CN107488734B - miR-19a-3p在制备前列腺癌骨转移诊断试剂和治疗药物中的应用 - Google Patents
miR-19a-3p在制备前列腺癌骨转移诊断试剂和治疗药物中的应用 Download PDFInfo
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Abstract
本发明公开了miR‑19a‑3p在制备前列腺癌骨转移诊断试剂和治疗药物中的应用。本发明研究发现miR‑19a‑3p在前列腺癌骨转移中低表达，且miR‑19a‑3p低表达与前列腺癌骨转移相关，过表达miR‑19a‑3p能在体外抑制骨转移前列腺癌迁移和侵袭，在体内抑制前列腺癌骨转移，以上结果表明，miR‑19a‑3p可应用于制备前列腺癌骨转移诊断试剂、治疗药物中。
Description
技术领域
本发明属于分子生物学领域，更具体地涉及miR-19a-3p在制备前列腺癌骨转移诊断试剂和治疗药物中的应用。
背景技术
前列腺癌(Prostate Cancer,PCa)是男性泌尿生殖系统最常见的恶性肿瘤之一。癌症转移会大大增加癌症患者的死亡率，骨是癌症转移的第二最常见部位，常见骨转移的癌症有乳腺癌、前列腺癌、肺癌和肾癌。在我国超过80％的前列腺癌患者在确诊时已经发生骨转移，而骨转移是导致前列腺癌患者死亡的重要原因，因此，对骨转移的早期诊断，是临床诊疗和预后的关键。此外，对于中晚期前列腺癌患者，手术治疗的效果差，大多数采用药物治疗。根据不同的病情可以采用化学药物治疗、外放射治疗、放射性核素内放射治疗以及各种疗法的综合应用等。然而，放化疗药物不仅作用于肿瘤，还可以作用于肿瘤周围健康的组织，因而在杀伤肿瘤的同时，也给机体带来了很大的副作用，最终影响对肿瘤的治疗效果。因此，本领域迫切需要开发早期诊断前列腺癌骨转移的特异性标志物及抑制前列腺癌骨转移的靶向药物。
miRNA是具有18～24个核苷酸的非编码RNA，其结合信使RNA的3’未翻译区域中的互补性位点且抑制它们的翻译。单一miRNA能够靶向若干mRNA且调控细胞通路和细胞命运。越来越多研究表明，miRNA与许多生物过程相关，如增殖、分分化、细胞周期、细胞凋亡等，还调节各类肿瘤的进展与转移。已有研究表明，一些miRNA会促进肿瘤细胞转移，如脑癌中的miR-10b、结肠直肠癌中的miR-21、前列腺癌中的miR-184。也有发现抑制前列腺癌骨转移的一些miRNA，如miR-143、miR-145和miR-203。目前尚没有miR-19a-3p与前列腺癌骨转移之间的报道。
发明内容
本发明的目的在于提供miR-19a-3p在制备前列腺癌骨转移诊断试剂和治疗药物中的应用。
本发明所采取的技术方案是：
miR-19a-3p在制备前列腺癌骨转移诊断试剂盒中的应用。
作为上述应用的优选，所述试剂含有能定量检测miR-19a-3p和/或其编码基因的试剂。
作为上述应用的优选，所述试剂含有miR-19a-3p的特异性引物、探针及芯片中的一种或多种。
作为上述应用的优选，所述miR-19a-3p选自pri-miRNA、pre-miRNA、成熟miRNA、dsmiRNA及其片段或变体中的一种或几种。
诊断前列腺癌骨转移的试剂盒，含有能定量检测miR-19a-3p和/或其编码基因的试剂。
miR-19a-3p在制备抑制前列腺癌骨转移的药物中的应用。
作为上述应用的优选，所述药物含有以下物质中的一种或多种：1)能促进miR-19a-3p表达的物质；2)能增强miR-19a-3p活性的物质；3)能增长miR-19a-3p有效作用时间的物质；4)能增强miR-19a-3p稳定性的物质；5)模拟miR-19a-3p结构的物质。
作为上述应用的优选，所述药物含有以下物质中的一种或多种：1)miR-19a-3p分子；2)miR-19a-3p修饰物；3)miR-19a-3p模拟物；4)编码miR-19a-3p的DNA；5)表达miR-19a-3p的载体或病毒。
作为上述应用的优选，所述miR-19a-3p选自pri-miRNA、pre-miRNA、成熟miRNA、dsmiRNA及其片段或变体中的一种或几种。
抑制前列腺癌骨转移的药物，所述药物含有以下物质中的一种或多种：1)能促进miR-19a-3p表达的物质；2)能增强miR-19a-3p活性的物质；3)能增长miR-19a-3p有效作用时间的物质；4)能增强miR-19a-3p稳定性的物质；5)模拟miR-19a-3p结构的物质。
本发明的有益效果是：
本发明研究发现miR-19a-3p在前列腺癌骨转移中低表达，且miR-19a-3p低表达与前列腺癌骨转移相关，过表达miR-19a-3p能在体外抑制骨转移前列腺癌迁移和侵袭，在体内抑制前列腺癌骨转移，以上结果表明，miR-19a-3p可应用于制备前列腺癌骨转移诊断试剂、治疗药物中。
附图说明
图1：临床样品及细胞系中miR-19a-3p的表达情况；其中，图A为前列腺癌骨转移组织和无骨转移组织中miR-19a-3p的表达情况；图B为miR-19a-3p表达量与骨转移状态的病例统计图，图C为良性前列腺细胞及前列腺癌细胞中miR-19a-3p的表达情况；图中，miR-19a-3p-H为miR-19a-3p高表达组，miR-19a-3p-L为miR-19a-3p低表达组；
图2：构建的表达质粒转染到细胞中的miR-19a-3p表达量；图中，vector表示转染了阴性对照质粒pMSCV-puro、miR-19a-3p表示转染了miR-19a-3p表达质粒、NC表示转染了阴性对照质粒pH1、anti-miR-19a-3p表示转染了anti-miR-19a-3p表达质粒；
图3：miR-19a-3p过表达在体外抑制前列腺癌细胞迁移和侵袭结果；其中，图A为侵袭实验结果，图B为迁移实验结果；图中，vector表示转染了阴性对照质粒pMSCV-puro、miR-19a-3p表示转染了miR-19a-3p表达质粒、NC表示转染了阴性对照质粒pH1、anti-miR-19a-3p表示转染了anti-miR-19a-3p表达质粒；
图4：miR-19a-3p过表达在体内抑制前列腺癌骨转移的结果；其中，图A为骨X射线图，图B为X射线检测骨质破坏程度评分结果；图C为骨HE染色图，图中，vector表示对照组，miR-19a-3p表示miR-19a-3p组；L表示左胫骨，R表示右胫骨。
具体实施方式
现结合具体实验例进一步阐述本发明，但并不局限于此。下述实验例中所使用的实验方法如无特殊说明，均为常规方法，具体条件未注明的，按常规操作或厂家所建议的条件，下述实验例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
本发明的人前列腺癌细胞系22RV1、PC-3、VCaP、DU145、LNCaP和正常的人前列腺上皮细胞RWPE-1由ATCC细胞库提供；人前列腺癌细胞C4-2B购自MD安德森癌症中心；以上细胞系培养方法按本领域常规操作；121例前列腺癌组织(包括76例无骨转移和45例骨转移)由广州市第一人民医院提供，以上临床标本均来自手术过程或穿刺活检，经临床病理学确诊。
实验例1、miR-19a-3p在骨转移前列腺癌组织中低表达
方法：
RNA提取、逆转录和实时定量RT-PCR检测miR-19a-3p：利用TRIzol裂解液(LifeTechnologies公司)提取所有组织和细胞的总RNA，按照Revert Aid First StrandcDNA SynthesisKit(Thermo Fisher公司)说明书进行逆转录，利用SYBR Green I试剂和ABI7500HT实时定量PCR系统(Biosystems公司)，选用2-ddCt相对定量法进行荧光定量检测，以U6为内参，求算miR-19a-3p的相对表达量，miR-19a-3p和U6的引物由广州市锐博生物科技有限公司设计、合成和纯化，miR-19a-3p序列参考miRBase编号MIMAT0000073。
结果：
临床标本和细胞系中miR-19a-3p表达量结果如图1所示，图1A表明，与非骨转移前列腺癌对比，骨转移前列腺癌中miR-19a-3p表达量更低，按前列腺癌组织中miR-19a-3p表达量的中位值将患者划分为miR-19a-3p低表达组和miR-19a-3p高表达组，统计miR-19a-3p表达量与前列腺癌患者骨转移状态的病例数，如图1B所示，前列腺癌骨转移患者中，miR-19a-3p低表达比例高于miR-19a-3p高表达比例，而前列腺癌无骨转移患者中，miR-19a-3p高表达比例高于miR-19a-3p低表达比例，可见miR-19a-3p的表达量与前列腺癌骨转移相关；进一步的，参照图1C，对比正常的人前列腺上皮细胞系RWPE-1，前列腺癌细胞系22RV1和脑转移前列腺癌细胞系DU145中miR-19a-3p高表达，而骨转移前列腺癌细胞系PC-3、C4-2B、VCaP和淋巴结转移前列腺细胞系LNCaP中miR-19a-3p低表达，以上结果一致表明，miR-19a-3p在骨转移前列腺癌组织/细胞中低表达，因此，miR-19a-3p可作为前列腺癌骨转移的生物标志物，可应用于制备前列腺癌骨转移的诊断试剂。
实验例2、miR-19a-3p过表达能在体外抑制前列腺癌迁移和侵袭
方法：
将人miR-19a-3p(miRBase编号MIMAT0000073)基因克隆到pMSCV-puro逆转录病毒载体(Clontech公司)中，获得miR-19a-3p表达质粒，人miR-19a-3p的反义核苷酸(anti-miR-19a-3p)序列克隆到pH1质粒中，由上海吉凯基因化学技术有限公司合成，分别将miR-19a-3p表达质粒、anti-miR-19a-3p表达质粒，及其对应的阴性对照质粒转染到PC-3细胞、C4-2B细胞中，采用Transwell小室法检测细胞迁移和侵袭情况，采用荧光定量检测miR-133b的表达情况
结果：
构建的表达质粒转染到PC-3细胞和C4-2B细胞中，miR-19a-3p的表达量如图2所示，可见miR-19a-3p表达质粒和anti-miR-19a-3p表达质粒分别能上调和下调miR-19a-3p的表达。
迁移和侵袭实验结果如图3所示，与相应的阴性对照质粒相比，转染了miR-19a-3p表达质粒的PC-3细胞和C4-2B细胞，相对迁移率和相对侵袭率均显著降低，而转染了anti-miR-19a-3p表达质粒的细胞，相对迁移率和相对侵袭率显著提高，以上结果说明，miR-19a-3p过表达能在体外抑制前列腺癌迁移和侵袭，反之，miR-19a-3p下调表达能在体外促进前列腺癌迁移和侵袭。
实验例3、miR-19a-3p过表达能在体内抑制前列腺癌骨转移
实验分组及动物模型构建：
①miR-19a-3p组
将实验例2构建的miR-19a-3p转染到PC-3-细胞中，形成稳定高表达miR-19a-3p的PC-3细胞，将其通过胫骨内注射到6只3～4周龄的雄性SCID小鼠的右胫骨中。
②对照组
将实验例2的阴性对照质粒pMSCV-puro转染到PC-3细胞中，形成质粒对照的PC-3细胞，将其通过胫骨内注射到6只3～4周龄的雄性SCID小鼠的左胫骨中。
影像学检测：
X光拍片检查骨转移处骨质破坏情况，根据骨质破坏程度进行评分，0：无骨质破坏，1：轻微骨质破坏；2：少量骨质破坏；3：显著的骨质破坏，伴随胫骨边缘轻微断裂，4：显著的骨质破坏，伴随胫骨边缘大量断裂；随后进行HE染色小鼠胫骨肿瘤切片，进行组织病理学鉴定。
结果：
影像学检测前列腺癌骨转移情况如图4所示，其中，左右胫骨X光图及骨质破坏程度得分如图4A和4B显示，与对照组(左胫骨)相比，miR-19a-3p组小鼠(右胫骨)的骨质破坏程度更小，HE染色小鼠胫骨肿瘤切片结果如图4C，显示miR-19a-3p组小鼠(右胫骨)骨中的肿瘤更小，骨转移更少；以上结果均说明，miR-19a-3p过表达能在体内抑制前列腺癌骨转移，该结果预示着miR-19a-3p可应用于制备抑制前列腺癌骨转移的药物。
Claims (1)
1.miR-19a-3p在制备抑制前列腺癌骨转移的药物中的应用；所述药物含有以下物质中的一种或多种： 1）miR-19a-3p分子；2）miR-19a-3p修饰物；3）miR-19a-3p模拟物；4）编码miR-19a-3p的DNA；5）表达miR-19a-3p的载体或病毒。
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