CN116121377A - 一种食管鳞癌外泌体富含的miRNA作为诊断食管鳞癌的标志物中的应用 - Google Patents
一种食管鳞癌外泌体富含的miRNA作为诊断食管鳞癌的标志物中的应用 Download PDFInfo
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Abstract
本发明提供了一种食管鳞癌外泌体富含的miRNA作为诊断食管鳞癌的标志物中的应用,属于生物医药技术领域,所述miRNA为has‑miR‑1246,该应用以食管鳞癌外泌体富含的miRNA has‑miR‑1246作为诊断标志物,可用于在患癌早期进行食管鳞癌的诊断,辅助提升患者预后生存率。
Description
技术领域
本发明属于生物医药技术领域,特别涉及一种食管鳞癌外泌体富含的miRNA作为诊断食管鳞癌的标志物中的应用。
背景技术
食管癌(Esophageal cancer,EC)是世界上高发病率和高死亡率的恶性肿瘤之一。我国食管癌的病理类型以食管鳞癌(Esophageal squamous cell cancer,ESCC)为主。由于患者早期缺乏特异症状,首次就诊时已经为中晚期,而且缺乏有效的治疗手段,导致临床预后极差,5年总体生存率仅为10%~15%,III-IV期患者5年生存率仅有5%。因此,研究食管癌癌变机理对于提高患者生存至关重要。
MicroRNAs(MiRNAs)是一类高度保守的组织特异性非蛋白编码小RNA,通过负基因调控维持细胞内环境的稳定。平衡的生理环境需要适当调控miRNA的表达,因为这些小分子几乎影响从细胞周期检查点、细胞增殖到细胞凋亡的每一条遗传途径,并具有广泛的靶基因。MiRNAs调控基因表达的机制之一是其“种子”序列主要与靶基因转录的mRNA的3‘端相互作用,基因内miRNA表达的变化可能是由于编码miRNA的宿主基因的表达发生变化。一些研究表明miRNA的表达与多种恶性肿瘤中的转录因子、宿主基因和mRNAs靶标有关。许多研究表明,外泌体miRNA可作为肿瘤的生物标记物,且已在多种肿瘤中得到证实。
发明内容
为了获得一种新的食管鳞癌诊断或治疗途径,本发明提供了一种食管鳞癌外泌体富含的miRNA作为诊断食管鳞癌的标志物中的应用,该应用以食管鳞癌外泌体富含的miRNAhas-miR-1246作为诊断标志物,可用于在患癌早期进行食管鳞癌的诊断,辅助提升患者预后生存率。
本发明通过以下技术方案实现:
本发明提供一种食管鳞癌外泌体富含的miRNA作为诊断食管鳞癌的标志物中的应用,所述miRNA为has-miR-1246。
进一步的,所述食管鳞癌外泌体的直径为30-150nm,表面标志物为Calnexin。
基于同一发明构思,本发明还提供has-miR-1246或其检测试剂在制备食管鳞癌诊断试剂盒或在制备食管鳞癌用药效果评价体系中的应用。
基于同一发明构思,本发明还提供has-miR-1246的抑制剂或食管鳞癌外泌体抑制剂在制备治疗食管鳞癌药物中的应用,所述has-miR-1246的抑制剂包括has-miR-1246转录抑制剂、has-miR-1246转录后加工抑制剂和has-miR-1246的功能抑制剂中的至少一种。
基于同一发明构思,本发明还提供has-miR-1246的靶基因的表达促进剂在制备抑制或减缓食管鳞癌细胞转移药物中的应用,所述has-miR-1246的靶基因为基因LZTFL1。
基于同一发明构思,本发明还提供has-miR-1246和/或基因LZTFL1作为靶标物在筛选抑制或减缓食管鳞癌细胞转移药物中的应用。
基于同一发明构思,本发明还提供一种用于诊断食管鳞癌的试剂盒,包括检测食管鳞癌外泌体的试剂和/或检测has-miR-1246的试剂。
基于同一发明构思,本发明还提供一种治疗食管鳞癌的药物,所述药物包含has-miR-1246的抑制剂、食管鳞癌外泌体分泌抑制剂和基因LZTFL1的表达促进剂中的至少一种,所述has-miR-1246的抑制剂包括has-miR-1246转录抑制剂、has-miR-1246转录后加工抑制剂和has-miR-1246的功能抑制剂中的至少一种。
本发明实施例中的一个或多个技术方案,至少具有如下技术效果或优点:
本发明一种食管鳞癌外泌体富含的miRNA作为诊断食管鳞癌的标志物中的应用,所述miRNA为has-miR-1246,has-miR-1246在食管鳞癌外泌体中高表达且高于细胞内表达,该应用以食管鳞癌外泌体富含的miRNA has-miR-1246作为诊断标志物,可用于在患癌早期进行食管鳞癌的诊断,辅助提升患者预后生存率。
附图说明
为了更清楚地说明本发明实施例中的技术方案,下面将对实施例描述中所需要使用的附图作一简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其它的附图。
图1为食管鳞癌细胞上清对Het-1A细胞的形态和增殖迁移能力影响图:A.食管鳞癌细胞培养基(KYSE150)对正常食管上皮细胞(Het-1A)形态的影响;B.KYSE150细胞外泌体促进Het-1A细胞的增殖能力;C.KYSE150细胞外泌体促进Het-1A细胞的迁移能力;D:PKH67染色,证实外泌体可以被食管鳞癌细胞吸收。
图2为食管鳞癌细胞外泌体的形态尺寸和特异标志蛋白图:A.透射电镜(TEM)观察食管鳞癌细胞来源外泌体形态;图2B.纳米颗粒追踪分析检测外泌体浓度和粒径大小;图2C.Western Blot检测外泌体特异标志蛋白Calenxin。
图3为食管鳞癌外泌体中高表达且高于细胞内表达的miRNA筛选示意图:3A.miRNA测序筛选KYSE150和KYSE450外泌体中高表达miRNA;3B.食管鳞癌细胞和外泌体内miRNA热图测序分析发现novel-has-miR294-3p、novel-has-miR14-5p、novel-has-miR181-3p、has-miR-1246、has-miR-451a、has-miR-619-5p、novel-has-miR116-5pnovel-has-miR283-5p在食管鳞癌外泌体中表达较高。
图4为miR-1246细胞功能学研究结果:4A.miR-1246过表达后增殖能力增强;4B.miR-1246过表达后迁移能力增强。
图5为miR-1246的靶基因筛选示意图:图5A.生物信息学分析miR-1246的靶基因可能是是LZTFL1;图5B.LZTFL1与miR-1246可能的结合位点示意图;图5C.双荧光素酶实验验证二者相关性;图5D.过表达miR-1246后,LZTFL1表达降低。
具体实施方式
下文将结合具体实施方式和实施例,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,这些具体实施方式和实施例是用于说明本发明,而非限制本发明。
在整个说明书中,除非另有特别说明,本文使用的术语应理解为如本领域中通常所使用的含义。因此,除非另有定义,本文使用的所有技术和科学术语具有与本发明所属领域技术人员的一般理解相同的含义。若存在矛盾,本说明书优先。
除非另有特别说明,本发明中用到的各种原材料、试剂、仪器和设备等,均可通过市场购买得到或者可通过现有方法制备得到。
本发明整体思路如下:
亮氨酸拉链转录因子样1(LZTFL1)是调节癌症转移的关键基因之一,LZTFL1作为肿瘤抑制因子,在胃癌和肺癌中被下调。虽然LZTFL1最初被认为是一种转录因子,基于其与转录因子bHLH家族的序列相似性,它是一种细胞质蛋白,被证明与其他细胞质蛋白相互作用,调节纤毛运输和控制β-catenin核信号传导。
本发明收集了食管鳞癌细胞培养上清,处理正常食管上皮细胞,发现Het-1A细胞形态发生变化,且促进了其增殖能力和迁移能力;因此收集食管鳞癌细胞和其分泌的外泌体进行了miRNA测序结果进行分析,novel-has-miR294-3p、novel-has-miR14-5p、novel-has-miR181-3p、has-miR-1246、has-miR-451a、has-miR-619-5p、novel-has-miR116-5pnovel-has-miR283-5p在食管鳞癌细胞外泌体表达高;在细胞功能实验中,过表达miR-1246促进细胞的增殖和迁移能力;数据库预测LZTFL1是miR-1246的靶基因并预测了其结合位点,双荧光素酶实验验证两者相关性。
本申请发现,食管鳞癌细胞条件培养基可以导致正常食管上皮细胞形态发生变化,且促进其增殖和迁移能力;miR-1246在食管鳞癌外泌体中表达较高;过表达miR-1246促进食管鳞癌细胞的增殖和迁移能力;miR-1246的靶基因是LZTFL1。
下面将结合实施例及实验数据对本申请一种食管鳞癌外泌体富含的miRNA作为诊断食管鳞癌的标志物中的应用进行详细说明。
实施例1
食管鳞癌细胞上清对正常食管上皮细胞Het-1A的影响
收集条件培养基,培养Het-1A细胞,观察其形态变化:当食管鳞癌细胞密度生长至60%左右时,PBS清洗数次,更换为无血清的培养基,48小时后,收集培养基并以300g离心10分钟和2000g离心10分钟以除去细胞和大碎片,然后以10,000g离心30分钟以除去小碎片。上清液经0.45μm无菌过滤器过滤后贮存于-80℃(即获得KYSE150条件培养基)。用含有10%FBS、50U/mL青霉素和50U/mL链霉素的新鲜DMEM以1:1稀释条件培养基进行进一步的细胞培养,显微镜下观察细胞形态的变化。收集150条件培养基培养Het-1A细胞,分别在第一天、第三天、第六天、第十天观察Het-1A形态的变化,显微镜下观察并拍照记录,结果表明食管鳞癌细胞KYSE150条件培养基对正常食管上皮细胞Het-1A细胞形态变化有影响,结果如图1A所示。
收集条件培养基,培养Het-1A细胞,检测其增殖能力:分别选取完全培养基、Het-1A条件培养基(Het-1A条件培养基是按照上述所说的方法收集,完全培养基是用含有10%FBS、50U/mL青霉素和50U/mL链霉素的新鲜DMEM配置)、KYSE150条件培养基培养的Het-1A,MTT法检测其增殖能力的变化,每日每组设置5个副孔,检测5天。结果如图1B所示,从图1B可知,KYSE150条件培养基培养的Het-1A(图1B中KYSE150-条件培养基)增殖能力显著高于完全培养基(control)和Het-1A条件培养基(Het-1A-exsomes)培养的Het-1A。
收集条件培养基,培养Het-1A细胞,检测其迁移能力:分别选取完全培养基、Het-1A条件培养基、KYSE150条件培养基培养的Het-1A,Transwell实验检测Het-1A的迁移能力。结果如图1C所示。从图1C可知,KYSE150条件培养基培养的Het-1A迁移能力显著高于完全培养基和Het-1A条件培养基培养的Het-1A。
使用PKH67染料荧光标记KYSE450衍生的外泌体:将450μL外泌体重悬于500μL稀释剂DiluentC中,然后与在1mL稀释剂DiluentC中稀释的4μL PKH67染料混合,然后在室温下温育5分钟,加入含有1% BSA的2mL PBS以终止反应,并通过超速离心法重新分离标记的外泌体。将1×105个细胞铺在置于12孔板中的玻片上,将PKH67标记的外泌体加入到每个孔中,并将细胞在37℃和5% CO2下培养24小时。用PBS洗涤细胞,并在室温下用4%的多聚甲醛固定20分钟。将大约0.2μg/mL的DAPI加入到核染色中,然后用共聚焦激光扫描显微显示PKH67标记的外泌体。结果如图1D所示,图1D中DAPY代表细胞核染色、PKH67染色外泌体分别拍照,MERGE代表将两次拍的照片合并,从图1D可以得出食管鳞癌细胞分泌的外泌体可以被其他细胞吸收。
实施例2
食管鳞癌外泌体特异miRNA的筛选
外泌体的提取
食管鳞癌细胞融合度为50%左右时,弃培养液,PBS洗涤细胞数次,加入新鲜的RPMI1640或DMEM,37℃,5% CO2培养箱中培养48h后收集细胞培养液,300×g离心10min,祛除细胞;2 000×g离心10min,祛除死细胞;10 000×g离心30min祛除细胞碎片;100000×g离心60min,弃去上清,用PBS重悬外泌体。
外泌体的鉴定
对外泌体进行电镜观察:将5-10μL外泌体溶液滴加到铜网上,室温吸附10min左右,用滤纸小心吸去多余的液体,然后向铜网上滴加10μL 2%的磷钨酸溶液,在室温下对外泌体染色处理2min,小心用滤纸吸去多余的染色液,将铜网在室温下晾干,上机观察,电压为120kV。结果如图2A所示。
对外泌体进行粒径分析:是对每个颗粒的布朗运动进行追踪和分析,结合Stockes-Einstein方程式计算出纳米颗粒的流体力学直径和浓度。结果如图2B所示。
对外泌体进行Western Blot分析,通过BCA法检测外泌体浓度,鉴定外泌体标志物Calnexin的表达。结果如图2C表示。
通过对食管鳞癌外泌体miRNA测序分析,发现在外泌体中高表达且高于食管鳞癌细胞内的一组miRNA。
通过高通量测序分析食管鳞癌外泌体miRNA的含量,发现一组食管鳞癌外泌体高表达的miRNA,miR-1246(即has-miR-1246)在Het-1A外泌体中低表达,在KYSE450和KYSE150外泌体中均高表达,提示miR-1246在ESCC细胞外泌体中显著上调。结果如图3A所示。
将食管鳞癌细胞内miRNA含量进行高通量测序分析发现,筛选出在食管鳞癌外泌体中高表达且高于细胞内表达的miRNA,即miR-1246。结果如图3B所示。
实施例3
miR-1246细胞功能学研究
检测食管鳞癌细胞中miR-1246的内源性表达,选取低表达的细胞转染miR-1246mimic,MTT实验结果表明,与对照组相比,过表达miR-1246后,细胞增殖能力增强。结果如图4A所示。划痕实验表明,与NC组相比,过表达miR-1246后能够改变细胞的迁移能力,且迁移能力增强。结果如图4B所示。
实施例4
外泌体miR-1246促进细胞形态学变化的关键基因是LZTFL1
为了进一步了解外泌体miR-1246促进食管鳞癌细胞增殖和迁移的潜在机制,我们使用3个数据库预测miR-1246的潜在靶基因,分别是TargetScan Site Type、RNAhybridMFE、miRanda MFE,最后将三个数据库合并分析筛选出可能的候选基因。结果如图5A所示。使用miRDB数据库分析,结果表明LZTFL1是miR-1246的潜在靶基因,及其可能的结合位点示意图。结果如图5B所示。
双荧光素酶报告实验验证miR-1246与LZTFL1的关系,结果如图5C所示。
此外在食管鳞癌细胞转染miR-1246-mimic后,利用实时荧光定量PCR实验验证miR-1246和靶基因LZTFL1的表达水平,结果显示,过表达miR-1246后,LZTFL1表达降低。结果如图5D所示。
最后,还需要说明的是,术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已描述了本发明的优选实施例,但本领域内的技术人员一旦得知了基本创造性概念,则可对这些实施例作出另外的变更和修改。所以,所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
Claims (8)
1.一种食管鳞癌外泌体富含的miRNA作为诊断食管鳞癌的标志物中的应用,其特征在于,所述miRNA为has-miR-1246。
2.根据权利要求1所述的应用,其特征在于,所述食管鳞癌外泌体的直径为30-150nm,表面标志物为Calnexin。
3.has-miR-1246或其检测试剂在制备食管鳞癌诊断试剂盒或在制备食管鳞癌用药效果评价体系中的应用。
4.has-miR-1246的抑制剂或食管鳞癌外泌体抑制剂在制备治疗食管鳞癌药物中的应用,其特征在于,所述has-miR-1246的抑制剂包括has-miR-1246转录抑制剂、has-miR-1246转录后加工抑制剂和has-miR-1246的功能抑制剂中的至少一种。
5.has-miR-1246的靶基因的表达促进剂在制备抑制或减缓食管鳞癌细胞转移药物中的应用,其特征在于,所述has-miR-1246的靶基因为基因LZTFL1。
6.has-miR-1246和/或基因LZTFL1作为靶标物在筛选抑制或减缓食管鳞癌细胞转移药物中的应用。
7.一种用于诊断食管鳞癌的试剂盒,其特征在于,包括检测食管鳞癌外泌体的试剂和/或检测has-miR-1246的试剂。
8.一种治疗食管鳞癌的药物,其特征在于,所述药物包含has-miR-1246的抑制剂、食管鳞癌外泌体分泌抑制剂和基因LZTFL1的表达促进剂中的至少一种,所述has-miR-1246的抑制剂包括has-miR-1246转录抑制剂、has-miR-1246转录后加工抑制剂和has-miR-1246的功能抑制剂中的至少一种。
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