CN110527682B - 一种长链非编码rna及其应用 - Google Patents

一种长链非编码rna及其应用 Download PDF

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CN110527682B
CN110527682B CN201810513771.9A CN201810513771A CN110527682B CN 110527682 B CN110527682 B CN 110527682B CN 201810513771 A CN201810513771 A CN 201810513771A CN 110527682 B CN110527682 B CN 110527682B
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钱政江
杨海洋
李燕娇
韦命余
李伟丽
李翔
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Shenzhen Institute of Advanced Technology of CAS
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Abstract

本发明提供了一种长链非编码RNA及其应用，所述长链非编码RNA为LncRNA00705，位于KLF6基因下游，本发明通过设计精密实验，逐步筛查，发现并确定了该基因的用途，将LncRNA00705作用miR‑181a和KLF6异常引起的疾病的调节因子，用于制备相关疾病的药物，具有广阔的应用前景和市场价值。

Description

一种长链非编码RNA及其应用
技术领域
本发明涉及生物医学领域，尤其涉及一种长链非编码RNA及其应用。
背景技术
长链非编码RNA(Long non-coding RNA，LncRNA)是长度大于200个核苷酸的非编码RNA。LncRNAs通常具有mRNA样结构，经过剪接，具有polyA尾巴与启动子结构，分化过程中有动态的表达与不同的剪接方式。大多数的LncRNAs在组织分化发育过程中，都具有明显的时空表达特异性，在肿瘤与其他疾病中有特征性的表达方式。研究表明，LncRNA在剂量补偿效应、表观遗传调控、细胞周期调控和细胞分化调控等众多生命活动中发挥重要作用，成为遗传学研究热点。目前已有大量报道LncRNAs作用的机制，包括：1)结合到特定蛋白质上，改变该蛋白质的细胞定位，2)作为结构组分与蛋白质形成核酸蛋白质复合体，3)与编码蛋白基因的转录本形成互补双链，在Dicer酶的作用下产生内源性siRNA，4)与编码蛋白基因的转录本形成互补双链，干扰mRNA的剪切，形成不同的剪切形式，5)与小分子RNA(如miRNA、piRNA)特异性结合，调节小分子RNA的活性及功能。
KLF6(Kruppel-like factor 6)基因是一种抑癌基因，它的失活或表达异常参与了多种肿瘤的发生发展。KLF6基因全长约7kb，含有4个外显子，分别为218、574、124、525bp，突变常发生在第2、3外显子，mRNA长1383bp。KLF6表达产物是含283个氨基酸的蛋白，在它的C端有三个KLF6家族共有的连续的C2H2锌指结构，能与多种基因启动子区域的富含GC或CACCC序列结合，从而与DNA相互作用。富含脯氨酸和丝氨酸等酸性氨基酸残基的区域，与转录活性有关；而富含丝氨酸与苏氨酸的区域则与转录或翻译后调节途径有关。研究表明，KLF6基因与生长发育、细胞的分化、生长相关的细胞信号转导、细胞增殖、凋亡以及血管形成、损伤应答等过程都有关系，它的缺失、突变以及失活与多种肿瘤的发生、发展有密切关系。因此，KLF6基因极具潜力作为治疗相关疾病的药物新靶点。已经有众多研究表明LncRNA对小分子RNA如miRNA的表达具有调节作用，并且在各种疾病发生中发挥重要的调控功能。然而针对调节miR-181a的LncRNA研究相对较少，由于已有文献报道miR-181a能够通过与KLF6的3’-UTR结合，从而在转录后水平对KLF6表达进行调控，miR-181a通过靶向KLF6在不同肿瘤中具有重要的调控功能，然而，是否存在LncRNA特异的对miR-181a进行调节继而影响KLF6的表达，最终导致下游细胞功能的改变仍然不清楚。
因此，现有研究进展及技术还有待于深入完善和发展，需研发能够特异调控miR-181a及下游KLF6表达的机制，将对研发相关肿瘤发生的靶向药物具有指导作用。
发明内容
针对现有技术的不足及实际的需求，本发明提供一种长链非编码RNA及其应用，所述长链非编码RNA为LncRNA00705，位于KLF6基因下游，将LncRNA00705作用miR-181a和KLF6异常引起的疾病的调节因子，用于制备相关疾病的药物，具有广阔的应用前景和市场价值。
为达此目的，本发明采用以下技术方案：
第一方面，本发明提供一种长链非编码RNA，所述长链非编码RNA的基因为Lnc00705，其核苷酸序列如SEQ ID No.1所示，其转录本的核苷酸序列如SEQ ID No.2所示。
SEQ ID No.1如下：
GCGGAATATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGCCACCTTTGATGTCTAGAATCAGGGGATCCGAATGTTATTTAATGGTGCCAATGATGTTGGAGGATACCTGTGGGAACCTGGATTAGAAGCATGTGCTGCTCTCAGACCTGGCCTACAGAGAAGAACTGACACAGACTTCCTTAGATGTGTTGTCTTCCTGTCACTGGTGCATTCCTTATCAATGTGGGTGGCTCCGCACTCAGCCCTGTGATCACCTGAGTAATGGGAAGTCAGTGAGTGCAGTGCCTGCAGAGGGAGCGTGCTTGCCAATCACCATCACCCACTTTTGCACTTGTGCCAGAGGATGTGCCCAAGCCAGCCTGAGAAAGATGAAAGACACATGGCTCAGACACTCCATTACCCCATCCCAGGGCCATTAACCTGCCAGGCATAATCCAAAATTCTTATCAAGTTATCTTCAATCTATGCAAGGAAAATATTTATCCCAAAGGAGGATTTTCAGAAGATGACACATGAGGGGCATTCATCACAGTCAAGTCTTTTGTTTTGAAATACGGAATCTGCATACCTACTGCTTTCCAAGTTTTGTAAAAAAGAAAAAAGAAAAATGCTCTGAGAATTATCTAATTCATTTTTGCATAAAGAATTTCCATTTTCTATTTTTCCGTTGTCTAATTCACTTGCATAAAGAATTTTCTTTTTCATGTTTCAATTCACATCTAACAAATGTTATCATACTTTTTCAAATACTCTTTATTTCATTCACTGTAATTCCAATTGGCTTAGTTAATAATCTTACATGTGTAAACCATCCTCAGCCTTTAAATAAGGTTCTTGTTCTGGGATTAAAGAAAAATTTTAATCAATCAATTAATTACTAATTGCATTTTGCATTTAGCACCTACTGCCTCTGAATTTTTTAACTGAAAATATACCTACTCTTATATTTTTATTTTAGCGTTTATTTTTCAAGTCTGCTAGTGACAATCCAAATTATATTTTTTAATCCAGATATTTTTAATTTAGAAATTAGGAGAGGAATTTTGGTAAGCCACACTATCCTCTGAGGAGCAACTGAATATATTAGAATTCTAAATGGTACCACACGATGGGAAAAAAATGGCAAAAATAATCCCAAAGTCTATATTATGTTGAAAACTTCAAATGAAATTCTAAGTCTTCACTAACTGAATGGACTCATTTTTGGCCCAGCGGATGCCAGAAAACCCTTAAGATTGAATTCCTGGACATGACAGGATGGGCGGTGAGGTATGGCTCATTATACTCCCTCACTTTTGGGGTTTAGACACAACTGACCAGCATAGATGTTAAAATACAGATTATAAGAATGGCAGAACAGACTCTCTGACAATACGATATCAAATTATAAACAGAACCTAAACCCATGGCAGGCGAGGTTTAAGTCACTTACCTACCCTTGAAGAATAAGCTTGTTCTCACTGCCATAGCTTTTTTTTTTTTTTGTCTTTCTCTAGCAGCTAAATAAGCACTGGCCTGGAGATAAGCACTAGTGAAAGAATTTGCAGCTCATCCACTGCTGGACCCTGATTAAGTGACCCCCTGTTCCACAAGCCATGACTCCAGCTTTGACTGGACAAGAGACTGATTTCAGTAGCTTTTTTTCATATAAGAAGACCATTGACTGTGGAATGATTCCAGAGGCTGCACACTGTCAGGACTTCATGTCCTGAAAAGACCTTTTGACATGTACGGCCTGATTGCAATGCATTTGAATGTTGTCTCCACCCTAAAGTGAACATGGATCATATGTTACATGCACGTTTGTTCAATGCCCATGTGGCAAGACCCCTTGATGAATATTCGTAACTCCTCCTGTAACCTGTTGAATATGTATGTCCTACCCCAACCCCTCCTCCTTCATGGTGCCTGTCTTTGGTCTTGACTAGAGGCCAACCTGCAGGTTGGCCACCTGGCAGGCTGTAACCCTAAGAAATAAAAACTTCTTTTCTCCTTTTCAAAATGTATAAATTGTGATTTCTATTAAGTCAACAATGTCCAGGAATAATGTTATTCTTGAGACAGTTTTATGATCTGAGCTTTGTTCAAAAACTGGAAAAGTTACAAAGTAAAACCATAATTTTCTTTAGTGGGTTAACTGTGTGCCAATTTAAATTTAATTTTTTCAGCTGTGTTGAGGAAAAATTTATGCCTCCGAAGCTGCACATGTAGAAATTTCACAATTTGAAGAATTTTCACATATGTATGCTGGGTGAAACCACACCATAATCAAGATACCCAACATTTCTACCTTCCCCAAAAGTTTCTTCACACCGTTGTGCATTCATCTCTTCATTCAACCCTTTCTCTGCAATCAATGGTTTACTTCTCGTCAGTATGGATTAATTTGCAATTTCAAGAATGTTGTATAAATGAAATTATAGAGCAGTACGTTTTTGTTTGATTTTATTCACTCACAGAATTATTTCAAGATCCATCCATATTGTTGCATGATTCAGAAGGCTGCTCTTTTTATATTGCTGAGTAGTTTTCAATGGAATATACTACATTTTGTATATCCATAAGCCTATCATGTGCATTTTGGAATGTTTCCATTTTTTTGGTAATTGCAAATAAAACTGCAATGAACACTCACTCGAGTCTAGAGCCCCCGTATCCCGCC.
SEQ ID No.2如下：
GAATGTTATTTAATGGTGCCAATGATGTTGGAGGATACCTGTGGGAACCTGGATTAGAAGCATGTGCTGCTCTCAGACCTGGCCTACAGAGAAGAACTGACACAGACTTCCTTAGATGTGTTGTCTTCCTGTCACTGGTGCATTCCTTATCAATGTGGGTGGCTCCGCACTCAGCCCTGTGATCACCTGAGTAATGGGAAGTCAGTGAGTGCAGTGCCTGCAGAGGGAGCGTGCTTGCCAATCACCATCACCCACTTTTGCACTTGTGCCAGAGGATGTGCCCAAGCCAGCCTGAGAAAGATGAAAGACACATGGCTCAGACACTCCATTACCCCATCCCAGGGCCATTAACCTGCCAGGCATAATCCAAAATTCTTATCAAGTTATCTTCAATCTATGCAAGGAAAATATTTATCCCAAAGGAGGATTTTCAGAAGATGACACATGAGGGGCATTCATCACAGTCAAGTCTTTTGTTTTGAAATACGGAATCTGCATACCTACTGCTTTCCAAGTTTTGTAGAAAAAAAAAAGAAAAAAGAAAAATGCTCTGAGAATTATCTAATTCATTTTTGCATAAAGAATTTCCATTTTCTATTTTTCCGTTGTCTAATTCACTTGCATAAAGAATTTTCTTTTTCATGTTTCAATTCACATCTAACAAATGTTATCATACTTTTTCAAATACTCTTTATTTCATTCACTGTAATTCCAATTGGCTTAGTTAATAATCTTACATGTGTAAACCATCCTCAGCCTTTAAATAAGGTTCTTGTTCTGGGATTAAAGAAAAATTTTAATCAATCAATTAATTACTAATTGCATTTTGCATTTAGCACCTACTGCCTCTGAATTTTTTAACTGAAAATATACCTACTCTTATATTTTTATTTTAGCGTTTGTTTTTCAAGTCTGCTAGTGACAATCCAAATTATATTTTTTAATCCAGATATTTTTAATTTAGAAATTAGGAGAGGAATTTTGGTAAGCCACACTATCCTCTGAGGAGCAACTGAATATATTAGAATTCTAAATGGTACCACACGATGGGAAAAAAATGGCAAAAATAATCCCAAAGTCTATATTATGTTGAAAACTTCAAATGAAATTCTAAGTCTTCACTAACTGAGTGGACTCATTTTTGGCCCAGCGGATGCCAGAAAACCCTTAAGATTGAATTCCTGGACATGACAGGATGGGCGGTGAGGTATGGCTCATTATACTCCCTCACTTTTGGGGTTTAGACACAACTGACCAGCATAGATGTTAAAATACAGATTATAAGAATGGCAGAACAGACTCTCTGACAATACGATATCAAATTATAAACAGAACCTAAACCCATGGCAGGCGAGGTTTAAGTCACTTACCTACCCTTGAAGAATAAGCTTGTTCTCACTGCCATAGCTTTTTTTTTTTTTTTTTTTTTGTCTTTCTCTAGCAGCTAAATAAGCACTGGCCTGGAGATAAGCACTAGTGAAAGAATTTGCAGCTCATCCACTGCTGGACCCTGATTAAGTGACCCCCTGTTCCACAAGCCATGACTCCAGCTTTGACTGGACAAGAGACTGATTTCAGTAGCTTTTTTTCATATAAGAAGACCATTGACTGTGGAATGATTCCAGAGGCTGCACACTGTCAGGACTTCATGTCCTGAAAAGACCTTTTGACATGTACGGCCTGATTGCAATGCATTTGAATGTTGTCTCCACCCTAAAGTGAACATGGATCATATGTTACATGCACGTTTGTTCAATGCCCATGTGGCAAGACCCCTTGATGAATATTCGTAACTCCTCCTGTAACCTGTTGAATATGTATGTCCTACCCCAACCCCTCCTCCTTCATGGTGCCTGTCTTTGGTCTTGACTAGAGGCCAACCTGCAGGTTGGCCACCTGGCAGGCTGTAACCCTAAGAAATAAAAACTTCTTTTCTCCTTTTCAAAATGTATAAATTGTGATTTCTATTAAGTCAACAATGTCCAGGAATAATGTTATTCTTGAGACAGTTTTATGATCTGAGCTTTGTTCAAAAACTGGAAAAGTTACAAAGTAAAACCATAATTTTCTTTAGTGGGTTAACTGTGTGCCAATTTAAATTTAATTTTTTCAGCTGTGTTGAGGAAAAATTTATGCCTCCGAAGCTGCACATGTAGAAATTTCACAATTTGAAGAATTTTCACATATGTATGCTGGGTGAAACCACACCATAATCAAGATACCCAACATTTCTACCTTCCCCAAAAGTTTCTTCACACCGTTGTGCATTCGTCTCTTCATTCAACCCTTTCTCTGCAATCAATGGTTTACTTCTCGTCAGTATGGATTAATTTGCAATTTCAAGAATGTTGTATAAATGAAATTATAGAGCAGTACGTTTTTGTCTGATTTTATTCACTCACAGAATTATTTCAAGATCCATCCATATTGTTGCATGATTCAGAAGGCTGCTCTTTTTATATTGCTGAGTAGTTTTCAATGGAATATACTACATTTTGTATATCCATAAGCCTATCATGTGCATTTTGGAATGTTTCCATTTTTTTGGTAATTGCAAATAAAACTGCAATGAACACTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGAAAAAAAAAAAAAAAAAA.
本发明中，发明人根据UCSC在线数据库(http://genome.ucsc.edu/)，在KLF6基因下游分析发现一个LncRNA，命名为Lnc00705，进一步分析发现Lnc00705的转录本有miR-181a的四个结合位点；利用荧光素酶报告系统分析Lnc00705对miR-181a的结合作用；针对Lnc00705设计siRNA序列，并利用胃粘膜细胞系GES-1细胞分析抑制Lnc00705之后对miR-181a和KLF6表达以及细胞增殖、和凋亡的影响；最终确定了一种内源性、特异性靶向miR-181a，进而调控KLF6表达的长链非编码RNA。
优选地，所述长链非编码RNA的基因位于KLF6基因下游。
优选地，所述长链非编码RNA的基因的转录本具有与miR-181a结合的四个结合位点。
优选地，所述四个结合位点分别对应于Lnc00705基因的第1-7位、第1696-1703位、第2344-2351位和第2529-2536位。
本发明的鉴定方法如下：
1)根据UCSC在线数据库(http://genome.ucsc.edu/)，在KLF6基因下游分析发现一个LncRNA，命名为Lnc00705，进一步分析发现Lnc00705的转录本有miR-181a的四个结合位点；
2)针对Lnc00705设计siRNA序列。利用荧光素酶报告系统分析Lnc00705对miR-181a的结合作用，并利用胃粘膜细胞系GES-1细胞分析抑制Lnc00705之后对miR-181a以及KLF6表达的影响；
3)利用胃粘膜细胞系GES-1细胞分析抑制Lnc00705之后对细胞增殖、和凋亡的影响。
第二方面，本发明一种如第一方面所述的长链非编码RNA用作miR-181a和/或KLF6基因调节因子的用途。
第三方面，本发明提供一种重组载体，所述载体包含第一方面所述的长链非编码RNA。
第四方面，本发明提供一种宿主细胞，所述细胞包含第一方面所述的长链非编码RNA；
优选地，所述宿主细胞包括GES-1、MGC-803、SGC-7901或BGC823中的任意一种或至少两种的组合，优选为GES-1细胞。
第五方面，本发明提供一种siRNA，所述siRNA由Lnc00705设计得到，其核苷酸序列如SEQ ID No.3所示。
SEQ ID No.3如下：CUGACCAGCAUAGAUGUUA.
第六方面，本发明提供一种如第三方面所述的重组载体、第四方面所述的宿主细胞或第五方面所述的siRNA用作miR-181a调节因子的用途。
第七方面，本发明提供一种如如第三方面所述的重组载体、第四方面所述的宿主细胞或第五方面所述的siRNA用作KLF6基因调节因子的用途。
第八方面，本发提供一种如如第三方面所述的重组载体、第四方面所述的宿主细胞或第五方面所述的siRNA用于制备治疗前列腺疾病、神经系统疾病和恶性肿瘤的药物和/或试剂。
优选地，所述恶性肿瘤包括胃癌、列腺癌、肺癌或肝癌中的任意一种或至少两种的组合。
与现有技术相比，本发明具有如下有益效果：
本发明初次发现Lnc00705位于KLF6基因下游，转录产物具有miR-181a的四个结合位点；本发明发现Lnc00705对抑制miR-181a的表达，进而影响KLF6的表达；本发明提供的长链非编码RNA可作为一种新的用于治疗由miR-181a以及KLF6异常表达所引起的疾病，因此，本发明的长链非编码RNA可作为一种新的调节因子用于制备治疗炎症性疾病、前列腺、神经系统疾病及各种恶性肿瘤等的药物。
附图说明
图1为本发明的在线数据库分析结果图，其中图1(A)为Lnc00705在KLF6基因中的位置，图1(B)为Lnc00705转录本中miR-181a的四个结合位点示意图；
图2为本发明的荧光素酶报告分析结果图；
图3为本发明的抑制Lnc00705后miR-181a及KLF6的表达图，其中图3(A)为miR-181a的表达图，图3(B)为KLF6转录水平的表达图，图3(C)为KLF6蛋白水平的表达图；
图4为本发明的抑制Lnc00705表达后对GES-1细胞增殖及凋亡的影响图，其中图4(A)为细胞增殖折线图，图4(B)为细胞凋亡流式图，图4(C)为细胞凋亡柱状图。
具体实施方式
为更进一步阐述本发明所采取的技术手段及其效果，以下结合附图并通过具体实施方式来进一步说明本发明的技术方案，但本发明并非局限在实施例范围内。
实施例1长链非编码RNA的鉴定及荧光素酶活性分析
利用UCSC在线基因组数据库分析，发现在KLF6下游存在一系列潜在的长链非编码RNA，见图1(A)，因此可能存在长链非编码RNA对KLF6进行反馈调节；
基于以往报道，miR-181a对KLF6具有靶向调节作用；因此，对长链非编码RNA转录产物进行分析，发现其中一个长链非编码RNA，Lnc00705的转录产物有miR-181a的四个结合位点，见图1(B)；
Lnc00705d核苷酸序列如SEQ ID No.1所示，其转录本的核苷酸序列如SEQ IDNo.2所示。
SEQ ID No.1如下：
GCGGAATATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGCCACCTTTGATGTCTAGAATCAGGGGATCCGAATGTTATTTAATGGTGCCAATGATGTTGGAGGATACCTGTGGGAACCTGGATTAGAAGCATGTGCTGCTCTCAGACCTGGCCTACAGAGAAGAACTGACACAGACTTCCTTAGATGTGTTGTCTTCCTGTCACTGGTGCATTCCTTATCAATGTGGGTGGCTCCGCACTCAGCCCTGTGATCACCTGAGTAATGGGAAGTCAGTGAGTGCAGTGCCTGCAGAGGGAGCGTGCTTGCCAATCACCATCACCCACTTTTGCACTTGTGCCAGAGGATGTGCCCAAGCCAGCCTGAGAAAGATGAAAGACACATGGCTCAGACACTCCATTACCCCATCCCAGGGCCATTAACCTGCCAGGCATAATCCAAAATTCTTATCAAGTTATCTTCAATCTATGCAAGGAAAATATTTATCCCAAAGGAGGATTTTCAGAAGATGACACATGAGGGGCATTCATCACAGTCAAGTCTTTTGTTTTGAAATACGGAATCTGCATACCTACTGCTTTCCAAGTTTTGTAAAAAAGAAAAAAGAAAAATGCTCTGAGAATTATCTAATTCATTTTTGCATAAAGAATTTCCATTTTCTATTTTTCCGTTGTCTAATTCACTTGCATAAAGAATTTTCTTTTTCATGTTTCAATTCACATCTAACAAATGTTATCATACTTTTTCAAATACTCTTTATTTCATTCACTGTAATTCCAATTGGCTTAGTTAATAATCTTACATGTGTAAACCATCCTCAGCCTTTAAATAAGGTTCTTGTTCTGGGATTAAAGAAAAATTTTAATCAATCAATTAATTACTAATTGCATTTTGCATTTAGCACCTACTGCCTCTGAATTTTTTAACTGAAAATATACCTACTCTTATATTTTTATTTTAGCGTTTATTTTTCAAGTCTGCTAGTGACAATCCAAATTATATTTTTTAATCCAGATATTTTTAATTTAGAAATTAGGAGAGGAATTTTGGTAAGCCACACTATCCTCTGAGGAGCAACTGAATATATTAGAATTCTAAATGGTACCACACGATGGGAAAAAAATGGCAAAAATAATCCCAAAGTCTATATTATGTTGAAAACTTCAAATGAAATTCTAAGTCTTCACTAACTGAATGGACTCATTTTTGGCCCAGCGGATGCCAGAAAACCCTTAAGATTGAATTCCTGGACATGACAGGATGGGCGGTGAGGTATGGCTCATTATACTCCCTCACTTTTGGGGTTTAGACACAACTGACCAGCATAGATGTTAAAATACAGATTATAAGAATGGCAGAACAGACTCTCTGACAATACGATATCAAATTATAAACAGAACCTAAACCCATGGCAGGCGAGGTTTAAGTCACTTACCTACCCTTGAAGAATAAGCTTGTTCTCACTGCCATAGCTTTTTTTTTTTTTTGTCTTTCTCTAGCAGCTAAATAAGCACTGGCCTGGAGATAAGCACTAGTGAAAGAATTTGCAGCTCATCCACTGCTGGACCCTGATTAAGTGACCCCCTGTTCCACAAGCCATGACTCCAGCTTTGACTGGACAAGAGACTGATTTCAGTAGCTTTTTTTCATATAAGAAGACCATTGACTGTGGAATGATTCCAGAGGCTGCACACTGTCAGGACTTCATGTCCTGAAAAGACCTTTTGACATGTACGGCCTGATTGCAATGCATTTGAATGTTGTCTCCACCCTAAAGTGAACATGGATCATATGTTACATGCACGTTTGTTCAATGCCCATGTGGCAAGACCCCTTGATGAATATTCGTAACTCCTCCTGTAACCTGTTGAATATGTATGTCCTACCCCAACCCCTCCTCCTTCATGGTGCCTGTCTTTGGTCTTGACTAGAGGCCAACCTGCAGGTTGGCCACCTGGCAGGCTGTAACCCTAAGAAATAAAAACTTCTTTTCTCCTTTTCAAAATGTATAAATTGTGATTTCTATTAAGTCAACAATGTCCAGGAATAATGTTATTCTTGAGACAGTTTTATGATCTGAGCTTTGTTCAAAAACTGGAAAAGTTACAAAGTAAAACCATAATTTTCTTTAGTGGGTTAACTGTGTGCCAATTTAAATTTAATTTTTTCAGCTGTGTTGAGGAAAAATTTATGCCTCCGAAGCTGCACATGTAGAAATTTCACAATTTGAAGAATTTTCACATATGTATGCTGGGTGAAACCACACCATAATCAAGATACCCAACATTTCTACCTTCCCCAAAAGTTTCTTCACACCGTTGTGCATTCATCTCTTCATTCAACCCTTTCTCTGCAATCAATGGTTTACTTCTCGTCAGTATGGATTAATTTGCAATTTCAAGAATGTTGTATAAATGAAATTATAGAGCAGTACGTTTTTGTTTGATTTTATTCACTCACAGAATTATTTCAAGATCCATCCATATTGTTGCATGATTCAGAAGGCTGCTCTTTTTATATTGCTGAGTAGTTTTCAATGGAATATACTACATTTTGTATATCCATAAGCCTATCATGTGCATTTTGGAATGTTTCCATTTTTTTGGTAATTGCAAATAAAACTGCAATGAACACTCACTCGAGTCTAGAGCCCCCGTATCCCGCC.
SEQ ID No.2如下：
GAATGTTATTTAATGGTGCCAATGATGTTGGAGGATACCTGTGGGAACCTGGATTAGAAGCATGTGCTGCTCTCAGACCTGGCCTACAGAGAAGAACTGACACAGACTTCCTTAGATGTGTTGTCTTCCTGTCACTGGTGCATTCCTTATCAATGTGGGTGGCTCCGCACTCAGCCCTGTGATCACCTGAGTAATGGGAAGTCAGTGAGTGCAGTGCCTGCAGAGGGAGCGTGCTTGCCAATCACCATCACCCACTTTTGCACTTGTGCCAGAGGATGTGCCCAAGCCAGCCTGAGAAAGATGAAAGACACATGGCTCAGACACTCCATTACCCCATCCCAGGGCCATTAACCTGCCAGGCATAATCCAAAATTCTTATCAAGTTATCTTCAATCTATGCAAGGAAAATATTTATCCCAAAGGAGGATTTTCAGAAGATGACACATGAGGGGCATTCATCACAGTCAAGTCTTTTGTTTTGAAATACGGAATCTGCATACCTACTGCTTTCCAAGTTTTGTAGAAAAAAAAAAGAAAAAAGAAAAATGCTCTGAGAATTATCTAATTCATTTTTGCATAAAGAATTTCCATTTTCTATTTTTCCGTTGTCTAATTCACTTGCATAAAGAATTTTCTTTTTCATGTTTCAATTCACATCTAACAAATGTTATCATACTTTTTCAAATACTCTTTATTTCATTCACTGTAATTCCAATTGGCTTAGTTAATAATCTTACATGTGTAAACCATCCTCAGCCTTTAAATAAGGTTCTTGTTCTGGGATTAAAGAAAAATTTTAATCAATCAATTAATTACTAATTGCATTTTGCATTTAGCACCTACTGCCTCTGAATTTTTTAACTGAAAATATACCTACTCTTATATTTTTATTTTAGCGTTTGTTTTTCAAGTCTGCTAGTGACAATCCAAATTATATTTTTTAATCCAGATATTTTTAATTTAGAAATTAGGAGAGGAATTTTGGTAAGCCACACTATCCTCTGAGGAGCAACTGAATATATTAGAATTCTAAATGGTACCACACGATGGGAAAAAAATGGCAAAAATAATCCCAAAGTCTATATTATGTTGAAAACTTCAAATGAAATTCTAAGTCTTCACTAACTGAGTGGACTCATTTTTGGCCCAGCGGATGCCAGAAAACCCTTAAGATTGAATTCCTGGACATGACAGGATGGGCGGTGAGGTATGGCTCATTATACTCCCTCACTTTTGGGGTTTAGACACAACTGACCAGCATAGATGTTAAAATACAGATTATAAGAATGGCAGAACAGACTCTCTGACAATACGATATCAAATTATAAACAGAACCTAAACCCATGGCAGGCGAGGTTTAAGTCACTTACCTACCCTTGAAGAATAAGCTTGTTCTCACTGCCATAGCTTTTTTTTTTTTTTTTTTTTTGTCTTTCTCTAGCAGCTAAATAAGCACTGGCCTGGAGATAAGCACTAGTGAAAGAATTTGCAGCTCATCCACTGCTGGACCCTGATTAAGTGACCCCCTGTTCCACAAGCCATGACTCCAGCTTTGACTGGACAAGAGACTGATTTCAGTAGCTTTTTTTCATATAAGAAGACCATTGACTGTGGAATGATTCCAGAGGCTGCACACTGTCAGGACTTCATGTCCTGAAAAGACCTTTTGACATGTACGGCCTGATTGCAATGCATTTGAATGTTGTCTCCACCCTAAAGTGAACATGGATCATATGTTACATGCACGTTTGTTCAATGCCCATGTGGCAAGACCCCTTGATGAATATTCGTAACTCCTCCTGTAACCTGTTGAATATGTATGTCCTACCCCAACCCCTCCTCCTTCATGGTGCCTGTCTTTGGTCTTGACTAGAGGCCAACCTGCAGGTTGGCCACCTGGCAGGCTGTAACCCTAAGAAATAAAAACTTCTTTTCTCCTTTTCAAAATGTATAAATTGTGATTTCTATTAAGTCAACAATGTCCAGGAATAATGTTATTCTTGAGACAGTTTTATGATCTGAGCTTTGTTCAAAAACTGGAAAAGTTACAAAGTAAAACCATAATTTTCTTTAGTGGGTTAACTGTGTGCCAATTTAAATTTAATTTTTTCAGCTGTGTTGAGGAAAAATTTATGCCTCCGAAGCTGCACATGTAGAAATTTCACAATTTGAAGAATTTTCACATATGTATGCTGGGTGAAACCACACCATAATCAAGATACCCAACATTTCTACCTTCCCCAAAAGTTTCTTCACACCGTTGTGCATTCGTCTCTTCATTCAACCCTTTCTCTGCAATCAATGGTTTACTTCTCGTCAGTATGGATTAATTTGCAATTTCAAGAATGTTGTATAAATGAAATTATAGAGCAGTACGTTTTTGTCTGATTTTATTCACTCACAGAATTATTTCAAGATCCATCCATATTGTTGCATGATTCAGAAGGCTGCTCTTTTTATATTGCTGAGTAGTTTTCAATGGAATATACTACATTTTGTATATCCATAAGCCTATCATGTGCATTTTGGAATGTTTCCATTTTTTTGGTAATTGCAAATAAAACTGCAATGAACACTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGAAAAAAAAAAAAAAAAAA.
由图1(B)可知，所述四个结合位点分别对应于Lnc00705基因的第1-7位、第1696-1703位、第2344-2351位和第2529-2536位。
利用HEK293A细胞(购自ATCC，Manassas，VA)，在含10％胎牛血清的DMEN培养基培养中培养，待细胞密度达70％时用磷酸钙法转染细胞，共转染KLF6基因3’-UTR双荧光素酶报告载体、Lnc00705的过表达载体、miR-181a结合位点突变的载体、miR-181a类似物以及相应的对照，转染两天后进行荧光素酶报告活性检测，结果如图2所示；
由图2可知，与对照相比，转染miR-181a后降低了荧光素酶活性，而共转染miR-181a以及Lnc00705后荧光素酶活性恢复；对Lnc00705转录本miR-181a结合位点的突变则部分恢复了荧光素酶活性，Lnc00705能够抑制miR-181a对KLF6的靶向调控。
实施例2长链非编码RNA靶向miR-181a抑制KLF6的表达
利用为粘膜细胞系GES-1进行常规培养，当细胞密度达到70％时，通过Lipofectamine 2000(Invitrogen)转染Lnc00705siRNA，转染6小时后更换培养基，继续培养48小时用于实验；
Lnc00705siRNA的核苷酸序列如SEQ ID No.3所示：
CUGACCAGCAUAGAUGUUA.
mRNA和蛋白表达水平的检测分别利用qRT-PCR以及western blot进行，结果如图3(A)-图3(C)所示；
由图3(A)-图3(C)可知，抑制Lnc00705表达后，miR-181a表示上天，而KLF6在mRNA和蛋白水平表达均下调。
实施例3长链非编码RNA对GES-1细胞增殖和凋亡的影响
利用MTT方法以及流式细胞仪对抑制Lnc00705后，GES-1细胞增殖和细胞凋亡的影响，结果如图4(A)-图4(C)所示；
由图4(A)-图4(C)可知，与对照相比，抑制Lnc00705后促进了GES-1细胞的增殖，而抑制了细胞凋亡。
综上所述，本发明提供一种长链非编码RNA及其应用，所述长链非编码RNA为LncRNA00705，位于KLF6基因下游，将LncRNA00705作用miR-181a和KLF6异常引起的疾病的调节因子，用于制备相关疾病的药物，具有广阔的应用前景和市场价值。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。
SEQUENCE LISTING
<110> 中国科学院深圳先进技术研究院
<120> 一种长链非编码RNA及其应用
<130> 2018
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 2684
<212> DNA
<213> 人工合成
<400> 1
gcggaatata cgactcacta tagggagacc caagctggct agcgtttaaa cttaagcttg 60
ccacctttga tgtctagaat caggggatcc gaatgttatt taatggtgcc aatgatgttg 120
gaggatacct gtgggaacct ggattagaag catgtgctgc tctcagacct ggcctacaga 180
gaagaactga cacagacttc cttagatgtg ttgtcttcct gtcactggtg cattccttat 240
caatgtgggt ggctccgcac tcagccctgt gatcacctga gtaatgggaa gtcagtgagt 300
gcagtgcctg cagagggagc gtgcttgcca atcaccatca cccacttttg cacttgtgcc 360
agaggatgtg cccaagccag cctgagaaag atgaaagaca catggctcag acactccatt 420
accccatccc agggccatta acctgccagg cataatccaa aattcttatc aagttatctt 480
caatctatgc aaggaaaata tttatcccaa aggaggattt tcagaagatg acacatgagg 540
ggcattcatc acagtcaagt cttttgtttt gaaatacgga atctgcatac ctactgcttt 600
ccaagttttg taaaaaagaa aaaagaaaaa tgctctgaga attatctaat tcatttttgc 660
ataaagaatt tccattttct atttttccgt tgtctaattc acttgcataa agaattttct 720
ttttcatgtt tcaattcaca tctaacaaat gttatcatac tttttcaaat actctttatt 780
tcattcactg taattccaat tggcttagtt aataatctta catgtgtaaa ccatcctcag 840
cctttaaata aggttcttgt tctgggatta aagaaaaatt ttaatcaatc aattaattac 900
taattgcatt ttgcatttag cacctactgc ctctgaattt tttaactgaa aatataccta 960
ctcttatatt tttattttag cgtttatttt tcaagtctgc tagtgacaat ccaaattata 1020
ttttttaatc cagatatttt taatttagaa attaggagag gaattttggt aagccacact 1080
atcctctgag gagcaactga atatattaga attctaaatg gtaccacacg atgggaaaaa 1140
aatggcaaaa ataatcccaa agtctatatt atgttgaaaa cttcaaatga aattctaagt 1200
cttcactaac tgaatggact catttttggc ccagcggatg ccagaaaacc cttaagattg 1260
aattcctgga catgacagga tgggcggtga ggtatggctc attatactcc ctcacttttg 1320
gggtttagac acaactgacc agcatagatg ttaaaataca gattataaga atggcagaac 1380
agactctctg acaatacgat atcaaattat aaacagaacc taaacccatg gcaggcgagg 1440
tttaagtcac ttacctaccc ttgaagaata agcttgttct cactgccata gctttttttt 1500
ttttttgtct ttctctagca gctaaataag cactggcctg gagataagca ctagtgaaag 1560
aatttgcagc tcatccactg ctggaccctg attaagtgac cccctgttcc acaagccatg 1620
actccagctt tgactggaca agagactgat ttcagtagct ttttttcata taagaagacc 1680
attgactgtg gaatgattcc agaggctgca cactgtcagg acttcatgtc ctgaaaagac 1740
cttttgacat gtacggcctg attgcaatgc atttgaatgt tgtctccacc ctaaagtgaa 1800
catggatcat atgttacatg cacgtttgtt caatgcccat gtggcaagac cccttgatga 1860
atattcgtaa ctcctcctgt aacctgttga atatgtatgt cctaccccaa cccctcctcc 1920
ttcatggtgc ctgtctttgg tcttgactag aggccaacct gcaggttggc cacctggcag 1980
gctgtaaccc taagaaataa aaacttcttt tctccttttc aaaatgtata aattgtgatt 2040
tctattaagt caacaatgtc caggaataat gttattcttg agacagtttt atgatctgag 2100
ctttgttcaa aaactggaaa agttacaaag taaaaccata attttcttta gtgggttaac 2160
tgtgtgccaa tttaaattta attttttcag ctgtgttgag gaaaaattta tgcctccgaa 2220
gctgcacatg tagaaatttc acaatttgaa gaattttcac atatgtatgc tgggtgaaac 2280
cacaccataa tcaagatacc caacatttct accttcccca aaagtttctt cacaccgttg 2340
tgcattcatc tcttcattca accctttctc tgcaatcaat ggtttacttc tcgtcagtat 2400
ggattaattt gcaatttcaa gaatgttgta taaatgaaat tatagagcag tacgtttttg 2460
tttgatttta ttcactcaca gaattatttc aagatccatc catattgttg catgattcag 2520
aaggctgctc tttttatatt gctgagtagt tttcaatgga atatactaca ttttgtatat 2580
ccataagcct atcatgtgca ttttggaatg tttccatttt tttggtaatt gcaaataaaa 2640
ctgcaatgaa cactcactcg agtctagagc ccccgtatcc cgcc 2684
<210> 2
<211> 2642
<212> DNA
<213> 人工合成
<400> 2
gaatgttatt taatggtgcc aatgatgttg gaggatacct gtgggaacct ggattagaag 60
catgtgctgc tctcagacct ggcctacaga gaagaactga cacagacttc cttagatgtg 120
ttgtcttcct gtcactggtg cattccttat caatgtgggt ggctccgcac tcagccctgt 180
gatcacctga gtaatgggaa gtcagtgagt gcagtgcctg cagagggagc gtgcttgcca 240
atcaccatca cccacttttg cacttgtgcc agaggatgtg cccaagccag cctgagaaag 300
atgaaagaca catggctcag acactccatt accccatccc agggccatta acctgccagg 360
cataatccaa aattcttatc aagttatctt caatctatgc aaggaaaata tttatcccaa 420
aggaggattt tcagaagatg acacatgagg ggcattcatc acagtcaagt cttttgtttt 480
gaaatacgga atctgcatac ctactgcttt ccaagttttg tagaaaaaaa aaagaaaaaa 540
gaaaaatgct ctgagaatta tctaattcat ttttgcataa agaatttcca ttttctattt 600
ttccgttgtc taattcactt gcataaagaa ttttcttttt catgtttcaa ttcacatcta 660
acaaatgtta tcatactttt tcaaatactc tttatttcat tcactgtaat tccaattggc 720
ttagttaata atcttacatg tgtaaaccat cctcagcctt taaataaggt tcttgttctg 780
ggattaaaga aaaattttaa tcaatcaatt aattactaat tgcattttgc atttagcacc 840
tactgcctct gaatttttta actgaaaata tacctactct tatattttta ttttagcgtt 900
tgtttttcaa gtctgctagt gacaatccaa attatatttt ttaatccaga tatttttaat 960
ttagaaatta ggagaggaat tttggtaagc cacactatcc tctgaggagc aactgaatat 1020
attagaattc taaatggtac cacacgatgg gaaaaaaatg gcaaaaataa tcccaaagtc 1080
tatattatgt tgaaaacttc aaatgaaatt ctaagtcttc actaactgag tggactcatt 1140
tttggcccag cggatgccag aaaaccctta agattgaatt cctggacatg acaggatggg 1200
cggtgaggta tggctcatta tactccctca cttttggggt ttagacacaa ctgaccagca 1260
tagatgttaa aatacagatt ataagaatgg cagaacagac tctctgacaa tacgatatca 1320
aattataaac agaacctaaa cccatggcag gcgaggttta agtcacttac ctacccttga 1380
agaataagct tgttctcact gccatagctt tttttttttt tttttttttg tctttctcta 1440
gcagctaaat aagcactggc ctggagataa gcactagtga aagaatttgc agctcatcca 1500
ctgctggacc ctgattaagt gaccccctgt tccacaagcc atgactccag ctttgactgg 1560
acaagagact gatttcagta gctttttttc atataagaag accattgact gtggaatgat 1620
tccagaggct gcacactgtc aggacttcat gtcctgaaaa gaccttttga catgtacggc 1680
ctgattgcaa tgcatttgaa tgttgtctcc accctaaagt gaacatggat catatgttac 1740
atgcacgttt gttcaatgcc catgtggcaa gaccccttga tgaatattcg taactcctcc 1800
tgtaacctgt tgaatatgta tgtcctaccc caacccctcc tccttcatgg tgcctgtctt 1860
tggtcttgac tagaggccaa cctgcaggtt ggccacctgg caggctgtaa ccctaagaaa 1920
taaaaacttc ttttctcctt ttcaaaatgt ataaattgtg atttctatta agtcaacaat 1980
gtccaggaat aatgttattc ttgagacagt tttatgatct gagctttgtt caaaaactgg 2040
aaaagttaca aagtaaaacc ataattttct ttagtgggtt aactgtgtgc caatttaaat 2100
ttaatttttt cagctgtgtt gaggaaaaat ttatgcctcc gaagctgcac atgtagaaat 2160
ttcacaattt gaagaatttt cacatatgta tgctgggtga aaccacacca taatcaagat 2220
acccaacatt tctaccttcc ccaaaagttt cttcacaccg ttgtgcattc gtctcttcat 2280
tcaacccttt ctctgcaatc aatggtttac ttctcgtcag tatggattaa tttgcaattt 2340
caagaatgtt gtataaatga aattatagag cagtacgttt ttgtctgatt ttattcactc 2400
acagaattat ttcaagatcc atccatattg ttgcatgatt cagaaggctg ctctttttat 2460
attgctgagt agttttcaat ggaatatact acattttgta tatccataag cctatcatgt 2520
gcattttgga atgtttccat ttttttggta attgcaaata aaactgcaat gaacactcaa 2580
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaggaaaaaa aaaaaaaaaa 2640
aa 2642
<210> 3
<211> 19
<212> RNA
<213> 人工合成
<400> 3
cugaccagca uagauguua 19

Claims (2)

1.一种长链非编码RNA的siRNA在制备调节细胞中miR-181a和/或KLF6基因的试剂中的应用；
所述长链非编码RNA的核苷酸序列如SEQ ID No.1所示，其转录本的核苷酸序列如SEQID No.2所示；
所述siRNA的核苷酸序列如SEQ ID No.3所示；
所述siRNA调节miR-181a基因表达上调，KLF6基因表达下调；
所述细胞为HEK293A细胞或粘膜细胞系GES-1。
2.一种长链非编码RNA的过表达载体在制备调节细胞中miR-181a和/或KLF6基因的试剂中的应用；
所述长链非编码RNA的核苷酸序列如SEQ ID No.1所示，其转录本的核苷酸序列如SEQID No.2所示；
所述过表达载体调节miR-181a基因表达下调，KLF6基因表达上调；
所述细胞为HEK293A细胞或粘膜细胞系GES-1。
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