CN111718959B - 一种rbm8a基因影响胶质母细胞瘤迁移和侵袭的分子机制及预警应用 - Google Patents

一种rbm8a基因影响胶质母细胞瘤迁移和侵袭的分子机制及预警应用 Download PDF

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CN111718959B
CN111718959B CN202010486774.5A CN202010486774A CN111718959B CN 111718959 B CN111718959 B CN 111718959B CN 202010486774 A CN202010486774 A CN 202010486774A CN 111718959 B CN111718959 B CN 111718959B
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rbm8a
glioblastoma
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邹东华
陈子龙
张春丽
林燕
李荣杰
胡北泉
覃刚
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First Peoples Hospital of Nanning
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Abstract

本发明涉及了一种RBM8A基因影响胶质母细胞瘤迁移和侵袭的分子机制及预警应用。1)Western blot分析胶质母细胞瘤细胞系RBM8A表达，2)慢病毒介导敲除RBM8A基因对胶质母细胞瘤细胞迁移和侵袭的影响，3)过表达RBM8A基因对胶质母细胞瘤细胞迁移和侵袭的影响，4)Western blot分析检测干扰RBM8A基因表达后的细胞系中Notchl、磷酸化STAT3(p‑STAT3)、总STAT3(STAT3)、磷酸化H3(p‑H3)、总H3(H3)和肌动蛋白，发现Notch/STAT3通路是RBM8A介导的胶质母细胞瘤细胞迁移和侵袭的机制。本发明提供了一种胶质母细胞瘤治疗的新思路，为研究胶质母细胞瘤药物治疗靶点提供了新途径。

Description

一种RBM8A基因影响胶质母细胞瘤迁移和侵袭的分子机制及 预警应用
技术领域
本发明属于基因工程技术领域，它涉及一种干扰RBM8A基因表达引起胶质母细胞瘤迁移和侵袭的分子机制及预警方法。
背景技术
胶质母细胞瘤是一种内在的脑肿瘤，可能发生在任何年龄，并被认为是由于影响神经胶质干细胞或祖细胞基因改变而引起的[Weller M, Wick W,Aldape K,et al.Glioma[J].Nature Reviews Disease Primers, 2015:15017.]。高侵袭性及预后不佳是胶质母细胞瘤的特征，尽管在治疗胶质母细胞瘤上取得了重大进展，但胶质母细胞瘤患者的生存率并没有得到改善[Quinn,T,Ostrom,et al.CBTRUS Statistical Report: Primary Brainand Central Nervous System Tumors Diagnosed in the United States in 2008-2012.[J].Neuro Oncology,2015.]。因此，寻找介导胶质母细胞瘤侵袭的机制将会为治疗胶质母细胞瘤提供新的见解。 RNA结合基序蛋白8A(RBM8A)是一个无义介导的mRNA降解(NMD)因子，RBM8A基因在细胞质和细胞核中大量表达,它有助于调节RNA转录、翻译、调节细胞周期和凋亡[Salicioni A M,Xi M, Vanderveer L A,et al.Identification andStructural Analysis of Human RBM8A and RBM8B:Two Highly Conserved RNA-BindingMotif Proteins That Interact with OVCA1,a Candidate Tumor Suppressor[J].Genomics,2000,69(1):0-62.]，其中NMD功能异常也会促进肿瘤生长和侵袭[The UPF1 RNAsurveillance gene is commonly mutated in pancreatic adenosquamous carcinoma[J].Nature Medicine,2014, 20(6):596-598.]。RBM8A是外显子连接复合物(EJC)的核心因子，并且EJC是真核生物的转录后调控网络中的一个节点。RBM8A在几种肿瘤中均异常表达，它能与转录因子STAT3结合，促进其DNA结合，从而上调靶基因的表达[Muromoto R,TairaN,Ikeda O,et al.The exon-junction complex proteins,Y14 and MAGOHregulate STAT3activation[J].Biochemical&Biophysical Research Communications,2009,382(1):0-68.]。然而，RBM8A影响胶质母细胞瘤细胞迁移和侵袭的机制尚未明确。
综上所述，目前极少研究分析RBM8A促进胶质母细胞瘤细胞迁移和侵袭的机制。因此我们的研究团队以人T98G和U251为细胞来源，建立了自己的实验技术平台及方法，进一步优化了RBM8A序列设计构建慢病毒质粒的方法，并鉴定其对胶质母细胞瘤迁移和侵袭的影响，通过在Transwell、Matrigel-Transwell实验过程中优化其干扰胶质母细胞瘤迁移和侵袭的功能，并取得了可喜的研究成果，为RBM8A应用于基因工程、肿瘤学及在临床上胶质母细胞瘤的诊断指标和治疗靶标提供可靠的理论依据。解决现存的问题将会为RBM8A在胶质母细胞瘤进展中的作用提供了新的见解，并有助于确定该疾病的潜在治疗靶点。
发明内容
针对现存的技术问题，本发明提供了一种干扰RBM8A基因表达引起胶质母细胞瘤迁移和侵袭的分子机制及预警方法。
本发明是这样实现的，一种影响RBM8A基因表达引起胶质母细胞瘤迁移和侵袭的分子机制及预警方法包括：
第一步：Westernblot分析胶质母细胞瘤细胞系RBM8A表达；
第二步：慢病毒介导RBM8A敲除；
第三步：慢病毒介导RBM8A过表达；
第四步：Transwell、Matrigel-Transwel检测干扰RBM8A基因表达后，胶质母细胞瘤细胞的迁移和侵袭情况观察；
第五步：Westernblot分析检测干扰RBM8A基因表达后的细胞系中的Notchl、磷酸化STAT3(p-STAT3)、总STAT3(STAT3)、磷酸化 H3(p-H3)、总H3(H3)和肌动蛋白，发现Notch/STAT3通路是RBM8A 影响的胶质母细胞瘤细胞迁移和侵袭的机制。
Westernblot分析的方法包括：
用兔抗人RBM8A抗体和小鼠抗β-肌动蛋白抗体。用RIPA缓冲液溶解组织，其中含有蛋白酶抑制剂。将裂解物样品在 8-12％SDS-PAGE凝胶上分离，转移到聚偏氟乙烯膜上，在4℃下与初级抗体孵育过夜，然后与辣根过氧化物酶结合的二级抗体孵育。一个ECL试剂盒用于检测结合抗体。
根据引物设计原则，设计3个能够特异敲除RBM8A的引物， RBM8A-shRNA：5‘-AGAGCATTCACAAACAACTGAA-3’(RBM8A-KD1)和5‘-CATCAGCGTT GGTGTGTGT-3’ (RBM8A-KD2)。制备重组RBM8A-shRNA慢病毒和阴性对照(N C) 慢病毒。在U251-MG细胞感染敲除RBM8A基因的慢病毒载体 (RBM8A-KD)，获得稳定感染敲除RBM8A表达的细胞系和对照细胞系。
Transwell和Matrigel-Transwell方法包括：
细胞被镀在Transwell分析插入物的上室，在无血清RPMI1640 培养基中加入Matrigel基质液并加入下室。用FBS清洗。24小时后用无菌棉签擦洗插入物的顶层，以去除剩余的细胞。用0.1％结晶紫染色底层侵入细胞1小时，用数字显微镜检查、计数和成像。对每个腔室的五个随机视野的细胞数进行计数和平均。
综上所述，本发明的优点及积极效果在于：
RBM8A被证实是EJC的核心因子，有助于调节RNA转录、翻译、调节细胞周期和凋亡。本发明提供一种影响RBM8A基因表达引起胶质母细胞瘤增殖的分子机制及预警方法。为研究胶质母细胞瘤药物治疗靶点提供了新途径。
附图说明
图1是本发明实施提供的影响RBM8A基因表达引起胶质母细胞瘤迁移和侵袭的分子机制及预警方法流程图
图2是本发明实施提供的敲除RBM8A后，胶质母细胞瘤的迁移和侵袭情况
图3是本发明实施提供的过表达RBM8A后，胶质母细胞瘤的迁移和侵袭情况
图4是本发明实施提供的Notch/STAT3通路参与RBM8A介导的胶质母细胞瘤细胞迁移和侵袭
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
现有技术中，RBM8A促进胶质母细胞瘤迁移和侵袭的具体机制尚不明确。为解决上述技术问题，下面结合具体分析对本发明作详细描述。对于RBM8A促进胶质母细胞瘤迁移和侵袭的具体机制尚未见明确报道。因此，本发明选择RBM8A基因为主要分析内容。本发明初步确定RBM8A在胶质母细胞瘤中表达，在此基础上，本发明拟通过如下实验进一步阐明RBM8A促进胶质母细胞瘤迁移和侵袭的具体机制：
人胶质母细胞瘤细胞系U251-MG用于构建敲除RBM8A基因的细胞系，人胶质母细胞瘤细胞系T98G用于构建过表达RBM8A基因的细胞系，观察干扰RBM8A基因表达后胶质母细胞瘤细胞迁移和侵袭情况。
本发明旨在从体外细胞水平和体内水平共同探索RBM8A促进胶质母细胞瘤迁移和侵袭的具体机制，确认RBM8A作为一种新型标志物在胶质母细胞瘤的迁移和侵袭的临床意义，也为今后以RBM8A为靶点设计药物，对胶质母细胞瘤增殖的靶向治疗提供依据。
下面结合具体方案对本发明作进一步描述。
如图1所示，本发明实施例提供的一种影响RBM8A基因表达引起胶质母细胞瘤迁移和侵袭的分子机制及预警方法包括：
1)Westernblot分析胶质母细胞瘤细胞系RBM8A表达
a.用兔抗人RBM8A抗体和小鼠抗β-肌动蛋白抗体。用RIPA缓冲液溶解组织，其中含有蛋白酶抑制剂。将裂解物样品在 8-12％SDS-PAGE凝胶上分离，转移到聚偏氟乙烯膜上，在4℃下与初级抗体孵育过夜，然后与辣根过氧化物酶结合的二级抗体孵育。一个ECL试剂盒用于检测结合抗体。
2)细胞学水平分析敲除RBM8A基因对胶质母细胞瘤细胞迁移和侵袭的影响：
a.根据引物设计原则，设计3个能够特异敲除RBM8A的引物， RBM8A-shRNA： 5‘-AGAGCATTCACAAACAACTGAA-3’(RBM8A-KD1)和 5‘-CATCAGCGTTGGTGTGTGT-3’(RBM8A-KD2)。制备重组 RBM8A-shRNA慢病毒和阴性对照(NC)慢病毒。在U251-MG细胞感染敲除RBM8A基因的慢病毒载体(RBM8A-KD)，获得稳定感染的敲除RBM8A细胞系和对照细胞系。
b.细胞被镀在Transwell分析插入物的上室，在无血清RPMI1640 培养基中加入Matrigel基质液并加入下室。用FBS清洗。24小时后用无菌棉签擦洗插入物的顶层，以去除剩余的细胞。用0.1％结晶紫染色1小时底层侵入细胞，用数字显微镜检查、计数和成像。对每个腔室的五个随机视野的细胞数进行计数和平均。
3)细胞学水平分析过表达RBM8A基因对胶质母细胞瘤细胞迁移和侵袭的影响：
a.利用pMSCV-IRES-GFP载体构建了标记RBM8A的表达质粒，并将该质粒或相应的空载体(NC)转染293T细胞。利用这些细胞的重组逆转录病毒多重感染感染T98G细胞，使细胞感染过表达RBM8A 基因的慢病毒载体(RBM8A-OE)，以获得稳定感染过表达RBM8A 基因的细胞系和对照细胞系。
b.细胞被镀在Transwell分析插入物的上室，在无血清RPMI1640 培养基中加入Matrigel基质液并加入下室。用FBS清洗。24小时后用无菌棉签擦洗插入物的顶层，以去除剩余的细胞。用0.1％结晶紫染色1小时底层侵入细胞，用数字显微镜检查、计数和成像。对每个腔室的五个随机场中的细胞数进行计数和平均。
4)干扰RBM8A基因表达后，对细胞系中的Notchl、磷酸化STAT3 (p-STAT3)、总STAT3(STAT3)、磷酸化H3(p-H3)、总H3(H3)和肌动蛋白进行Westernblot分析。
下面结合效果对本发明作进一步描述。
在本发明中，图2所示，在细胞水平敲除RBM8A基因可减少胶质母细胞瘤细胞的迁移和侵袭。
图3所示，在细胞水平过表达RBM8A基因可促进胶质母细胞瘤细胞的迁移和侵袭。
图4所示，干扰RBM8A基因表达后，对胶质母细胞瘤细胞系中Notchl、磷酸化STAT3(p-STAT3)、总STAT3(STAT3)、磷酸化H3(p-H3)、总 H3(H3)和肌动蛋白进行Westernblot分析，Notch/STAT3通路可能参与RBM8A影响胶质母细胞瘤迁移和侵袭的机制。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (4)

1.一种RBM8A基因影响胶质母细胞瘤迁移和侵袭的非疾病治疗目的的应用。
2.根据权利要求1所述的应用，其特征在于构建慢病毒载体介导RBM8A基因敲除。
3.根据权利要求1所述的应用，其特征在于构建慢病毒载体介导RBM8A基因过表达。
4.一种检测RBM8A基因表达量的物质在制备检测胶质母细胞瘤的产品中的应用。
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