2×105个/孔接种于12孔板细胞培养板中，37℃，5％CO2培养箱常规培养过夜。第二天待细胞完全贴壁，且密度约为70％左右即可转染质粒进行过表达。取1.5ml EP管，按照每个孔50μl 150nM的氯化钠溶液，分别加入1μl质粒和2μl的转染试剂
(Polyplus Transfection，货号：101-10N)转染试剂，分别混匀，将50μl含有
的氯化钠溶液加入到50μl含有质粒的氯化钠溶液中，混匀，室温静置25min。转染4-6h后，换新鲜的完全培养基，过表达24h后即可进行后续处理。
(9)收取细胞，进行免疫印迹检测Coro1a蛋白的表达情况，以评估过表达效果，方法同(6)所述。
干扰组免疫印迹结果如图1A所示，Coro1a的干扰效果良好，Coro1a干扰之后，MC38和B16-F10细胞上清中的外泌体蛋白含量经BCA检测，酶标仪测量吸光度，以OD值计算其质量，可以观察到Coro1a干扰组的外泌体含量明显降低(图1B)。
过表达组免疫印迹结果如图1C所示，Coro1a的过表达效果良好，Coro1a过表达之后，MC38和B16-F10细胞上清中的外泌体蛋白含量经BCA检测，可以观察到Coro1a过表达组的外泌体含量明显升高(图1D)。
二、体外分别在HEK293细胞(人胚肾细胞)过表达Coro1a(人Coro1a基因的GeneID：11151)，检测相应细胞外泌体的分泌水平。实验步骤如下：
(1)HEK293细胞提前按照2×105个/孔接种于12孔板细胞培养板中，37℃，5％CO2培养箱常规培养过夜。第二天待细胞完全贴壁，且密度约为70％左右即可转染质粒进行过表达，方法同(8)所述。
(2)收取细胞，进行免疫印迹检测Coro1a蛋白的表达情况，以评估过表达效果，方法同(6)所述。收取相应上清，提取外泌体，使用BCA对获得的外泌体浓度进行测定。方法同(7)所述。
免疫印迹结果如图1E所示，Coro1a的过表达效果良好，Coro1a过表达之后，HEK293细胞上清中的外泌体蛋白含量经BCA检测，可以观察到Coro1a过表达组的外泌体含量明显升高(图1F)。
实施例2
构建敲除Coro1a的MC38和B16-F10细胞株及对其分泌外体水平的检测，实验步骤如下：
(1)使用CRISPR-Cas9系统，在MC38和B16-F10细胞中稳定表达Cas9和装载Coro1asgRNA的目的质粒，方法如下：设计sgRNA序列，sgRNA的编码序列为：
mCoro1a：5’-GTAGACAAGAACGTGCCCCTGG-3’；
MC38和B16-F10细胞3×105个/孔接种于12孔板细胞培养板中，37℃，5％CO2培养箱常规培养过夜。第二天取1.5ml EP管，按照每个孔50μl 150nM的氯化钠溶液，分别加入1μgCas9质粒和1μg目的质粒以及4μl的
转染试剂，分别混匀，将50μl含有
转染试剂的氯化钠溶液加入到50μl含有质粒的氯化钠溶液中，混匀，室温静置25min。转染4-6h后，换新鲜的完全培养基，过表达24h后即可收集细胞进行分选。
(2)使用流式细胞分选仪(Beckman moflo Astrios EQ)，获得阳性细胞，体外培养，获得单克隆细胞，经免疫印迹鉴定，得到MC38-Coro1a-/-和B16-F10-Coro1a-/-细胞系。
(3)收集细胞培养上清按照300g，10min；2000g，20min；10000g，30min的梯度转速离心，之后取上清液，100000g，4℃离心1h。
(4)弃上清，300μl/管重悬，使用BCA Protein Assay Kit(Thermo Fisher，货号：23225)对获得的外泌体蛋白浓度进行测定。
免疫印迹结果显示如图2A所示，Coro1a的敲除效果良好，Coro1a敲除之后，MC38-Coro1a-/-和B16-F10-Coro1a-/-细胞中的上清中的外泌体蛋白含量经BCA法检测，酶标仪测量吸光度，以OD值计算其质量，可以观察到Coro1a敲除组的外泌体含量明显降低(图2B)。
实施例3
以雌性SPF级C57BL/6为实验对象，利用MC38和MC38-Coro1a-/-细胞或B16-F10和B16-F10-Coro1a-/-小鼠细胞系构建荷瘤小鼠模型，采用ELISA法检测血清中CD9阳性外泌体的含量，实验步骤如下：
(1)皮下接种MC38和MC38-Coro1a-/-细胞或B16-F10和B16-F10-Coro1a-/-细胞，皮下注射1×106个细胞，从皮下接种为第0天计算，第1天开始回输对应外泌体，20μg/只/次，连续两周隔天回输MC38-EXO或MC38-Coro1a-/--EXO。第5天开始测量肿瘤体积，第5天取荷瘤小鼠外周血或保留小鼠观察生存率。
(2)取小鼠外周血于1.5ml EP管，室温静置2小时后，4℃过夜，第二天4000rpm，20min离心，吸取上清。
(3)以ELISA包被缓冲液为介质，抗CD63抗体(Purified anti-mouse CD63Antibody BioLegend，货号：143901)作为包被抗体，抗体终浓度是4μg/ml，4℃包被96孔酶标板过夜。
(4)含0.05％PBST洗板4遍后，用含10％胎牛血清的PBS缓冲液，室温封闭1h。
(5)PBST洗板4次后，加入100μl待检血清，37℃孵育过夜。
(6)PBST洗板4次后，以含10％胎牛血清的PBS缓冲液为介质，抗CD9抗体(Biotinanti-mouse CD9 Antibody BioLegend，货号：124803)为检测抗体，终浓度为4μg/ml，37℃孵育1h。
(7)PBST洗板4次后，加入100μl的Avidin-HRP(eBioscience，货号18-4100)室温孵育1h。
(8)PBST洗板6次后，加入0.3mg/ml的二甲基联苯胺底物室温反应15min。
(9)加入50μl 1N的H2SO4终止反应，450nm测量吸光度,间接评价血清外泌体的含量。
MC38和MC38-Coro1a-/-或B16-F10和B16-F10-Coro1a-/-肿瘤小鼠的生长和生存率曲线如图3A，3B所示,相比于MC38或B16-F10肿瘤小鼠，发现MC38-Coro1a-/-或B16-F10-Coro1a-/-荷瘤小鼠的肿瘤体积明显偏小(图3A)，其生存率也大幅提高(图3B)。此外，与MC38或B16-F10肿瘤小鼠相比，MC38-Coro1a-/-或B16-F10-Coro1a-/-荷瘤小鼠血清外泌体的水平明显降低(图3C)。
实施例4
依照实施例3方法构建MC38或MC38-Coro1a-/-小鼠荷瘤模型，流式分析皮下接种MC38和MC38-Coro1a-/-细胞的肿瘤小鼠第20天肿瘤输出淋巴结中相应CD8+T细胞亚群的比例，实验步骤如下：
(1)取腋下，腹股沟淋巴结，研磨，过滤膜，离心1500rpm，5min，破红液破红2min，2倍体积1640培养基中和，离心1500rpm，5min，PBS重悬。
(2)表染：膜蛋白，FITC-FVD(eBioscienc，货号：65-0863-14)，PB-CD45(BioLegend，货号：103126)，APC-CD3(BioLegend，货号：100235)，PerCP-CD8(eBioscience，货号：45-0081-82)，PE-PD1(BioLegend，货号：135205)，PE-Tim3(BioLegend，货号：119703)，室温避光30min，PBS洗两遍，200μl重悬上机。
(3)破膜：IC-fixcation buffer固定，200μl/test，室温避光20min；1×permebilication洗液清洗2次，离心，400g，5min，PE-GzmB(eBioscience，货号：12-8898-82)染色，4℃避光，30min；同上的方法清洗2次，200μl洗液，重悬上机。
(4)破核：破核液1ml，4℃避光1h。离心，400g，5min,弃上清，洗液补至100μl，PE-Ki67(BioLegend，货号：15120)染色，室温避光30min，洗液洗2次，200μl重悬上机。
流式检测结果如图4所示，与对照组相比，在MC38-Coro1a-/-荷瘤小鼠中，检测到相应CD8+T细胞耗竭标记减少，活化和增殖标记增加。
序列表
<110> 浙江大学
<120> Coro1a基因作为靶点在制备抗肿瘤药物中的应用
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Claims (1)
1.Coro1a基因在非疾病治疗为目的的调控外泌体分泌中的应用，
通过敲除Coro1a基因或者降低Coro1a基因表达来抑制外泌体分泌，或者通过Coro1a基因的过表达来提高外泌体分泌，
其中，Coro1a基因为人源，人Coro1a基因的Gene ID：11151。
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