CN114634984A - 一种脑胶质瘤生物标记物mlkl基因及其应用 - Google Patents
一种脑胶质瘤生物标记物mlkl基因及其应用 Download PDFInfo
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- CN114634984A CN114634984A CN202210115084.8A CN202210115084A CN114634984A CN 114634984 A CN114634984 A CN 114634984A CN 202210115084 A CN202210115084 A CN 202210115084A CN 114634984 A CN114634984 A CN 114634984A
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Abstract
本发明属于生物医药技术领域，具体为一种脑胶质瘤生物标记物MLKL基因及其应用。本发明研究表明：MLKL在脑胶质瘤中显著高表达；高表达的MLKL基因和蛋白与胶质瘤的不良预后相关；降低MLKL表达可明显抑制恶性胶质瘤细胞的生长；因而MLKL基因和其表达产物可作为预后评估和治疗效果的分子标志物；MLKL还可作为治疗胶质瘤的靶标。本发明还包括：脑胶质瘤诊断、筛查和/或预后的检测试剂盒，siRNA核酸‑脂质颗粒，MLKL基因表达抑制剂siRNA，GalNAc‑siRNA缀合物，以及它们在脑胶质瘤诊断、筛查和/或预后的检测中的应用。
Description
技术领域
本发明属于生物医药技术领域，具体涉及一种潜在的脑胶质瘤生物标记物MLKL基因及其应用，包括MLKL在恶性胶质瘤诊断、治疗以及预后中的应用。
背景技术
胶质瘤是成年人最常见的中枢神经系统原发性肿瘤，约占恶性的脑及其他中枢神经系统肿瘤的50％，具有恶性程度高、进展快、易复发等特点。根据传统的世界卫生组织(WHO)分级，胶质瘤根据组织学特征大致分为四级，包括较低级别胶质瘤(Lower GradeGlioma,LGG)及IV级胶质母细胞瘤(Glioblastoma,GBM)。我国脑胶质瘤的年发病率为5-8/10万，5年病死率在全身肿瘤中仅次于胰腺癌和肺癌。GBM是胶质瘤中恶性程度最高的类型，综合发病高峰在30-40岁，平均进展时间6.9个月，患者总生存期平均仅有14.6个月，5年生存率不足3％，给个人、家庭、社会带来沉重的负担。
目前的临床治疗手段仍然仅限于手术切除后放疗与化疗。手术切除是治疗脑胶质瘤的首要策略，但因胶质瘤浸润性生长特点，与正常脑组织边界模糊，几乎难以切除干净，手术切除后的复发概率仍高达95％以上，并最终造成患者死亡。替莫唑胺(temozolomide,TMZ)是一种口服烷化剂，具有广谱的抗肿瘤活性，在安全有效切除原发胶质瘤后，其可以穿过血脑屏障直接作用于病灶，进一步抑制肿瘤复发率，但TMZ耐药问题严峻。近年来，新的治疗方法陆续出现，包括免疫治疗和分子靶向治疗，但这些治疗方法并没有明显提高胶质瘤患者的生存率，主要是由于胶质瘤的遗传异质性。因此，寻找有效的生物标志物和治疗靶点对胶质瘤患者的诊断和治疗十分重要。
细胞程序性坏死(necroptosis)在人类免疫反应中起着重要作用。受体相互作用蛋白RIPK1和RIPK3是启动necroptosis的两个关键蛋白，而RIPK3的作用底物混合系列蛋白激酶样结构域蛋白(MLKL)，是细胞程序性坏死的特异性执行蛋白。MLKL磷酸化后发生构建变化，从坏死复合体中解离后转移质膜，造成质膜的不稳定，胞内渗透压升高，导致胞内物质从破裂的细胞膜中释放，细胞最终呈现坏死状态。研究发现，使用选择性MLKL磷酸化抑制剂或抑制MLKL蛋白的表达均可抑制程序性细胞坏死的进程。程序性细胞坏死最先被发现于脑缺血损伤的病理表现中。越来越多的研究表明，程序性细胞坏死广泛参与感染类疾病、神经系统相关疾病以及肿瘤的发生发展。
发明内容
本发明的目的在于提供一种能够用于恶性胶质瘤的辅助诊断的脑胶质瘤的生物标志物及其应用。
本发明意外发现MLKL在脑胶质瘤中显著高表达，能够用于恶性胶质瘤的辅助诊断；高表达的MLKL基因和蛋白与胶质瘤的不良预后相关，提示MLKL基因和其表达产物能够作为预后评估和治疗效果的分子标志物。进一步地，降低MLKL表达可明显抑制恶性胶质瘤细胞的生长，提示MLKL还可作为治疗胶质瘤的靶标。本发明还公开了MLKL基因表达抑制剂siRNA，对MLKL基因的表达具有良好的干扰效果，具有临床基因治疗的应用前景。
在第一方面，本发明提供了一种脑胶质瘤标志物MLKL基因和/或MLKL基因的表达产物在制备用于脑胶质瘤诊断、筛查和/或预后的检测试剂盒中的应用。
本发明通过生物信息学分析方法，检测MLKL基因在胶质瘤组织(Tumor)及正常组织(Normal)中的mRNA表达量的方法，显示MLKL基因在33种癌组织(Tumor)与正常组织(Normal)的mRNA表达存在差异，MLKL在癌症组织的表达水平均显著高于正常组织。
采用Cox回归方法分析MLKL mRNA水平与患者总生存期的相关性，发现MLKL高表达的胶质瘤患者总生存期(OS)显著低于MLKL基因低表达患者的总生存期，提示MLKL基因的表达产物能作为胶质瘤的预后标志物，通过检测MLKL基因的表达，可以评估不同级别胶质瘤患者的预后、提示患者的潜在生存时间，同时可以用于评估胶质瘤的治疗效果。更有意义的是，MLKL的高表达与其它29种癌症的不良预后无关，提示MLKL作为胶质瘤检测与预后标志物的专一性特点。通过检测MLKL基因表达，能够及时评估低级胶质瘤和高级别胶质瘤患者的预后，对调整临床治疗策略，进一步提高临床治疗效果，改善患者生存质量具有指导意义。
因此，MLKL可作为一种新的胶质瘤预后标志物，为不同亚型的胶质瘤患者的生存期提供评估依据。结合ROC曲线分析发现，MLKL基因应用于胶质瘤诊断的AUC值高于0.7，显示其作为胶质瘤诊断和预后标志物具有高特异性和高灵敏度，是胶质瘤早期分子诊断与患病分险筛查的理想靶点。
可选择地，上述的脑胶质瘤包括低级别胶质瘤(LGG)与高级别胶质瘤(GBM)。优选地，所述的肿瘤为高级别胶质瘤，即胶质母细胞瘤。
所述脑胶质瘤标志物MLKL基因的表达产物包括MLKL mRNA和/或MLKL蛋白。
在第二方面，本发明还提供用于胶质瘤诊断和预后的试剂盒，所述的试剂盒使用MLKL基因和/或MLKL基因的表达产物作为检测靶标。
本发明制备的用于胶质瘤诊断和预后的试剂盒，所述的试剂盒包含如下的组分：
可用于特异性扩增MLKL基因的引物对；和/或
特异识别/结合MLKL基因的表达产物的抗体。
人源MLKL基因的序列可参考美国国家生物技术信息中心Gene ID:197259(NM_152649.4)，具体序列为SEQ ID NO：1所示。
atggaaaatttgaagcatattatcacccttggccaggtcatccacaaacggtgtgaagagatgaaatactgcaagaaacagtgccggcgcctgggccaccgcgtcctcggcctgatcaagcctctggagatgctccaggaccaaggaaagaggagcgtgccctctgagaagttaaccacagccatgaaccgcttcaaggctgccctggaggaggctaatggggagatagaaaagttcagcaatagatccaatatctgcaggtttctaacagcaagccaggacaaaatactcttcaaggacgtgaacaggaagctgagtgatgtctggaaggagctctcgctgttacttcaggttgagcaacgcatgcctgtttcacccataagccaaggagcgtcctgggcacaggaagatcagcaggatgcagacgaagacaggcgagctttccagatgctaagaagagataatgaaaaaatagaagcttcactgagacgattagaaatcaacatgaaagaaatcaaggaaactttgaggcagtatttaccaccaaaatgcatgcaggagatcccgcaagagcaaatcaaggagatcaagaaggagcagctttcaggatccccgtggattctgctaagggaaaatgaagtcagcacactttataaaggagaataccacagagctccagtggccataaaagtattcaaaaaactccaggctggcagcattgcaatagtgaggcagactttcaataaggagatcaaaaccatgaagaaattcgaatctcccaacatcctgcgtatatttgggatttgcattgatgaaacagtgactccgcctcaattctccattgtcatggagtactgtgaactcgggaccctgagggagctgttggatagggaaaaagacctcacacttggcaagcgcatggtcctagtcctgggggcagcccgaggcctataccggctacaccattcagaagcacctgaactccacggaaaaatcagaagctcaaacttcctggtaactcaaggctaccaagtgaagcttgcaggatttgagttgaggaaaacacagacttccatgagtttgggaactacgagagaaaagacagacagagtcaaatctacagcatatctctcacctcaggaactggaagatgtattttatcaatatgatgtaaagtctgaaatatacagctttggaatcgtcctctgggaaatcgccactggagatatcccgtttcaaggctgtaattctgagaagatccgcaagctggtggctgtgaagcggcagcaggagccactgggtgaagactgcccttcagagctgcgggagatcattgatgagtgccgggcccatgatccctctgtgcggccctctgtggatgaaatcttaaagaaactctccaccttttctaagtag(SEQ ID NO：1)。
本领域技术人员通过已知的引物设计方法即可设计出适用于扩增MLKL的特异性引物。
更优选的，本发明制备的用于胶质瘤诊断和预后的试剂盒，所述的试剂盒包含可用于特异性扩增MLKL基因的引物对，所述的引物对包含如下的序列：
(1)上游引物序列：5’-TCACACTTGGCAAGCGCATGGT-3’(SEQ ID NO：2)；
下游引物序列5’-GTAGCCTTGAGTTACCAGGAAGT-3’(SEQ ID NO：3)；
(2)上游引物序列：5’-TGCAGAGGAAGACGGAAATGA-3’(SEQ ID NO：4)；
下游引物序列5’-CTCCTGTGTGGGTTTTAGTGAGC-3’(SEQ ID NO：5)；
(3)上游引物序列：5′-AGGAGGCTAATGGGGAGATAA-3′(SEQ ID NO：6)；
下游引物序列5′-TGGCTTGCTGTTAGAAACCTG-3′(SEQ ID NO：7)。
根据MLKL的基因序列，MLKL基因的表达产物如MLKL蛋白已广为人知，具体可参考UniProt数据库ID:Q8NB16，具体序列为SEQ ID NO：8所示：
MENLKHIITLGQVIHKRCEEMKYCKKQCRRLGHRVLGLIKPLEMLQDQGKRSVPSEKLTTAMNRFKAALEEANGEIEKFSNRSNICRFLTASQDKILFKDVNRKLSDVWKELSLLLQVEQRMPVSPISQGASWAQEDQQDADEDRRAFQMLRRDNEKIEASLRRLEINMKEIKETLRQYLPPKCMQEIPQEQIKEIKKEQLSGSPWILLRENEVSTLYKGEYHRAPVAIKVFKKLQAGSIAIVRQTFNKEIKTMKKFESPNILRIFGICIDETVTPPQFSIVMEYCELGTLRELLDREKDLTLGKRMVLVLGAARGLYRLHHSEAPELHGKIRSSNFLVTQGYQVKLAGFELRKTQTSMSLGTTREKTDRVKSTAYLSPQELEDVFYQYDVKSEIYSFGIVLWEIATGDIPFQGCNSEKIRKLVAVKRQQEPLGEDCPSELREIIDECRAHDPSVRPSVDEILKKLSTFSK(SEQ ID NO：8)。
此外，本领域技术人员掌握如何获得特异识别/结合MLKL蛋白的抗体的方法，例如通过全长的MLKL蛋白或其部分蛋白序列作为抗原如免疫动物如老鼠、兔子或羊驼等，可以获得特异性识别MLKL蛋白的抗体(亲和力达到10-9nM或更高)。所述抗体是单克隆抗体、多克隆抗体或纳米抗体，优选单克隆抗体。抗体可以自行制备，或者可以购买商用的识别MLKL抗体，例如Abcam公司的anti-MLKL抗体(货号：ab184718)。
更优选的，本发明制备了用于胶质瘤诊断和预后的试剂盒，所述的试剂盒包含可以用于特异扩增MLKL基因的引物对，或特异扩增MLKL基因表达产物的引物对，或能特异识别/结合MLKL蛋白的抗体。所述的抗体具有如下的特征：可识别人源全长MLKL蛋白，亲和力达到10-9nM或更高。
在更优选的实施方案中，可以通过基因手段，如多种PCR手段，例如荧光实时定量PCR，使用所述引物进行检测。也可以通过免疫手段，如蛋白质印迹、酶联免疫反应、免疫组化、免疫荧光等检测MLKL蛋白。
第三方面，本发明发现降低MLKL表达可明显抑制恶性胶质瘤细胞的生长，提示MLKL还可作为治疗胶质瘤的靶标。
本发明人发现MLKL基因或蛋白水平表达越高，胶质瘤患者的生存率越低，即MLKL基因或蛋白过表达明确指示患者较差的生存预后，说明MLKL基因和/或其表达产物能够作为胶质瘤的预后标志物，用于预测患者的预后情况。根据本发明人的研究，抑制内源性MLKL可显著降低恶性胶质瘤细胞的增殖，促使MLKL可成为治疗胶质瘤的有效靶标。
因此，本发明公开了MLKL基因作为药物或制剂针对脑胶质瘤细胞的作用靶标在筛选脑胶质瘤治疗药物中的用途；MLKL基因作为药物或制剂针对脑胶质瘤细胞的作用靶标具体是指：将MLKL基因作为药物或制剂针对脑胶质瘤细胞产生RNA干扰作用的靶标，从而能降低脑胶质瘤细胞中MLKL基因的表达水平；将MLKL基因作为药物或制剂针对脑胶质瘤细胞的作用靶标应用于筛选脑胶质瘤治疗药物具体是指：将MLKL基因作为作用对象对药物或制剂进行筛选，以找到可以抑制MLKL基因表达的药物作为脑胶质瘤治疗备选药物；所述靶标的序列如SEQ ID NO：1所示。
基于这种认识，本发明还制备了MLKL基因表达抑制剂siRNA，对MLKL基因的表达具有良好的干扰效果，具有临床基因治疗的应用前景。所述MLKL基因表达抑制剂siRNA可用于制备治疗胶质瘤的药物，辅助胶质瘤的临床治疗，提高胶质瘤的治疗效果。因此，本发明还提供一种MLKL基因的表达抑制剂，所述表达抑制剂包括基于所述MLKL基因设计的siRNA，其中所述siRNA包含15-30个核苷酸。siRNA(Small interfering RNA；小干扰RNA)与靶基因MLKL转录后的mRNA结合，通过RNA介导的沉默复合体RISC(RNA-induce siliencingcomplex)介导mRNA的降解。siRNA长度更优选为21-23nt，被RISC识别结合之后，siRNA发生解旋。RISC在siRNA的反义链指导下，寻找具有同源序列的内源性的mRNA，并在距离5’端10-11位碱基之间切割mRNA，从而导致转录后基因沉默。已知siRNA有多个在线设计工具，例如siDirect设计网站(http://sidirect2.rnai.jp)、DSIR设计网站(http://biodev.extra.cea.fr/DSIR/DSIR.html)、invivogen设计网站(http://www.invivogen.com/sirnawizard/siRNA.php)和thermofisher设计网站(https://rnaidesigner.thermofisher.com)等，可供参考。
优选的，本发明还提供一种MLKL基因的表达抑制剂，所述表达抑制剂包括基于所述MLKL基因设计的siRNA，所述siRNA核苷酸序列正义链如：
SEQ ID NO.9(5’-GAAGCAUAUUAUCACCCUUTT-3’)或
SEQ ID NO.10(5’-GCAAUAGAUCCAAUAUCUGTT-3’)或
SEQ ID NO.11(5′-GAACCUGCCCGAUGACAUU-3′)等所示。
在另一优选实施方案中，上述siRNA包括至少一个修饰的核苷酸。所述修饰的siRNA通常具有比相应未修饰siRNA序列更小的免疫刺激性，且保持针对目的靶基因的RNAi活性。在一些实施方案中，修饰的siRNA含有至少一个2’OMe嘌呤或嘧啶核苷酸诸如2’OMe-鸟苷、2’OMe-尿苷、2’OMe-腺苷，和/或2’OMe-胞嘧啶核苷酸(参考M.M.Zhang et al.，Biochemical Pharmacology 189(2021)114432)。在优选的实施方案中，修饰的核苷酸可以存在于siRNA的一条链(即，有义或反义)或两条链中。siRNA序列可以具有突出端(例如3’或5’突出端)或可以缺少突出端(即具有平端)。
连接RNA磷酸骨架的磷酸二酯键是核酸酶作用的化学键，而磷原子是核酸酶攻击的中心，对该原子的修饰能够影响酶的降解作用，从而提高小干扰核酸抗核酸酶的能力，增加其稳定性。在另一优选实施方案中，通过将磷酸酯键(PO)换成硫代磷酸酯(PS)，所述硫代磷酸修饰是用一个硫原子取代磷酸二酯键的非桥氧原子，即P-S键替代P-O键，这种修饰通常会增加核酸对核酸酶的稳定性。
本发明中，所述siRNA的制备方法没有特别的限制，可以通过化学合成得到，或者通过质粒和/或病毒载体的表达而得到。根据不同的修饰方式，可以使用经过修饰核苷酸替代相应位置中未修饰的核苷酸，或者进行磷酸二酯键的硫代磷酸修饰等。选择性地，也可以委托专门从事核酸合成的生物技术公司合成本发明的siRNA。一般来说，用于合成siRNA的方法包括以下四个过程：(1)寡聚核糖核苷酸的合成；(2)脱保护；(3)纯化分离；(4)脱盐等。
siRNA因为含有多个磷酸酯键而带有大量的负电荷，这样的极性分子很难自行穿过细胞膜；此外siRNA暴露血液会有稳定性问题并造成免疫原性。因此，在另一优选实施方案中，需要将上述siRNA用载体(如LNP)包裹或者和特定配体(如GalNac)连接以有效避免肾消除，而且还能靶向性地富集在特定的组织中。
在另一优选实施方案中，本发明制备了一种siRNA核酸-脂质颗粒，包含：
(a)siRNA核酸，所述的siRNA核苷酸序列正义链如SEQ ID NO.9、SEQ ID NO.10或SEQ ID NO.11所示；
(b)阳离子脂质，其占所述颗粒中存在的总脂质的50mol％-65mol％；
(c)非-阳离子脂质，其包括磷脂和胆固醇或其衍生物的混合物，其中所述磷脂占所述颗粒中存在的总脂质的3mol％-15mol％，所述胆固醇或其衍生物占所述颗粒中存在的总脂质的30mol％-40mol％；和
(d)抑制颗粒聚集的缀合脂质，其占所述颗粒中存在的总脂质的0.5mol％-2mol％；其中，其中所述阳离子脂质包括1，2-二亚油基氧基-N，N-二甲基氨基丙烷(DLinDMA)、1，2-二亚麻基氧基-N，N-二甲基氨基丙烷(DLenDMA)或其混合物。
其中，所述磷脂包括二棕榈酰磷脂酰胆碱(DPPC)、二硬脂酰磷脂酰胆碱(DSPC)或其混合物；
其中，所述抑制颗粒聚集的缀合脂质包括聚乙二醇(PEG)-脂质缀合物，选自PEG-二酰甘油(PEG-DAG)缀合物、PEG-二烷氧基丙基(PEG-DAA)缀合物或其混合物。
在另一优选实施方案中，本发明了制备了一种GalNAc-siRNA缀合物，即将如SEQID NO.9、SEQ ID NO.10或SEQ ID NO.11所示siRNA缀合至N-乙酰半乳糖胺(N-Acetylgalactosamine，GalNAc)形成GalNAc-siRNA缀合物。
其中GalNAc配体可以结合肝细胞表达的去唾液酸糖蛋白受体(Asialoglycoprotein receptor，ASGPR)并将siRNA靶向递送至肝细胞。可以皮下长循环给药是GalNAc-siRNA的一个巨大优势，更适合用于皮下给药。
本发明还提供一种制备GalNAc-siRNA缀合物的方法，即在固相寡核苷酸合成过程中，使用GalNAc负载固体载体，从而将GalNAc与siRNA正义链的3'末端通过三元间隔子相连接；随后进行核苷酸脱保护和乙酸半乳糖胺脱保护，除去固体载体，可得到GalNAc-siRNA缀合物。在一些实施方案中，所述siRNA可附接至一个或多个(例如，两个、三个、四个或更多个)GalNAc衍生物。所述附接可以不经由接头，或经由一个或多个接头(例如，两个、三个、四个或更多个接头)。在一些实施方案中，本文所述的接头是多价(例如，二价、三价或四价)支化接头。在一些实施方案中，所述一个或多个GalNAc衍生物附接至所述dsRNA的有义链的3'端、反义链的3′端、有义链的5′端和/或反义链的5′端。有关GalNAc-siRNA缀合物的合成可参考WO2009/073809、WO2011/104169、WO2012/083046、WO2014/118267和WO2014/179620等，在此全部引入作为参考。
利用本发明制备的MLKL表达抑制剂siRNA转染恶性胶质瘤细胞后，免疫印迹实验显示MLKL的表达水平显著降低，同时细胞周期蛋白依赖激酶2(CDK2)和细胞周期蛋白A2(cyclin A2)表达量伴随MLKL的下降而降低。在机体，CDKs主要与周期蛋白(cyclin)结合形成激活态的蛋白酶体复合物，从而催化底物丝氨酸/苏氨酸残基磷酸化，促进细胞周期不断进行。其中CDK2通过与cyclin A形成蛋白酶体复合物，调控细胞G1期到S期的转变和S期的进行。CDK2-cyclin A2表达量降低，提示敲低MLKL可能抑制恶性胶质瘤细胞的增殖速率，提示MLKL基因表达抑制剂siRNA可用于制备治疗胶质瘤的药物，辅助胶质瘤的临床治疗，提高胶质瘤的治疗效果。
因此，本发明还提供了上述MLKL表达抑制剂siRNA在制备治疗脑胶质瘤药物中的用途。
综上，本发明为胶质瘤治疗提供新的理论依据和全新的靶标，本发明还为胶质瘤提供了新的辅助诊断和预后诊断的方法以及新的治疗药物。
附图说明
图1为本发明实施例1提供的MLKL mRNA在33种癌症组织(Tumor)和正常组织(Normal)中表达对比。
图2为本发明实施例2提供的MLKL基因表达水平对29种癌症患者总生存期(OS)影响的生存分析森林图。
图3为本发明实施例3提供的与其它3个文献报道的胶质瘤标志物NUSAP1、GINS2和TRIM21相比，MLKL基因表达水平与高级别胶质瘤(GBM)患者的生存率相关性更显著。
图4为本发明实施例4提供的MLKL基因表达水平对低级别胶质瘤(LGG)和高级别胶质瘤(GBM)总生存期(OS)(a)、疾病特异性生存期(DSS)(b)以及无进展生存期(PFI)(c)影响的生存分析曲线。
图5为本发明实施例5提供的MLKL基因表达水平用于诊断胶质瘤的ROC曲线。
图6为本发明实施例6提供的MLKL基因在各个分子亚型胶质瘤中的表达水平。其中，(a)为WHO级别；(b)为IDH突变；(c)为1p/19q基因联合缺失；(d)临床病理分型。
图7为本发明实施例7提供的MLKL基因表达水平对各个分子亚型胶质瘤生存期影响的生存分析曲线。其中，(a)为WHO级别；(b)为IDH突变；(c)为临床病理分型。
图8为本发明实施例8提供的在临床胶质瘤样本中，胶质瘤恶性程度越高的MLKL蛋白表达量越高(a)；定量分析显示MLKL蛋白在高级别胶质瘤(b)和IDH1野生型胶质瘤中(c)表达量显著升高；
图9为本发明实施例8提供的MLKL蛋白表达水平与胶质瘤病人总生存期(a)；IDH1突变(b)以及肿瘤增殖标志物Ki67(c)相关性分析图；
图10为本发明实施例9提供的MLKL mRNA水平检测试剂盒使用的电泳结果(a)以及定量分析图(b)；
图11为本发明实施例11提供的利用siRNA敲减MLKL的细胞与对照细胞(NC)的免疫印迹实验(a)及其定量分析(b)，以及细胞增殖率检测实验(c)。
具体实施方式
下面结合具体实施例和附图对本发明内容进一步阐述。下列实施例中未注明具体条件的实验方法，通常按照本技术领域常规技术，所述siRNA合成工作由上海吉玛制药技术有限公司完成，所有引物合成由金唯智生物科技有限公司完成，下述实施例中所涉及的细胞均购于上海生命科学研究所，所用到的试剂和原料均可由市场购得。
下述实施例中所涉及的试剂如下所示：
MLKL抗体购自abcam公司，Name:重组Anti-MLKL抗体(ab184718)。β-actin抗体购自Proteintech公司(No.66009)。抗兔IgG抗体购自CellSignalling公司(#7074)。增强型CCK-8试剂盒，购自碧云天生物技术公司，产品编号：C0042。ECL化学发光底物购自天能公司(编号：180-5001)Lipofectamine 2000转染试剂购自ThermoFisherScientific公司，货号11668-019。TRIzol试剂购自ThermoFisherScientific公司，货号15596026。PrimeScript1st strand cDNA合成试剂盒购自TaKaRa公司，货号6110A。ChamQ UniversalSYBR qPCR预混液购自Vazyme公司，货号Q711-03。
实施例1
供利用生物信息学分析方法，检测MLKL基因在胶质瘤组织(Tumor)及正常组织(Normal)中的mRNA表达量的方法，具体内容如下：
从TCGA数据库(https://portal.gdc.cancer.gov/)和GTEx数据库(http://gtexportal.org/home/)上下载33种癌症与正常组织中MLKLmRNA表达情况。通过Wilcoxonrank sum test检测MLKL在正常组织和癌组织中表达量的差异。
图1显示MLKL基因在33种癌组织(Tumor)与正常组织(Normal)的mRNA表达差异水平，由图1可知，MLKL在癌症组织的表达水平均显著高于正常组织。
实施例2
通过TCGA数据库获取的30种癌症组织中MLKLmRNA信息和病人信息，采用Cox回归方法分析MLKLmRNA水平与患者总生存期的相关性，得到如图2所述的生存分析森林图。
由图2可知，MLKL基因高表达的低级胶质瘤(LGG)和高级胶质瘤(GBM)患者，其总生存率的风险比(HR)明显高于MLKL基因地表达的前列腺患者(P<0.01)，即MLKL高表达的胶质瘤患者总生存期(OS)显著低于MLKL基因低表达患者的总生存期，提示MLKL基因的表达产物能作为胶质瘤的预后标志物，通过检测MLKL基因的表达，可以评估不同级别胶质瘤患者的预后、提示患者的潜在生存时间，同时可以用于评估胶质瘤的治疗效果。更有意义的是，MLKL的高表达与其它29种癌症的不良预后无关，提示MLKL作为胶质瘤检测与预后标志物的专一性特点。
实施例3
从TCGA数据库分别下载MLKL、NUSAP1、GINS2和TRIM21信息以及病人信息，采用COX回归方法对比分析MLKL和3个已发现的胶质瘤标志物，即NUSAP1、GINS2、TRIM21的基因表达水平与患者总生存期的相关性，得到如图3所述的生存分析热图。
由图3可知，虽然MLKL、NUSAP1、GINS2和TRIM21基因的高表达与低级别胶质瘤(LGG)的不良预后均存在显著相关性(P<0.001)，但仅有MLKL的高表达可以作为评估高级别胶质瘤(GBM)预后的分子标志物，进一步验证MLKL在不同级别的胶质瘤中均具有准确的诊断意义。
实施例4
利用实施案例2中从TCGA数据库获取的胶质瘤组织中MLKLmRNA信息和病人信息，通过Kaplan-Meier曲线和log-rank检测用于分析MLKL基因表达水平与患者总生存期(OS)、疾病特异性生存期(DSS)以及无进展生存期(PFI)的相关性，得到如图3所述的生存分析曲线。
由图4a-c可知，MLKL基因高表达的低级别胶质瘤患者(LGG)和高级别胶质瘤患者(GBM)，在同一生存时间下的患者总生存率(OS)、疾病特异性生存率(DSS)以及肿瘤无进展生存率(PFI)明显低于MLKL低表达的胶质瘤患者(P<0.01)，进一步验证MLKL基因表达的产物能够作为胶质瘤的预后标志物。通过检测MLKL基因表达，能够及时评估低级胶质瘤和高级别胶质瘤患者的预后，对调整临床治疗策略，进一步提高临床治疗效果，改善患者生存质量具有指导意义。
实施例5
本实施例旨在提供一种评估MLKL基因表达水平用于胶质瘤诊断价值的的方法，具体内容为：采用实施例2中从TCGA数据库获取的胶质瘤病人的信息通过软件进行编辑分析，获得时间依赖型ROC(receiver operator characteristic curve)曲线。
上述的软件是R(3.6.3版本)，其中统计分析所用的R包为timeROC(0.4版本)，数据可视化所用的R包为ggplot2(3.3.3版本)。
ROC曲线是显示真阳性率(灵敏度，或称敏感性，TPR)和假阳性率(FPR)之间折中的一种图形化方法。一个好的分类模型，在本发明中指一个好的癌症标志物，应该进可能靠近ROC曲线的左上角。同时，也可以采用曲线下面积(AUC)来表示一个模型的平均表现，AUC越大，诊断准确度越高。
图5所示MLKL基因表达水平用于诊断胶质瘤的ROC曲线，如图所示，预测年限为1年时，曲线下面积(AUC)为0.753，MLKL基因表达用于诊断胶质瘤的灵敏度为66.4％，特异性为74.8％。预测年限为5年时，曲线下面积(AUC)为0.738，MLKL基因表达用于诊断胶质瘤的灵敏度为65.3％，特异性为75.3％。预测年限为10年时，曲线下面积(AUC)为0.791，MLKL基因表达用于诊断胶质瘤的灵敏度为64.1％，特异性为84.2％。显示其作为胶质瘤诊断和预后标志物具有高特异性和高灵敏度，是胶质瘤早期分子诊断与患病分险筛查的理想靶点。
实施例6
利用实施案例2中从TCGA数据库获取的胶质瘤组织中MLKLmRNA信息和病人信息，通过Kruskal-Wallis test检测MLKL基因表达与胶质瘤临床不同分型，即WHO分级、IDH突变、1p/19q共缺失以及不同组织类型之间的相关性，得到如图6(a-b)所示的柱形图。
所述的WHO分级越高指向越差的生存率，而IDH突变和1p/19q联合缺失突变与较好的预后具有强相关性。图6(a)显示MLKL基因在III级胶质瘤中的表达量高于II级，而在IV级胶质瘤中表达量高于II级与III级。图6(b)显示MLKL基因在IDH野生型中的表达量显著高于其在IDH突变型中的表达量。图6(c)显示MLKL基因在不发生1p/19联合缺失的表达量显著高于其在1p/19q联合缺失的表达量。图6(d)显示MLKL基因在少突胶质细胞瘤(oligodendroglioma)、少突星型胶质细胞瘤(oligoastrocytoma)、星形胶质细胞瘤(astrocytoma)、胶质母细胞瘤(glioblastoma)的表达量逐步升高。以上结果共同说明胶质瘤的恶性程度越高，MLKL基因的表达量越高。由此可知，MLKL的表达与胶质瘤的级别、IDH突变、1p/19共缺失以及病理组织分型具有相关性，MLKL基因及其表达产物可作为临床判断胶质瘤级别和组织分型的标志物。
实施例7
利用实施案例2中从TCGA数据库获取的胶质瘤组织中MLKLmRNA信息和病人信息，通过Kaplan-Meier生存曲线检测MLKL表达在胶质瘤患者在不同亚型中患者总生存(OS)的预后价值。结果如图7a-c所示，MLKL在较高级别胶质瘤患者(G3和G4)(a)、IDH野生型患者(b)、以及3种较严重的组织分型(包括少突星形胶质细胞瘤、星形胶质细胞瘤、以及神经母细胞瘤)(c)患者中均具有预后意义。因此，MLKL可作为一种新的胶质瘤预后标志物，为不同亚型的胶质瘤患者的生存期提供评估依据。
实施例8
MLKL蛋白在高级别胶质瘤临床样本中蛋白表达高于低级别胶质瘤组织。
1.研究对象
研究对象选取81例胶质瘤患者临床样本，其中WHO分级II级17例，III级19例，IV级52例，本研究通过医院伦理委员会审查和批准，所有患者入组前均签署书面知情通知书。
2.免疫印迹实验检测MLKL蛋白在临床胶质瘤样本中的表达量。
用组织破碎仪将临床胶质瘤组织破碎为组织匀浆，进而通过RIPA细胞裂解液使各组分细胞破碎，蛋白溶解，在相同条件下对蛋白质进行定量调整后，进行western blot检测分析。具体步骤如下：首先通过SDS-PAGE电泳分离蛋白，并将蛋白转到硝酸纤维素膜上，然后采用5％的脱脂奶粉进行封闭实验，封闭结束后对膜进行清洗，并用分别与MLKL和Actin特异性结合的抗体与其充分结合，进一步用能与一抗特异性结合的二抗与其结合后，通过蛋白显色成像仪进行拍照，最后通过Image J软件对所显色的蛋白条带完成定量分析。
所获得的结果如图8a所示，对western blot定量分析结果表明MLKL在高级别胶质瘤(GBM)中的蛋白表达量显著高于在低级别胶质瘤中(LGG)的表达量(图8b)，并在IDH1野生型中的表达量显著高于其在IDH1突变型胶质瘤组织中的表达量(图8c)，说明MLKL在恶性程度高的胶质瘤中高表达，提示其蛋白表达量与病患的不良预后相关。
3.MLKL蛋白过表达的临床意义。
采用Kalpan-Meier方法绘制生存分析曲线，并通过log-rank方法检测其统计学意义。检测系数P<0.05统计学上有显著性差异。并且通过R包：survival包(3.2-10版本)用于生存资料的统计分析MLKL蛋白的表达与胶质瘤患者总生存率(Overall survival,OS)的关系。MLKL蛋白过表达和低表达按照MLKL蛋白表达水平的中位数来划定。同时结合临床胶质瘤病患的IDH1突变信息和免疫组化Ki67染色结果，应用GraphPad Prism 7软件的t检验，分析MLKL蛋白表达量与IDH1突变与Ki67指标之间的相关性。
结果如图9a所示，在81例胶质瘤组织中，MLKL蛋白过表达的胶质瘤病人比MLKL低表达的胶质瘤患者的总生存率(OS)明显降低。MLKL在2种分子亚型(即Ki67与IDH1)胶质瘤中的表达水平图显示，MLKL蛋白在野生型IDH1分子亚型(b)，以及高Ki67(>＝30)(c)的胶质瘤中特异性高表达。IDH1野生型与不良预后相关，提示MLKL蛋白水平与胶质瘤恶性程度正相关。Ki67是一种与细胞增殖相关的核内蛋白,可用于判断肿瘤增生指数。总体来说，Ki-67阳性标记数值越高，则组织分化越差，恶性程度越高，预后越差。综上，MLKL蛋白表达水平高，可作为胶质瘤不良预后的标志物。
实施例9
MLKL mRNA水平检测试剂盒使用示例
方法：采用RT-qPCR，在胶质瘤组织样本中检测MLKL的mRNA水平。
本实施例公开的一种胶质瘤MLKL诊断检测方法，采取正常脑组织或胶质瘤患者肿瘤组织，按照说明书操作步骤提取总RNA，使用TaKaRa逆转录酶将提好的总RNA逆转录为cDNA，并通过PCR分析MLKL的mRNA水平。具体条件如下：
1.胶质瘤组织总RNA的提取方法：
将组织称重后，每100mg组织加入1mLTrizol裂解液，低温超声破碎后放置在冰上，裂解5min；随后加入200μL氯仿，剧烈震荡数次后，冰浴10min。12000rpm 4℃低温离心10min后，小心转移最上层透明溶液至无RNA酶的离心管中，加入500μL异丙醇震荡数次，冰上放置20min后，12000rpm 4℃低温离心15min。小心吸去上清，加入1mL冰的75％乙醇，颠倒洗涤，不超过7500rpm离心5min，随后重复洗涤一次。小心吸去上清，加入20μL预冷的DEPC水，吹打混匀并测定总RNA浓度。
2.逆转录反应按Takara逆转录酶说明书进行操作，最终获得cDNA产物-80℃。
3.逆转录产物扩增方法参考Vazyme公司SYBR qPCR酶(货号：Q711-03)说明书，引物浓度为10μM，以GAPDH为内参，通过ImageJ软件定量分析MLKL mRNA水平。
MLKL基因水平拷贝数的引物序列为：
F:5’-TCACACTTGGCAAGCGCATGGT-3’，(SEQ ID NO：2)；
R:5′-GTAGCCTTGAGTTACCAGGAAGT-3’，(SEQ ID NO：3)；
GAPDH基因水平拷贝数的引物序列为：
F:5′-GGAGCGAGATCCCTCCAAAAT-3’,(SEQ ID NO：12)；
R:5′-GGCTGTTGTCATACTTCTCATGG-3’,(SEQ ID NO：13)。
结果：RT-PCR产物电泳结果及分析定量结果如图11所示。
实施例10
MLKL蛋白水平检测试剂盒使用示例
本实施例公开的一种胶质瘤MLKL诊断检测方法，采用WesternBlot方法，在胶质瘤组织样本中检测MLKL的蛋白表达水平，并通过定量分析MLKL的蛋白表达水平。
该试剂盒中包含能够特异性识别人源MLKL以及内参β-Actin的抗体，抗体结合示意图如图8a所示。
实施例11
MLKL蛋白促进胶质瘤细胞的恶性增殖。
1.免疫印迹实验检测敲低MLKL后细胞周期蛋白的表达量
为探索MLKL在胶质瘤中的生物学功能，利用siRNA技术特异性地敲低恶性胶质瘤细胞U87的MLKL。使用干扰序列siNC(5’–UUCUCCGAACGUGUCACGUTT–3’(SEQ ID NO.14)和siMLKL(#1：5’-GAAGCAUAUUAUCACCCUUTT-3’(SEQ ID NO.9)，#2：5’-GCAAUAGAUCCAAUAUCUGTT-3’(SEQ ID NO.10))转染细胞后，免疫印迹实验显示MLKL的表达水平显著降低(图11(a))，同时细胞周期蛋白依赖激酶2(CDK2)和细胞周期蛋白A2(cyclinA2)表达量伴随MLKL的下降而降低(图11(b))。在机体，CDKs主要与周期蛋白(cyclin)结合形成激活态的蛋白酶体复合物，从而催化底物丝氨酸/苏氨酸残基磷酸化，促进细胞周期不断进行。其中CDK2通过与cyclin A形成蛋白酶体复合物，调控细胞G1期到S期的转变和S期的进行。CDK2-cyclin A2表达量降低，提示敲低MLKL可能抑制U87细胞的增殖速率。
2.CCK8试剂盒检测恶性胶质瘤细胞的增殖速率
利用增强型CCK8试剂盒对不同组的细胞进行细胞数目统计，检测敲低MLKL蛋白的胶质瘤细胞生长速率的变化。结果如图11(c)显示，在细胞增殖曲线检测实验中，敲低MLKL后U87的生长收到了显著的抑制。以上实验说明MLKL可作为一种新型的促癌分子，参与胶质瘤的恶性生长及发展。
总结：本发明通过生物信息学分析发现了MLKL在胶质瘤，尤其是高级别胶质瘤中高表达，且其高表达水平与较差的胶质瘤患者生存期密切相关。同时在临床水平对比检测了MLKL在低级别胶质瘤(LGG)和高级别胶质瘤(GBM)组织中的表达情况，发现MLKL在GBM中显著高表达，且其蛋白表达量与Ki67和胶质瘤患者的总生存率相关，结果与生物信息学分析。为进一步研究MLKL是否参与调控胶质瘤细胞的增殖，利用siRNA干扰技术敲低人胶质瘤细胞U87的MLKL后，进行免疫印迹实验和细胞增殖速率检测实验，结果显示干扰MLKL可能通过降低细胞周期蛋白CDK2和cyclinA2抑制U87细胞的增殖速率。
另外，MLKL基因或其表达产物MLKL蛋白作为靶点在制备抗肿瘤药物中的用途也在本发明的保护范围之内。
本领域的普通技术人员可以理解，上述各实施方式是实现本发明的具体实施例，而在实际应用中，可以在形式上和细节上对其作各种改变，而不偏离本发明的精神和范围。
序列表
<110> 复旦大学
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Claims (32)
1.脑胶质瘤标志物MLKL基因和/或MLKL基因的表达产物在制备用于脑胶质瘤诊断、筛查和/或预后的检测试剂盒中的应用。
2.根据权利要求1所述的应用，其特征在于，所述的脑胶质瘤为低级别胶质瘤或高级别胶质瘤。
3.根据权利要求2所述的应用，其特征在于，所述的脑胶质瘤为胶质母细胞瘤。
4.根据权利要求1所述的应用，其特征在于，所述的脑胶质瘤标志物MLKL基因的表达产物为MLKL mRNA和/或MLKL蛋白。
5.用于胶质瘤诊断和预后的试剂盒，其特征在于，包含如下的组分：用于特异性扩增MLKL基因的引物对；和/或特异识别/结合MLKL基因的表达产物的抗体。
6.根据权利要求5所述的试剂盒，其特征在于，所述的引物对包含如下的序列：
(1)上游引物序列：SEQ ID NO：2；
下游引物序列：SEQ ID NO：3；和/或
(2)上游引物序列：SEQ ID NO：4；
下游引物序列：SEQ ID NO：5；和/或
(3)上游引物序列：SEQ ID NO：6；
下游引物序列：SEQ ID NO：7。
7.根据权利要求5所述的试剂盒，其特征在于，所述的抗体是单克隆抗体、多克隆抗体或纳米抗体。
8.根据权利要求7所述的试剂盒，其特征在于，所述的抗体为Abcam公司的anti-MLKL抗体。
9.根据权利要求5所述的试剂盒，其特征在于，包含用于特异扩增MLKL基因的引物对，或特异扩增MLKL基因表达产物的引物对，或能特异识别/结合MLKL蛋白的抗体；所述的抗体可识别人源全长MLKL蛋白，亲和力达到10-9nM或更高。
10.根据权利要求5所述的试剂盒，其特征在于，通过基因手段，包括荧光实时定量PCR，使用所述引物进行检测。
11.根据权利要求5所述的试剂盒，其特征在于：通过免疫手段，包括蛋白质印迹、酶联免疫反应、免疫组化或免疫荧光检测MLKL蛋白。
12.MLKL基因作为药物或制剂针对脑胶质瘤细胞的作用靶标在筛选脑胶质瘤治疗药物中的用途。
13.根据权利要求12所述的用途，其特征在于，MLKL基因作为药物或制剂针对脑胶质瘤细胞的作用靶标具体是指：将MLKL基因作为药物或制剂针对脑胶质瘤细胞产生RNA干扰作用的靶标，从而能降低脑胶质瘤细胞中MLKL基因的表达水平；将MLKL基因作为药物或制剂针对脑胶质瘤细胞的作用靶标应用于筛选脑胶质瘤治疗药物具体是指：将MLKL基因作为作用对象对药物或制剂进行筛选，以找到可以抑制MLKL基因表达的药物作为脑胶质瘤治疗备选药物；所述靶标的序列如SEQ ID NO：1所示。
14.一种MLKL基因的表达抑制剂，所述表达抑制剂包括基于所述MLKL基因设计的siRNA，其中所述siRNA包含15-30个核苷酸。
15.根据权利要求14所述的MLKL基因的表达抑制剂，其特征在于，所述的siRNA长度为21-23个核苷酸。
16.根据权利要求14所述的MLKL基因的表达抑制剂，其特征在于，所述表达抑制剂包括基于所述MLKL基因设计的siRNA，所述siRNA核苷酸序列正义链如SEQ ID NO.9，SEQ IDNO.10或SEQ ID NO.11所示。
17.根据权利要求14所述的MLKL基因的表达抑制剂，其特征在于，所述的siRNA包括至少一个修饰的核苷酸。
18.根据权利要求14所述的MLKL基因的表达抑制剂，其特征在于，所述修饰的siRNA含有至少一个2’OMe嘌呤或嘧啶核苷酸。
19.根据权利要求14所述的MLKL基因的表达抑制剂，其特征在于，修饰的核苷酸存在于siRNA的一条链或两条链中。
20.根据权利要求14所述的MLKL基因的表达抑制剂，其特征在于，所述的siRNA序列具有突出端或缺少突出端。
21.根据权利要求14所述的MLKL基因的表达抑制剂，其特征在于，将连接RNA磷酸骨架的磷酸二酯键换成硫代磷酸酯，所述硫代磷酸修饰是用一个硫原子取代磷酸二酯键的非桥氧原子，即P-S键替代P-O键。
22.一种siRNA核酸-脂质颗粒，包含：
(a)siRNA核酸，所述的siRNA核苷酸序列正义链为SEQ ID NO.10、SEQ ID NO.11或SEQID NO.12所示；
(b)阳离子脂质，其占所述颗粒中存在的总脂质的50mol％-65mol％；
(c)非-阳离子脂质，其包括磷脂和胆固醇或其衍生物的混合物，其中所述磷脂占所述颗粒中存在的总脂质的3mol％-15mol％，所述胆固醇或其衍生物占所述颗粒中存在的总脂质的30mol％-40mol％；和
(d)抑制颗粒聚集的缀合脂质，其占所述颗粒中存在的总脂质的0.5mol％-2mol％。
23.根据权利要求22所述的siRNA核酸-脂质颗粒，其特征在于，所述阳离子脂质包括1，2-二亚油基氧基-N，N-二甲基氨基丙烷、1，2-二亚麻基氧基-N，N-二甲基氨基丙烷或其混合物。
24.根据权利要求22所述的siRNA核酸-脂质颗粒，其特征在于，所述磷脂为二棕榈酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱或其混合物。
25.根据权利要求22所述的siRNA核酸-脂质颗粒，其特征在于，所述抑制颗粒聚集的缀合脂质包括聚乙二醇-脂质缀合物，选自PEG-二酰甘油缀合物、PEG-二烷氧基丙基缀合物或其混合物。
26.一种GalNAc-siRNA缀合物，是由如SEQ ID NO.10、SEQ ID NO.11或SEQ ID NO.12所示siRNA缀合至N-乙酰半乳糖胺形成的GalNAc-siRNA缀合物。
27.根据权利要求26所述的GalNAc-siRNA缀合物，其特征在于，所述siRNA可附接至一个或多个GalNAc衍生物。
28.根据权利要求27所述的GalNAc-siRNA缀合物，其特征在于，所述附接不经由接头，或经由一个或多个接头。
29.根据权利要求28所述的GalNAc-siRNA缀合物，其特征在于，所述的接头是多价支化接头。
30.根据权利要求26-30任一项所述的GalNAc-siRNA缀合物，其特征在于，所述一个或多个GalNAc衍生物附接至所述dsRNA的有义链的3'端、反义链的3′端、有义链的5′端和/或反义链的5′端。
31.制备权利要求26-31任一项所述的GalNAc-siRNA缀合物的方法，即在固相寡核苷酸合成过程中，使用GalNAc负载固体载体，从而将GalNAc与siRNA正义链的3'末端通过三元间隔子相连接；随后进行核苷酸脱保护和乙酸半乳糖胺脱保护，除去固体载体，得到GalNAc-siRNA缀合物。
32.权利要求14-26任一项所述的MLKL表达抑制剂siRNA在制备治疗脑胶质瘤药物中的用途。
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| WO2023147790A1 (zh) * | 2022-02-02 | 2023-08-10 | 复旦大学 | 一种脑胶质瘤生物标记物mlkl基因及其应用 |
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