CN110511910B - 一种LncRNA MALAT1的表达上调剂及其应用 - Google Patents

一种LncRNA MALAT1的表达上调剂及其应用 Download PDF

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CN110511910B
CN110511910B CN201910866784.9A CN201910866784A CN110511910B CN 110511910 B CN110511910 B CN 110511910B CN 201910866784 A CN201910866784 A CN 201910866784A CN 110511910 B CN110511910 B CN 110511910B
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姜霞
尹亚娟
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FIRST HOSPITAL OF HEBEI MEDICAL UNIVERSITY
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Abstract

本发明公开了一种LncRNA MALAT1的表达上调剂所述上调剂为毒胡萝卜素。毒胡萝卜素能够实现MALAT1表达的调控，解决细胞中MALAT1高表达的技术问题，降低成本，并且能够促进肿瘤细胞的转移，进而提高了细胞转移模型的造模成功率，对研究肿瘤的发表机制和肿瘤转移模型的建立具有重要意义。

Description

一种LncRNA MALAT1的表达上调剂及其应用
技术领域
本发明属于肿瘤技术领域，具体涉及一种促进LncRNA MALAT1在肿瘤细胞中表达的上调剂及其应用。
背景技术
人肺腺癌转移相关转录本1基因(Metastasis Associated Lung AdenocarcinomaTranscript 1，MALAT1)是一种长链非编码RNA，是最早被发现有功能的长链非编码RNA之一。MALAT1的功能近几年被多数研究所报道。在肿瘤领域，MALAT1的表达与多种恶性肿瘤(肺癌、乳腺癌、胃癌、大肠癌、前列腺癌等)细胞的增殖和转移正相关。最近还发现MALAT1参与帕金森、动脉粥样硬化及糖尿病血管病变的发生。但关于具体MALAT1调控疾病发生和进展的机制，目前尚未全面阐明。
人MALAT1基因被定位在11q13.1，具体位置信息为chr11:65,497,688-65,506,516(GRCh38/hg38)，长度为8,829bases。由于该基因长度较长，通过传统的构建表达质粒后，由于质粒较大超出10kb，普通的转染方法比较难实现细胞中MALAT1的过表达，只能通过高压电穿孔转染(电转)的方法进行转染，且转染后细胞损失大，超过半数的细胞会在电转的过程中死亡，仅约1/3的细胞能够在电转操作后存活下来。并且该方法需要使用电转仪和电转槽，转染成本很高。MALAT1细胞水平表达调控较为空难，这也是限制MALAT1调控机制研究的主要原因之一。
发明内容
本发明的目的在于提供一种促进LncRNA MALAT1在肿瘤细胞中表达的上调剂及其应用。
一种LncRNA MALAT1的表达上调剂，所述上调剂为毒胡萝卜素。
毒胡萝卜素是一种强有效的可穿透细胞的Ca2+-ATPase抑制剂，能够使Ca2+在细胞内的释放，进而诱导内质网应激。因此在科学研究中常被用作内质网应激激动剂。并且高剂量的毒胡萝卜素能够促进细胞凋亡，也常用于细胞凋亡模型制造。我们在研究中偶然发现毒胡萝卜素(Thapsigargin)低剂量处理肿瘤细胞，能够使MALAT1高表达，并且促进肿瘤细胞的转移，更好的促进肿瘤转移模型的建立。
所述毒胡萝卜素低剂量处理肿瘤细胞，能够使MALAT1高表达；高剂量处理肿瘤细胞能够促进细胞凋亡；所述低剂量处理的浓度为0.005-0.03μM，所述高剂量处理的浓度为大于0.1μM。
一种细胞迁移/侵袭促进剂试剂盒，包含促进剂和稀释液。
所述促进剂为浓度1μM的毒胡萝卜素，溶剂为DMSO。
所述稀释液成分中包括：HEPES 5958mg/L，碳酸氢钠3700mg/L，氯化钠3500mg/L，葡萄糖3000mg/L，L-谷氨酰胺511mg/L，氯化钾400mg/L，L-盐酸赖氨酸146mg/L，磷酸二氢钠108.5mg/L，L-亮氨酸105mg/L，L-异亮氨酸105mg/L，氯化钙100mg/L，硫酸镁97.7mg/L，L-苏氨酸95mg/L，L-缬氨酸94mg/L，L-盐酸精氨酸84mg/L，L-酪氨酸钠盐72mg/L，L-苯丙氨酸66mg/L，L-盐酸胱氨酸63mg/L，L-丝氨酸42mg/L，L-盐酸组氨酸42mg/L，L-甲硫氨酸30mg/L，甘氨酸30mg/L，肌醇30mg/L，L-色氨酸16mg/L，D-泛酸钙4mg/L，氯化胆碱4mg/L，烟酰胺4mg/L，盐酸吡哆辛4mg/L，盐酸硫胺4mg/L，叶酸4mg/L，维生素B12 2mg/L，维生素C 0.5mg/L，核黄素0.4mg/L，生物素0.2mg/L，溶剂为水。
本发明的有益效果：本发明拓展了毒胡萝卜素的应用领域，能够实现MALAT1表达的调控，解决细胞中MALAT1高表达的技术问题，降低成本，并且能够促进肿瘤细胞的转移，进而提高了细胞转移模型的造模成功率。毒胡萝卜素促进LncRNA MALAT1在肿瘤细胞中表达的方法及其应用，对研究肿瘤的发表机制和肿瘤转移模型的建立具有重要意义。
附图说明
图1为毒胡萝卜素对肿瘤细胞HT29生存率的影响。
图2为毒胡萝卜素对肿瘤细胞HCT116生存率的影响。
图3为毒胡萝卜素对肿瘤细胞HT29中MALAT1表达的促进作用。
图4为毒胡萝卜素对肿瘤细胞HCT116中MALAT1表达的促进作用。
图5为细胞迁移/侵袭促进剂试剂盒外观。
具体实施方式
下面结合附图和具体实施例对本发明做进一步说明。
实施例1毒胡萝卜素对肿瘤细胞生存率的影响
取对数生长期的细胞，胰酶消化并悬浮于完全培养基中计数。将细胞接种到96孔板中，每孔9000细胞。贴壁后更换含指定计量毒胡萝卜素的完全培养基，浓度分别为0μM(Control组)，0.1μM(10x组)，0.01μM(1x组)，每孔100μl。将96孔板放入37℃5％CO2的培养箱中培养24小时后，每孔中加入10μl Cell Counting Kit-8试剂，再次孵育3个小时，测定吸光度(450nm波长)，计算各组细胞的生存率。
如图1-2所示，0.01μM(1x组)毒胡萝卜素处理不会引起细胞凋亡，0.1μM(10x组)毒胡萝卜素处理能引起30％-40％细胞凋亡。
实施例2毒胡萝卜素对肿瘤细胞MALAT1表达的影响
取对数生长期的细胞，胰酶消化并悬浮于完全培养基中计数。将细胞接种到6孔板中，每孔5*105细胞。贴壁后更换含指定计量毒胡萝卜素的完全培养基，浓度分别为0μM(Control组)，0.01μM(1x组)，每孔2ml。将6孔板放入37℃5％CO2的培养箱中培养24小时后，提取总RNA并反转录成cDNA，Real-time PCR法检测MALAT1的相对表达量(内参基因为GAPDH)。
如图3-4所示，0.01μM(1x组)毒胡萝卜素处理能够显著促进MALAT1的表达。
实施例3细胞迁移/侵袭促进剂试剂盒的制备
细胞迁移/侵袭促进剂试剂盒，包含促进剂和稀释液。促进剂为浓度1uM的毒胡萝卜素，溶剂为DMSO，总体积为255μL，稀释液规格26mL，稀释液成分如表1所示。
表1稀释液成分表
Figure BDA0002201490640000041
Figure BDA0002201490640000051
细胞迁移/侵袭促进剂试剂盒中100X促进剂在-20℃保存，有效期一年，稀释液4℃保存，有效期一年，该试剂盒能够有效促进细胞的迁移，可用于迁移能力较差的细胞迁移和侵袭实验，实验操作过程如下：
(1)在24孔板中接种细胞悬液，将培养板放在培养箱中预培养12-24小时(培养箱37℃，5％CO2)；
(2)取445μL稀释液，加入胎牛血清50uL,颠倒混匀10次后，加入5uL 100X细胞迁移/侵袭促进剂，混匀备用；
(3)弃去24孔板中培养基，加入步骤(2)混好的稀释液，将培养板在培养箱内孵育12-24小时；
(4)弃去稀释液，PBS洗1-2次。
(5)执行迁移或侵袭实验。
采用本发明的试剂盒进行过表达MALAT1的成本及操作时间与传统方法相比，有明显的进步，具体表现见表2：
表2过表达MALAT1的成本及操作时间对比表
Figure BDA0002201490640000061

Claims (1)

1.低剂量毒胡萝卜素在上调肿瘤细胞LncRNA MALAT1的表达中的应用，其特征在于，所述肿瘤细胞为HT29或HCT116；所述低剂量毒胡萝卜素的浓度为0.005-0.03μM。
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