CN114807348B - 长链非编码rna lra-1及其干扰rna在治疗动脉粥样硬化中的应用 - Google Patents
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Abstract
本发明公开了一种长链非编码RNA LRA‑1及其干扰RNA在治疗动脉粥样硬化中的应用,属于生物医药技术领域。所述长链非编码RNA LRA‑1的核苷酸序列如SEQ ID NO.1所示。本发明首次发现LRA‑1与动脉粥样硬化疾病之间的关系,从RNA水平为动脉粥样硬化提供新的诊疗靶点,也为动脉粥样硬化的治疗提供了新的技术手段。
Description
技术领域
本发明涉及生物医药技术领域,具体涉及一种长链非编码RNA LRA-1及其干扰RNA在治疗动脉粥样硬化中的应用。
背景技术
动脉粥样硬化(atherosclerosis,AS)是冠心病、心肌梗死和脑梗死等心脑血管疾病发病的重要病理基础之一,且心血管疾病仍然是世界范围内死亡的主要原因。动脉粥样硬化与心血管疾病中的基本血管改变有关。以前的研究表明动脉粥样硬化是脂质紊乱和慢性炎症疾病的结合。血管内皮细胞凋亡是动脉粥样硬化发生和发展的关键环节,对人类健康造成巨大威胁。因此,抑制血管内皮细胞凋亡是动脉粥样硬化治疗的有效策略。
长非编码RNA(LncRNAs)属于一类长度大于200个核苷酸的非编码RNA,具有表观遗传调控潜力,在多种病理条件下经常被调控,在广泛的生物学过程中表现出多种功能。LncRNA在细胞中的分布能够部分指征其生物学功能:定位在细胞核中的LncRNA往往参与到基因转录的调控中,其中一种主要方式就是对其邻近基因的顺式转录调控;而在细胞质中也存在大量的有重要功能的LncRNA,其中以竞争性内源RNA(ceRNA)发挥功能的LncRNA就主要分布在细胞质中。目前,尽管有数千个LncRNAs被鉴定出来,但是,只有很少量的LncRNA的功能被阐明,大量的LncRNA的功能急需要我们搞清楚。现有研究表明,LncRNA可以通过调节血管内皮细胞、平滑肌细胞、脂类代谢、炎症及免疫影响动脉粥样硬化的形成。但LncRNA种类繁多,在不同物种间的保守性较低,表达上兼具时间特异性和组织特异性,因此,不同种类的LncRNA在功能上存在差异。目前仍有众多的LncRNA未被挖掘,其与AS关系也尚未明朗。对于LncRNA生物学功能和机制仍需进一步探究。
发明内容
针对上述现有技术,本发明的目的是提供一种长链非编码RNA LRA-1及其干扰RNA在治疗动脉粥样硬化中的应用。本发明首次发现LRA-1与动脉粥样硬化疾病之间的关系,从RNA水平为动脉粥样硬化提供新的诊疗靶点。
为实现上述目的,本发明采用如下技术方案:
本发明的第一方面,提供长链非编码RNA LRA-1作为靶点在如下(1)或(2)中的应用:
(1)制备诊断动脉粥样硬化的产品;
(2)制备治疗动脉粥样硬化的药物;
所述长链非编码RNA LRA-1的核苷酸序列如SEQ ID NO.1所示;具体如下:
TCAGTAAGGCCCTTTTTCCCTTAATAGATAGTTACATATGTAGATAGATAGACAGATAATCTGTCTAGAGCTACATATTTATCTACATATCCACAAACATAAATACATATAATAATGAATTATTATCACTATGTAGAGATATCCAACGATTTCTATTAGGGAAATAAATCTTTTATTGAAGTTCAGAGAGAAAAGCAATCTTT。
本发明的第二方面,提供用于检测上述长链非编码RNA LRA-1的试剂在制备诊断动脉粥样硬化的产品中的应用。
优选的,所述试剂为引物;更优选的,所述引物的序列如SEQ ID NO.2和SEQ IDNO.3所示;具体如下:
上游引物:5'-GCTTCTGGGTGTGCTGTGTA-3',(SEQ ID NO.2)
下游引物:5'-AGGTCCCTCTGCTAAGCACT-3'。(SEQ ID NO.3)
本发明的第三方面,提供抑制长链非编码RNA LRA-1的试剂在制备治疗动脉粥样硬化的药物中的应用。
上述应用中,所述抑制长链非编码RNA LRA-1的试剂选自微小RNA分子(microRNA,miRNA)、干扰小RNA(small interfering RNA,siRNA)或人工miRNA(artificial microRNA,amiRNA)中的一种或多种。
优选的,所述试剂为靶向长链非编码RNA LRA-1的siRNA;更优选的,所述siRNA的序列如SEQ ID NO.4和SEQ ID NO.5所示;具体如下:
正义链:GUAGAGAUAUCCAACGAUUTT;(SEQ ID NO.4)
反义链:AAUCGUUGGAUAUCUCUACTT。(SEQ ID NO.5)
本发明的有益效果:
本发明选择oxLDL处理血管内皮细胞来体外模拟动脉粥样硬化病理条件,通过对动脉粥样硬化HUVECs模型进行芯片测序,发现了一种长链非编码RNA(lncRNA)分子NONHSAT152179(NONCODE Gene ID:NONHSAG058073.1)受oxLDL诱导的HUVECs凋亡模型中表达显著上调,同时该lncRNA分子在动脉粥样硬化患者血清中高表达,将该lncRNA分子命名为LRA-1(Lncrna related to apoptosis-1)。LRA-1基因,长度约为203bp,位于人的1号染色体,目前,尚未报道这个lncRNA的功能。通过构建LRA-1的小干扰siRNA,对HUVECs凋亡进行干扰,实现了利用LRA-1治疗动脉粥样硬化。因此,本发明为LRA-1的生物学功能提供了重要的补充,为诊疗动脉粥样硬化提供了新的分子标记和干预靶点,也为动脉粥样硬化的治疗提供了新的技术手段。
附图说明
图1:LRA-1在动脉粥样硬化患者及健康对照的血清中的表达水平量化统计。通过对动脉粥样硬化患者及健康对照的血清LRA-1表达情况进行分析可知,动脉粥样硬化患者血清中LRA-1相对表达量为(29.98±0.65),而健康对照则为(10.67±0.46),**p<0.01,n=8。
图2:LRA-1在动脉粥样硬化斑块及正常正常血管内膜的表达水平量化统计。通过对动脉粥样硬化斑块及正常正常血管内膜LRA-1表达情况进行分析可知,动脉粥样硬化斑块中LRA-1相对表达量为(18.95±0.35),而健康对照则为(3.3±0.67),***p<0.001,n=8。
图3:LRA-1在oxLDL、CoCl2和高糖三个模型下诱导的血管内皮细胞凋亡中的相对表达量。**p<0.01,***p<0.001,n=3。
图4:siRNA转染效率(A)及在oxLDL、CoCl2和高糖三个模型下诱导的血管内皮细胞凋亡中LRA-1的相对表达量(B-D)。*p<0.05,**p<0.01,n=3。
图5:在oxLDL、CoCl2和高糖三个模型下诱导的血管内皮细胞中转染LRA-1的小干扰siRNA通过Hoechst33258染色观察HUVECs凋亡现象(A)以及HUVECs凋亡率(B)。
具体实施方式
应该指出,以下详细说明都是例示性的,旨在对本申请提供进一步的说明。除非另有指明,本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
如前所述,内皮细胞凋亡或炎症反应所引起的内皮细胞功能障碍是动脉血管发生粥样硬化的一个主要环节。内皮细胞(Ec)功能障碍被认为是早期动脉粥样硬化的重要标志,持续的不良刺激可能导致动脉血管的Ec损伤和细胞凋亡,而内皮损伤或损伤后的内皮修复是预防动脉粥样硬化的关键步骤。
目前虽有研究表明lncRNA在调节血管生成、脂质代谢、炎症反应、细胞增殖和凋亡中起重要作用。但由于我们对AS形成机制的了解有限,以及lncRNA的多样性和复杂性,lncRNA对As的调控机制还有很多未知。
基于此,为了从RNA水平为动脉粥样硬化提供新的诊疗靶点,本发明结合基因芯片和荧光定量PCR技术发现LncRNA NONHSAT152179(NONCODE Gene ID:NONHSAG058073.1)是在芯片测序中受凋亡调控上调倍数最大的基因。将该lncRNA分子命名为LRA-1(Lncrnarelated to apoptosis-1),长度约为203bp,位于人的1号染色体,目前,尚未报道这个lncRNA的功能。
为进一步验证这个lncRNA的功能,本发明使用50μg/mL oxLDL、100μM CoCl2和35mM高糖在HUVEC中建立模拟动脉粥样硬化的模型,通过设计有关LRA-1荧光定量PCR扩增的引物序列以及沉默LRA-1(siRNA)序列。荧光定量PCR实验进一步证明了基因芯片测序结果。结果表明:LRA-1与动脉粥样硬化相关,是动脉粥样硬化新的诊疗靶点。因此,本发明为LRA-1的生物学功能提供了重要的补充,为诊疗动脉粥样硬化提供了新的分子标记和干预靶点,也为动脉粥样硬化的治疗提供了新的技术手段,由此提出了本发明。
为了使得本领域技术人员能够更加清楚地了解本申请的技术方案,以下将结合具体的实施例详细说明本申请的技术方案。如果实施例中未注明的实验具体条件,通常按照常规条件,或者按照试剂公司所推荐的条件;下述实施例中所用的试剂、耗材等,如无特殊说明,均可通过商业途径获得。
实施例1:血清LncRNA分析
(1)标本采集:采集所有研究对象的空腹静脉血5mL(患者于手术前采集,体检健康者于体检时采集),置于含促凝胶的真空采血管中,2000r/min离心10分钟,吸取上层血清置于无RNase的1.5mL EP管中并于-80℃冰箱保存备用。
(2)RNA的提取:血清样品用TriZol吹打裂解细胞并收集;加入适量氯仿,剧烈震荡20s,静置2-3min;4℃下,12 000g,离心15min;取上层水相加入等体积的异丙醇,混匀于-20℃放置2h,产生RNA白色沉淀;离心12 000g,10min,弃上清,用DEPC水配制的75%酒精洗涤沉淀2次,分别离心7 500g,5min,吸去上清,空气干燥;待乙醇挥发后,取10-15μL DEPC水溶解RNA;取出1μL样品,用DEPC水1:50稀释后;用Nanodrop 2000对总RNA的浓度进行定量。
(3)反转录:
42℃,2min,将产物置于冰上;
反转录反应体系,(20μL体系):
反应条件:37℃,5min;85℃,5s;4℃保存。
(4)实时荧光定量PCR反应体系(20μL):
其中:
PCR Forward Primer:5'-GCTTCTGGGTGTGCTGTGTA-3',(SEQ ID NO.2)
PCR Reverse Primer:5'-AGGTCCCTCTGCTAAGCACT-3'。(SEQ ID NO.3)
反应条件:95℃预变性20s;95℃变性30s,58℃退火30s,72℃延伸30s,循环40次;72℃延伸5min。
结果分析:使用MJ Real-Time PCR仪进行实时荧光定量PCR,Opticon MonitorTMSoftware对实验结果进行分析。结果用2^(-ΔΔCt)法计算,做出柱形图。
结果表明:与正常人血清相比,LRA-1在动脉粥样硬化患者血清中的表达显著上调(图1)。
实施例2:组织LncRNA分析
(1)手术切除动脉粥样硬化患者主动脉斑块组织及正常血管内膜,将采集到的组织置于-80℃保存。
(2)实验时将组织取出,加入Trizol溶液500μL于4℃裂解30min,1000g离心10min。
(3)随后取上清液用苯酚-氯仿-异戊醇抽提,无水乙醇沉淀。待残留乙醇完全挥发后加入50μL去离子水溶解RNA。
(4)用逆转录试剂盒将RNA逆转录为cDNA,参照实施例1的荧光定量PCR的方法对LRA-1的表达量进行检测。分别检测动脉粥样硬化患者的斑块组织及其正常血管内膜中LRA-1表达水平,应用2^(-ΔΔCt)公式计算得到LRA-1的相对表达量。
结果表明:与正常血管内膜相比,斑块组织中LRA-1表达明显降低(图2)。
实施例3:qPCR检测LRA-1在oxLDL、CoCl2和高糖三个模型下诱导的血管内皮细胞凋亡中的水平。
Ox-LDL的动脉沉积与动脉粥样硬化相关的发病率有着密切的联系,为了模拟动脉粥样硬化血管内皮细胞损伤,我们以50μg/ml oxLDL建模进行以下实验。另外,CoCl2和高糖(HG)也是动脉粥样硬化血管内皮细胞损伤的诱导因子,我们使用100μM CoCl2和35mM高糖(葡萄糖浓度)在血管内皮细胞(HUVEC)中建立模拟动脉粥样硬化的模型。我们使用50μg/mLoxLDL、100μM CoCl2和35mM高糖三个模型处理细胞后,分别用1×PBS冲洗3次后吸尽;加入1mL TriZol吹打裂解细胞并收集后按实施例1所述方法对LRA-1的表达量进行检测。
结果表明,LRA-1在oxLDL、CoCl2和高糖三个模型下诱导的血管内皮细胞凋亡中的表达均上调(图3)。
实施例4:qPCR检测LRA-1干扰效率及其在oxLDL、CoCl2和高糖三个模型下诱导的血管内皮细胞凋亡中的干扰效率。
1、设计LRA-1siRNA序列信息为:
正义链:GUAGAGAUAUCCAACGAUUTT;(SEQ ID NO.4)
反义链:AAUCGUUGGAUAUCUCUACTT。(SEQ ID NO.5)
Scramble SiRNA的核苷酸序列为:UUC UCC GAA CGU GUC ACG UTT。(SEQ IDNO.6)
2、转染效率测定:
(1)50μg/mL oxLDL、100μM CoCl2和35mM高糖三个模型处理后的细胞,分别用1×PBS冲洗3次后吸尽;加入1mL TriZol吹打裂解细胞并收集;加入0.3mL氯仿,剧烈震荡20s,静置2-3min;4℃下,12 000g,离心15min;取上层水相加入等体积的异丙醇,混匀于-20℃放置2h,产生RNA白色沉淀;离心12 000g,10min,弃上清,用DEPC水配制的75%酒精洗涤沉淀2次,分别离心7 500g,5min,吸去上清,空气干燥;待乙醇挥发后,取10-15 L DEPC水溶解RNA;取出1L样品,用DEPC水1:50稀释后;用Nanodrop 2000对总RNA的浓度进行定量。
(2)用逆转录试剂盒将RNA逆转录为cDNA,使用实时荧光定量PCR试剂盒对LRA-1的表达量进行检测,配套使用荧光定量PCR仪进行检测。
(3)使用MJ Real-Time PCR仪进行实时荧光定量PCR,Opticon MonitorTMSoftware对实验结果进行分析。结果用2^(-ΔΔCt)法计算,做出柱形图。
采用LRA-1siRNA进行干扰,LRA-1siRNA分别设置了三个浓度梯度依次为:10nM、20nM、40nM。
(4)LRA-1siRNA的转染:
(1)转染前一天对HUVECs进行传代,使细胞汇合度为60%~70%;
(2)制备转染复合物:以24孔板为例,每孔加入1.25μL siRNA母液(20μM),稀释到100μL Opti-M199培养基为A液,取1μl LipofectamineTM RNAi MAX溶解于100μLOpti-M199培养基为B液,B液混合5min后,A液和B液混合,静置20min后加入到细胞培养板中;
(3)37℃含5%CO2培养箱孵育6h后,更换为原细胞生长培养基。
QPCR结果表明,与Scramble SiRNA相比,10、20和40nM LRA-1SiRNA均使LRA-1的表达显著下降,确定siRNAs对LRA-1具有靶向抑制作用,且转染有效。
进一步实验证明,与转染Scramble SiRNA组相比,转染SiRNA LRA-1(40nM)后均能抑制在oxLDL、CoCl2和高糖三个模型下诱导的血管内皮细胞凋亡中LRA-1的上调;ScrambleSiRNA,si-LRA-1转染组;数据以均值±标准差表示。P<0.001vs.Scramble SiRNA(图4)。
实施例5:在oxLDL、CoCl2和高糖三个模型下诱导的血管内皮细胞中转染LRA-1的小干扰siRNA通过Hoechst33258染色观察HUVECs凋亡现象。
取部分细胞在转染12h进行Hoechst33258染色,检测凋亡细胞。
(1)配染液:0.1mg/mL Hoechst33258储液用完全培养液稀释100倍,Hoechst33258染液终浓度为10μg/mL;
(2)吸出细胞转染后的完全培养基,用1×PBS洗两遍,加入2mL/皿Hoechst33258(10μg/mL)染液;
(3)37℃孵育15min;
(4)吸出染液,用1×PBS洗两遍,再加入500μL/皿PBS;
(5)倒置荧光显微镜下观察并拍照。
为了进一步验证LRA-1是否影响ox-LDL、CoCl2和高糖诱导的HUVECs的凋亡水平,本发明通过Hochest33258检测了si-RNA转染细胞24小时后的细胞凋亡水平,免疫荧光结果表明干扰掉LRA-1并加入50μg/mL ox-LDL、100μM CoCl2和35mM高糖处理,相比于ScrambleSiRNA干扰组,LRA-1干扰组内皮细胞凋亡率均是下降的(图5)。
以上所述仅为本申请的优选实施例而已,并不用于限制本申请,对于本领域的技术人员来说,本申请可以有各种更改和变化。凡在本申请的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本申请的保护范围之内。
SEQUENCE LISTING
<110> 济南大学
<120> 长链非编码RNA LRA-1及其干扰RNA在治疗动脉粥样硬化中的应用
<130> 2022
<160> 6
<170> PatentIn version 3.5
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<213> 长链非编码RNA LRA-1
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tcagtaaggc cctttttccc ttaatagata gttacatatg tagatagata gacagataat 60
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tattatcact atgtagagat atccaacgat ttctattagg gaaataaatc ttttattgaa 180
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Claims (4)
1.用于检测长链非编码RNA LRA-1的试剂在制备诊断动脉粥样硬化的产品中的应用;所述长链非编码RNA LRA-1的核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的应用,其特征在于,所述试剂为引物。
3.根据权利要求2所述的应用,其特征在于,所述引物的序列如SEQ ID NO.2和SEQ IDNO.3所示。
4.抑制长链非编码RNA LRA-1的试剂在制备治疗动脉粥样硬化的药物中的应用;所述长链非编码RNA LRA-1的核苷酸序列如SEQ ID NO.1所示;
所述抑制长链非编码RNA LRA-1的试剂为siRNA,所述siRNA的序列如SEQ ID NO.4和SEQ ID NO.5所示。
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