CN116440275A - Mobat1在制备治疗癌症的试剂中用途 - Google Patents
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Abstract
本发明涉及癌症的治疗领域,具体地,涉及MOBAT1在治疗癌症的用途。更具体地,涉及MOBAT1在治疗胰腺癌的用途。本发明提供了MOBAT1在制备治疗癌症的试剂中的用途。
Description
技术领域
本发明涉及癌症的治疗领域,具体地,涉及MOBAT1在治疗癌症的用途。更具体地,涉及MOBAT1在治疗胰腺癌的用途。
背景技术
胰腺癌(Pancreatic cancer)是一种早期表现隐匿、晚期进展迅速和预后极差的高度凶险的消化系统肿瘤,也被称为“癌中之王”。胰腺导管腺癌是其中最主要的病理类型,患者的中位生存期很短仅有六个月左右。从全球范围来看,胰腺癌的发病率和死亡率均逐年增加,美国癌症协会预测在2030年胰腺癌将成为肿瘤相关的第二大死因。在我国,最新的胰腺癌统计数据显示从1990到2017国内胰腺癌新发病例增长230.08%,标化死亡率增长47.51%。经过多年的投入与研究,尽管在胰腺癌的诊治手段方面取得了一定的进步,但是胰腺癌患者的预后并没有实质的提升。截至目前,胰腺癌五年的总体生存率仍不足10%。
因此,筛选新的治疗靶点是目前胰腺癌基础及转化研究的当务之急,具有十分重要的科学意义和应用价值。本领域需求这样新的治疗靶点,以便为胰腺癌的治疗提供新的选择。
发明内容
有鉴于此,第一方面,本发明提供了MOBAT1在制备治疗癌症的试剂中的用途。
根据本发明,所述MOBAT1基因来源于人,其Genbank登录号为NM_001080480。
进一步地,所述试剂为MOBAT1基因抑制剂。
根据本发明优选的,所述MOBAT1基因抑制剂是指以MOBAT1基因为作用靶标制备或筛选得到的对MOBAT1基因具有抑制效果的分子或制剂。所述抑制效果包括但不限于:抑制MOBAT1基因活性,或者抑制MOBAT1基因转录或表达。
根据本发明优选的,所述MOBAT1基因抑制剂为核酸分子、核酸构建体、慢病毒、抗体或小分子化合物。
进一步优选的,所述核酸分子为双链RNA或shRNA。
进一步优选的,所述核酸分子作用的MOBAT1基因靶序列如SEQ ID NO:1-5任一序列所示。
进一步优选的,所述shRNA的核苷酸序列如SEQ ID NO:6-11任一序列所示。
根据本发明,所述治疗癌药物具有以下功用之一种或多种:抑制癌细胞的增殖速率,改变癌细胞周期分布,促进癌细胞凋亡,抑制癌细胞克隆形成。
根据本发明优选的,所述治疗癌药物必然包括MOBAT1基因抑制剂,并以MOBAT1基因抑制剂作为前述功用的唯一有效成分或者有效成分之一。
根据本发明,所述癌为胰腺癌。
进一步地,所述癌为胰腺导管腺癌。
第二方面,提供一种核酸分子,所述核酸分子为MOBAT1基因抑制剂,可以降低胰腺癌细胞中MOBAT1基因的表达,包括:
a)双链RNA,所述双链RNA中含有能够在严紧条件下与MOBAT1基因杂交的核苷酸序列;或者
b)shRNA,所述shRNA中含有能够在严紧条件下与MOBAT1基因杂交的核苷酸序列。
根据本发明,所述双链RNA包含第一链和第二链,所述第一链和所述第二链互补共同形成RNA二聚体,并且所述第一链的序列与MOBAT1基因靶序列相同;所述shRNA包括正义链片段和反义链片段,以及连接所述正义链片段和反义链片段的茎环结构,所述正义链片段和所述反义链片段的序列互补,并且所述正义链片段的序列与MOBAT1基因靶序列相同。
根据本发明优选的,所述双链RNA为小干扰RNA(siRNA)。
根据本发明优选的,所述shRNA的核苷酸序列如SEQ ID NO:6-11任一序列所示。
根据本发明,所述shRNA经酶切加工后可成为siRNA,进而起到特异性沉默胰腺癌细胞中MOBAT1基因表达的作用。
本发明的第三方面,提供一种核酸构建体,所述核酸构建体为MOBAT1基因抑制剂,含有编码前述核酸分子中shRNA的基因片段,能表达所述shRNA。
根据本发明优选的,所述核酸构建体是将编码前述核酸分子中shRNA的基因片段克隆入载体获得的。
根据本发明优选的,所述载体为慢病毒载体。
根据本发明优选的,所述核酸构建体还含有启动子序列和/或编码肿瘤细胞中可被检测的标记物的核苷酸序列;进一步优选的,所述可被检测的标记物为绿色荧光蛋白(GFP)。
所述MOBAT1基因干扰核酸构建体经过病毒包装成为有感染力的病毒颗粒后,感染肿瘤细胞,进而转录出本发明所述的shRNA,通过酶切加工等步骤,最终获得siRNA,用于特异性沉默MOBAT1基因的表达。
本发明第四方面,提供一种慢病毒,所述慢病毒为MOBAT1基因抑制剂,由前述核酸构建体在慢病毒包装质粒、细胞系的辅助下,经过病毒包装而成。
本发明第五方面,提供一种治疗胰腺癌药物,包括上述MOBAT1基因抑制剂的一种或多种。
根据本发明优选的,所述MOBAT1基因抑制剂为治疗胰腺癌药物的唯一有效成分或有效成分之一。
所述治疗胰腺癌药物的形式无特殊限制,可以为固体、液体、凝胶、半流质或气雾的物质形式。其中,所述药物的剂型为临床上或药学上可接受的任一剂型。例如,但不限于,所述药物的剂型为粉剂、注射液、胶囊、口服液、片剂、滴丸、喷剂。
进一步地,所述药物还包括说明书。
在进一步地,所述说明书包含药物用于治疗癌症的使用信息,例如,为了治疗癌症所需要有效量,服用次数,间隔时间等。
本发明第六方面,提供一种胰腺癌联合治疗药物组合,包括上述一种或几种MOBAT1基因抑制剂和至少一种其他治疗胰腺癌药物。
进一步地,所述药物为紫杉醇、吉西他滨、各种靶向抗体等
更进一步地,所述药物为吉西他滨。
附图说明
图1A.人胰腺癌组织和癌旁组织中MBOAT1mRNA表达情况。B.正常小鼠胰腺组织、低级别胰腺导管上皮内瘤变、高级别胰腺导管上皮内瘤变进以及小鼠腺癌组织的H&E染色和MBOAT1免疫组化染色结果;标尺:100μm。C.人胰腺癌组织和配对的癌旁组织中MBOAT1蛋白表达情况。D.人胰腺癌组织芯片MBOAT1表达打分情况。E.在仁济医院胰腺癌患者队列中MBOAT1的表达与患者总体生存预后的关系。F.在TCGA胰腺癌患者队列中MBOAT1的表达与患者总体生存预后的关系;
图2A.腺癌细胞株AsPC-1、Capan-1、Patu8988、PANC-1以及对照细胞株HPNE和HPDE中MBOAT1的蛋白表达水平;B.AsPC-1和Capan-1细胞系MBOAT1表达干扰效率;C.MBOAT1干扰组和对照组胰腺导管腺癌AsPC-1和Capan-1细胞的细胞活力变化情况;D-E.MBOAT1干扰组和对照组AsPC-1和Capan-1细胞的克隆形成变化情况及统计结果;F.MBOAT1干扰组和对照组AsPC-1和Capan-1细胞的EdU荧光染料渗入变化情况。标尺为50μm;
图3A.MBOAT1干扰组和对照组胰腺导管腺癌AsPC-1细胞在裸鼠皮下的成瘤情况;标尺为1cm.B-C.MBOAT1干扰组和对照组AsPC-1细胞在裸鼠皮下的肿瘤生长曲线(B)和重量(C)统计情况;D-E MBOAT1干扰组和对照组皮下瘤MBOAT1(D)和PCNA(E)免疫组化染色情况;标尺为100μm.F.对照组小鼠、MBOAT1干扰组、吉西他滨化疗组和MBOAT1干扰联合化疗组C57BL6/J小鼠胰腺原位小动物成像结果和总体生存曲线。这些结果进一步明确了MBOAT1在胰腺导管腺癌的发生发展过程中发挥着非常重要的作用,提示干预MBOAT1的表达能够抑制胰腺癌的生长。
具体实施方式
下文将结合具体实施方案和实施例,具体阐述本发明,本发明的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解,这些具体实施方案和实施例是用于说明本发明,而非限制本发明。
实施例1、本发明MBOAT1高表达与胰腺癌不良预后呈正相关
申请人首先使用生信分析,寻找在胰腺导管腺癌中表达显著上调的基因,并发现了酰基转移蛋白酶MBOAT1。利用多个胰腺癌GEO数据库(GSE15471、GSE16515和GSE28735)和仁济胰腺癌测序数据进行验证,发现MBOAT1 mRNA水平在胰腺导管腺癌组织中表达相较于癌旁组织显著上调(图1A)。随后,用胰腺癌自发KPC模型小鼠来源的胰腺组织进行免疫组化染色,发现在小鼠低级别、高级别胰腺导管上皮内瘤变以及胰腺导管腺癌组织中MBOAT1蛋白表达水平相较于正常小鼠胰腺组织都有显著的上调(图1B),进一步通过免疫组化染色也发现MBOAT1蛋白表达在人胰腺癌组织中明显高于配对的癌旁组织(图1C)。利用仁济医建立的111例胰腺癌组织芯片,进行组化染色,依据MBOAT1不同表达强度分为低表达组(包括打分为-和+的样本)和高表达组(包括打分为++和+++的样本)(图1D)。对低表达组和高表达组临床病人的总体生存预后分析中,申请人发现MBOAT1高表达的胰腺癌患者,总体生存时间较短(图1E)。进一步对TCGA数据库中的胰腺癌患者队列依据MBOAT1表达进行分析,同样发现MBOAT1高表达预示患者预后不良(图1F)。
实施例2、干扰MBOAT1表达抑制胰腺癌细胞增殖
为了进一步研究MBOAT1在胰腺导管腺癌肿瘤细胞中的生物学功能,申请人首先通过WB检测胰腺癌细胞株AsPC-1、Capan-1、Patu8988、PANC-1以及对照细胞株HPNE和HPDE中MBOAT1的蛋白表达水平,结果显示胰腺癌细胞系中MBOAT1的蛋白表达水平明显高于胰腺癌对照细胞株(图2A)。随后,申请人从中选取了MBOAT1相对表达最高的AsPC-1和Capan-1细胞进行后续功能实验。通过稳定表达干扰MBOAT1 shRNA(SEQ ID NO.6和7)的慢病毒感染AsPC-1和Capan-1细胞,从而构建MBOAT1表达沉默的细胞系。通过WB进一步对MBOAT1干扰组细胞和对照组细胞中MBOAT1表达进行分析验证了shRNA的干扰效率(图2B)。然后通过CCK-8细胞活力实验发现,干扰MBOAT1表达的AsPC-1和Capan-1细胞活力明显低于对照组(图2C)。同样的,通过克隆形成实验结果也显示干扰MBOAT1的表达可以抑制胰腺癌细胞的克隆形成(图2D,E)。申请人进一步利用EdU荧光试剂盒检测了MBOAT1干扰组和对照组AsPC-1和Capan-1细胞摄取EdU染料速率的变化情况,并发现干扰MBOAT1可以明显抑制胰腺导管腺癌细胞的摄入EdU染料速率(图2F)。以上体外细胞实验的结果提示,MBOAT1与胰腺导管腺癌细胞的增殖、克隆形成和DNA复制密切相关。
实施例3、干扰MBOAT1表达抑制小鼠胰腺癌体内进展
基于之前细胞体外功能实验的结果,申请人进一步在动物体内研究了MBOAT1对于胰腺导管腺癌进展的影响。申请人首先构建了胰腺癌皮下瘤,通过皮下种植的方法把AsPC-1的对照细胞和MBOAT1干扰细胞接种到裸鼠腹股沟周围的皮下。7周后,取皮下瘤观察,发现MBOAT1干扰组的瘤体明显小于对照组(图3A)。通过绘制肿瘤体积变化曲线,发现MBOAT1干扰组的小鼠的肿瘤生长明显慢于对照组小鼠(图3B)。进一步对皮下瘤进行称重发现干扰组小鼠荷瘤肿瘤显著降低(图3C)。对肿瘤进行切片染色发现干扰组皮下瘤肿瘤细胞增殖标志物PCNA(Proliferating Cell Nuclear Antigen)染色也明显弱于对照组(图3D-E)。随后,申请人构建了胰腺癌原位模型,采用胰腺原位种植的方法,将小鼠胰腺癌细胞KPC1199的MBOAT1干扰组和对照组细胞通过胰岛素针分别接种于C57BL6/J小鼠的胰腺原位。之后定期通过小动物成像检测原位肿瘤的进展。结果显示干扰MBOAT1表达能够明显抑制原位胰腺癌的进展(图3F)。之后,将原位模型小鼠给予吉西他滨化疗药物(50mg/kg,每五天一次)处理,观察小鼠胰腺癌进展。发现干扰MBOAT1与吉他滨化疗能够呈现出协同效应,进一步抑制原位肿瘤的进展,延长小鼠的总体生存时间(图3F)。
Claims (10)
1.MOBAT1在制备治疗癌症的试剂中的用途。
2.根据权利要求1所述的用途,其特征在于,所述试剂为MOBAT1基因抑制剂。
3.根据权利要求2所述的用途,其特征在于,所述MOBAT1基因抑制剂为核酸分子、核酸构建体、慢病毒、抗体或小分子化合物。
4.根据权利要求1-3中任一项所述的用途,其特征在于,所述癌症为胰腺癌。
5.一种核酸分子,所述核酸分子为MOBAT1基因抑制剂,可以降低胰腺癌细胞中MOBAT1基因的表达,包括:
a)双链RNA,所述双链RNA中含有能够在严紧条件下与MOBAT1基因杂交的核苷酸序列;或者
b)shRNA,所述shRNA中含有能够在严紧条件下与MOBAT1基因杂交的核苷酸序列,所述shRNA如SEQ ID NO.6-11任一所示。
6.一种核酸构建体,所述核酸构建体为MOBAT1基因抑制剂,含有编码权利要求5所述的核酸分子的shRNA的基因片段,能表达所述shRNA。
7.一种慢病毒,所述慢病毒为MOBAT1基因抑制剂,由权利要求6所述核酸构建体在慢病毒包装质粒、细胞系的辅助下,经过病毒包装而成。
8.一种治疗胰腺癌药物,包括MOBAT1基因抑制剂的一种或多种。
9.根据权利要求8所述的药物,其中,所述药物还包括说明书;其中,所述说明书包括药物用于治疗癌症的使用信息。
10.一种胰腺癌联合治疗药物组合,包括MOBAT1基因抑制剂和至少一种其他治疗胰腺癌药物。
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