CN117442732B - lncRNA SCARNA2在抗病毒中的新用途 - Google Patents
lncRNA SCARNA2在抗病毒中的新用途 Download PDFInfo
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Abstract
本发明公开了lncRNA SCARNA2在抗病毒中的新用途,本发明首次发现lncRNA SCARNA2在抗病毒感染中的新用途,并通过实验进行了证实,本发明为制备用于治疗和/或预防各种病毒感染引起的感染性疾病的药物提供了新的思路和策略,为病毒感染的治疗提供了新的靶点,具有广泛的应用前景。
Description
技术领域
本发明属于生物医药技术领域,具体地,涉及lncRNA SCARNA2在抗病毒中的新用途。
背景技术
病毒感染一直以来是威胁人类健康的重要因素之一。病毒感染机体后试图大量复制、逃避免疫系统的监视进而实现病毒自身的存活和传播。天然免疫系统是机体抵抗病原体感染的第一道防线。天然免疫细胞通过表达模式识别受体(PRR)识别病原体表面的病原相关分子模式(PAMP)激活天然免疫信号通路,进而促进炎症和清除病原的相关基因的表达。其中,Ⅰ型干扰素介导的免疫反应在宿主抗病毒过程中发挥关键作用,因此,系统全面地了解Ⅰ型干扰素的调控可以更有效地指导机体对病原体的清除。虽然许多蛋白质在Ⅰ型干扰素调控过程中的重要作用已经得到了很好的阐明,但非编码RNA(ncRNA)在调节干扰素反应中的作用尚不清楚。
在各类ncRNA中,microRNA(miRNA)和长非编码RNA(lncRNA)在生物过程中的研究最广泛,lncRNA作为长度大于200 bp且缺乏蛋白质编码能力的一类数量庞大的非编码RNA,已被证明能够以物种和组织特异性的方式调节mRNA的转录,翻译和转录后的加工。lncRNA可以顺式改变邻近基因的表达,也可以反式与其他分子相互作用在整个细胞中执行各种功能。这些lncRNA可以在细胞生理和发育过程中表现出不同的作用,也可以在癌症、心血管疾病等疾病中表现出不同的作用。近年来,越来越多的研究表明lncRNA同样在免疫调控中通过多种机制发挥重要作用。
Ⅰ型干扰素下游JAK-STAT信号通路中,转录因子STAT1被激活后可以通过其核酸结合结构域与启动子DNA相互作用启动下游干扰素刺激基因(Interferon-stimulatedgenes,ISGs)的转录,ISG编码的蛋白已被报道通过多种机制包括抑制病毒转录、翻译和复制,病毒核酸的降解和细胞脂质代谢的改变来清除机体内的病毒。本申请的发明人首次发现了能与STAT1结合的lncRNA SCARNA2可以促进干扰素的效应,并进一步发现其在病毒感染中能够发挥抗病毒的功能。
发明内容
有鉴于此,本发明的目的在于提供lncRNA SCARNA2在抗病毒中的新用途。
为了实现上述目的,本发明采用了如下技术方案:
本发明的第一方面提供了过表达lncRNA SCARNA2的试剂在制备抗病毒药物中的应用。
进一步,所述病毒为水泡性口炎病毒(VSV)或脑心肌炎病毒(EMCV)。
进一步,所述试剂包括过表达lncRNA SCARNA2的载体、携带lncRNA SCARNA2的纳米颗粒、包裹lncRNA SCARNA2的蛋白微球、包裹lncRNA SCARNA2的脂质体和/或其任意组合。
进一步,所述lncRNA SCARNA2在细胞中的过表达能够显著促进ISG的表达。
进一步,所述lncRNA SCARNA2在细胞中的过表达能够显著抑制病毒mRNA水平的表达、抑制病毒的复制。
在本发明中,所述SCARNA2(small Cajal body-specific RNA 2)是一种lncRNA,在NCBI中的Gene ID为677766。所述SCARNA2具有本领域已知的序列或为其衍生分子。在一些实施方案中,所述SCARNA2为包括如下序列的分子:(a) 具有诸如Gene ID: 677766(人)所示序列的SCARNA2;(b) 在严格条件下与(a)限定的序列杂交的分子;(c) 与(a)或(b)中所示分子的序列具有70%以上(例如75%、80%、85%、90%、95%、98%、99%、99.5%以上,或其间的任何数值或数值范围)的序列同源性的分子,例如经密码子优化获得的SCARNA2。
在一些实施方案中,所述过表达lncRNA SCARNA2的试剂是指任何能够促进lncRNASCARNA2表达的物质,包括但不限于:能够促进lncRNA SCARNA2表达的天然纯化物质、经修饰的天然纯化物质、半合成物质、化学合成物质和/或其任意组合。
在一些实施方案中,所述过表达lncRNA SCARNA2的试剂包括但不限于:含有lncRNA SCARNA2的重组载体、含有lncRNA SCARNA2的纳米颗粒、含有lncRNA SCARNA2的蛋白微球、含有lncRNA SCARNA2的脂质体、含有lncRNA SCARNA2的PEG修饰蛋白、含有lncRNASCARNA2的细胞外囊泡和/或其任意组合。
在一些实施方案中,所述载体并无特别限制,只要能够用于递送本发明所述的lncRNA SCARNA2以过表达lncRNA SCARNA2的载体均在本发明的保护范围内,包括但不限于:DNA质粒载体、慢病毒载体、逆转录病毒载体、痘病毒载体、单纯疱疹病毒载体、腺病毒载体、腺相关病毒载体、结合DNA质粒的脂质体、结合DNA质粒的分子耦联体和/或结合DNA质粒的多聚物等。在本发明的具体实施方案中,所述载体为pSIF1-H1载体。
本发明的第二方面提供了一种抗病毒的药物组合物。
进一步,所述药物组合物包含过表达lncRNA SCARNA2的试剂。
进一步,所述试剂包括过表达lncRNA SCARNA2的载体、携带lncRNA SCARNA2的纳米颗粒、包裹lncRNA SCARNA2的蛋白微球、包裹lncRNA SCARNA2的脂质体和/或其任意组合。
在一些实施方案中,所述药物组合物还可包含药学上可接受的载体和/或辅料。所述载体和/或辅料的具体示例性例子包括但不限于:糖类,如乳糖、葡萄糖和蔗糖;淀粉,如玉米淀粉和土豆淀粉;纤维素及其衍生物,如羧甲基纤维素钠、乙基纤维素和甲基纤维素;西黄蓍胶粉末;麦芽;明胶;滑石;固体润滑剂,如硬脂酸和硬脂酸镁;硫酸钙;植物油,如花生油、棉籽油、芝麻油、橄榄油、玉米油和可可油;多元醇,如丙二醇、甘油、山梨糖醇、甘露糖醇和聚乙二醇;海藻酸;乳化剂,如润湿剂,如月桂基硫酸钠;着色剂;调味剂;压片剂、稳定剂;抗氧化剂;防腐剂;无热原水;等渗盐溶液;和磷酸盐缓冲液等。
在一些实施方案中,适合的药学上可接受的载体和/或辅料在Remington'sPharmaceutical Sciences(19th ed, 1995)中有详细的记载,这些物质根据需要用于帮助配方的稳定性或有助于提高活性或活性物质的生物有效性或在口服的情况下产生可接受的口感或气味。如此配制的药物组合物根据需要可选择本领域技术人员已知的任何适当的方式将药物进行给药,使用药物组合物时,是将安全有效量的本发明所述的药物组合物施用于人。
本发明的第三方面提供了一种抗病毒的药物制剂。
进一步,所述药物制剂包含本发明第二方面所述的药物组合物。
在一些实施方案中,本发明所述的药物组合物或药物制剂的适合给药剂量根据制剂化方法、给药方式、患者的年龄、体重、性别、病态、饮食、给药时间、给药途径、排泄速度及反应灵敏性之类的因素而可以进行多种处方,熟练的医生通常能够容易地决定处方及处方对所希望的治疗和/或预防有效的给药剂量。
本发明的第四方面提供了一种筛选抗病毒候选药物的方法。
进一步,所述方法包括如下步骤:
(1) 用待测试物质处理表达或含有lncRNA SCARNA2的体系;
(2) 检测所述体系中lncRNA SCARNA2的表达;
(3) 选择可提高lncRNA SCARNA2表达水平的测试物质为候选药物。
在一些实施方案中,所述lncRNA SCARNA2表达水平的检测可采用本领域技术人员所熟知的任何检测lncRNA表达水平的方法进行。在一些实施方案中,检测lncRNA转录物的方法包括测序技术、核酸杂交技术、核酸扩增技术。其中,核酸扩增技术选自聚合酶链式反应、逆转录聚合酶链式反应、转录介导的扩增、连接酶链式反应、链置换扩增和基于核酸序列的扩增,其中,聚合酶链式反应优选为实时荧光定量PCR反应。
在一些实施方案中,所述体系包括细胞体系、亚细胞体系、溶液体系、组织体系、器官体系和/或动物体系。在一些实施方案中,所述待测物质为任何可能对病毒感染具有治疗和/或预防作用的物质、或任何可能促进本发明所述lncRNA SCARNA2表达的物质,在本发明中,所述待测物质并无特别限制。
本发明的第五方面提供了如下任一方面应用:
(1) lncRNA SCARNA2在筛选抗病毒候选药物中的应用;
(2) 抑制lncRNA SCARNA2表达的试剂在制备用于治疗和/或预防自身免疫性疾病的药物中的应用;
(3) 过表达lncRNA SCARNA2的试剂在制备用于治疗和/或预防病毒感染相关疾病的药物中的应用。
此外,本发明还提供了一种治疗和/或预防病毒感染相关疾病的方法,所述方法包括如下步骤:给有需要的受试者施用有效量的本发明第一方面中所述的过表达lncRNASCARNA2的试剂、本发明第二方面所述的药物组合物和/或本发明第三方面所述的药物制剂。
在一些实施方案中,所述施用的方式包括但不限于:局部、经皮、肠胃外、静脉内、肌肉内、腹膜内、口服、鼻内滴注、腔内或膀胱内滴注、眼内、动脉内、病灶内或通过施用于诸如鼻、咽喉和支气管的粘膜来施用所述物质。
相对于现有技术,本发明具有的优点和有益效果如下:
本发明首次发现lncRNA SCARNA2在抗病毒感染中的新用途,本发明通过实验证实了在细胞中过表达lncRNA SCARNA2,可以在VSV和EMCV感染后显著促进ISG的表达,从而抑制病毒mRNA水平的表达、抑制病毒的复制,本发明为制备用于治疗和/或预防各种病毒感染引起的感染性疾病的药物提供了新的思路和策略,为病毒感染的治疗提供了新的靶点,具有广泛的应用前景。
附图说明
图1:STAT1可以与lncRNA SCARNA2结合对应的结果图;
图2:干扰SCARNA2的表达水平可显著抑制IFNα效应对应的结果图,其中,A图:RT-qPCR分析HepG2细胞中ASO介导的SCARNA2敲低效率,B图:RT-qPCR分析HepG2细胞中ISG15、IFIT1、MX2 mRNA表达水平,C图:RT-qPCR分析A549细胞中ASO介导的SCARNA2敲低效率,D图:RT-qPCR分析A549细胞中RSAD2、IFIT1、MX2 mRNA表达水平;
图3:在敲除SCARNA2后可以显著抑制IFNα处理后的ISG的mRNA表达水平对应的结果图;
图4:敲除SCARNA2可以显著抑制IFNγ处理后诱导的CXCL10、CXCL11和IL18BP的mRNA表达水平对应的结果图;
图5:敲除SCARNA2基因的A549细胞系构建对应的结果图,其中,A图:基因组水平,B图:转录水平;
图6:敲除SCARNA2可以促进VSV、EMCV病毒的复制对应的结果图;
图7:过表达SCARNA2可以抑制VSV、EMCV病毒的复制对应的结果图。
具体实施方式
下面结合具体实施例,进一步阐述本发明,具体实施例仅用于解释本发明,而不能理解为对本发明的限制。本领域的普通技术人员可以理解为:在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由权利要求及其等同物限定。下列实施例中未注明具体条件的实验方法,通常按照常规条件或按照厂商所建议的条件实施检测。下述实施例中所用的试剂、生物材料等,如无特殊说明,均可从商业途径得到。
实施例1 lncRNA SCARNA2促进干扰素效应
1、实验材料
人非小细胞肺癌细胞系A549,购自于ATCC;人胚肾细胞系HEK293T,购自于ATCC;人肝癌细胞系HepG2,购自于ATCC;SCARNA2敲除的A549细胞系,于本课题中构建。
2、实验方法
2.1 载体构建以及质粒抽提
(1)pGL3-U6-sgRNA-EGFP和pSpCas9(BB)-2A-GFP (PX458)载体分别使用BsaI-HF和BbsI-HF酶切:载体 1 µg,BsaI-HF或BbsI-HF(10 U/µL) 1 µL,10×cutsmart buffer 2µL,Nuclease-Free water补至20 µL。反应条件:37℃,2h;酶切产物使用1%琼脂糖胶分离,再使用Zymoclean Gel DNA Recovery试剂盒按照说明书进行胶回收。用T4 PNK将sgRNA磷酸化与复性:sgRNA -F (100 µM) 1 µL,sgRNA-R (100 µM) 1 µL,T4 DNA ligasereaction buffer (10 x) 1 µL,T4 PNK 1 µL,Nuclease-Free water 6 µL,反应条件:37℃ 30 min;95℃ 5min;ramp:10℃/min降温至25℃。将磷酸化后的sgRNA和酶切后的载体用quick连接酶连接:载体 100 ng,1:200稀释的sgRNA磷酸化后的复性产物 1 µL,2×quickligase buffer 5 µL,Nuclease-Free water补至10 µL,反应条件:室温15 min。将连接好的质粒转入感受态大肠杆菌DH5α中转化(冰浴30 min→42℃ 90 s→冰浴2 min),加入300µL LB-,于37℃中摇菌45 min,涂板。
(2)pSIF1-H1载体使用BamHI-HF和EcoRI-HF进行酶切后1%琼脂糖胶分离回收。用细胞提取的cDNA做模板,PCR扩增含有酶切位点的SCARNA2:模板cDNA 100 ng,PrimeSTARHS 25 µL,引物(F/R , 10 μM) 1µL,Nuclease-Free water补至50 µL,反应条件:95℃ 5min;(95℃ 10 s;60℃ 15 s;72℃ 30 s)×35个循环;72℃ 5 min。按同样方法进行酶切后将其与载体连接、转化并涂板,于37℃培养箱中12 h。挑出其中的单克隆菌株,摇菌6 h以上,保菌后(800 µL菌液+200 µL 50%甘油),将菌液送至生工公司测序,选择序列正确的载体进行后续实验。
(3)质粒提取:使用SanPrep柱式质粒DNA小量抽提试剂盒。将摇好的菌液8000×g2 min离心,弃上清培养基。在沉淀中加入250 µL已加入RNaseA的Buffer P1,彻底悬浮菌体,加入250 µL Buffer P2,立即温和颠倒离心管5-10次混匀,室温静置2 min,再加入350µL Buffer P3,立即温和颠倒5-10次混匀。12000×g离心10 min,将上清液转移到吸附柱内,8000×g离心30 s,加入500 µL wash buffer离心清洗柱子两次,空吸附柱9000×g离心1 min,在吸附膜中央加入50 µL Elution Buffer,室温静置后离心回收DNA,nanodrop测定浓度。使用Genpure Plasmid Maxi Kit进行质粒的大量提取。将摇好的菌液在大离心机内4000 rpm离心10 min弃上清。加入12 mL含有P2的P1重悬菌液,转移到50 mL离心管中,加入12 mL P3混匀室温静置3 min,再加入12 mL P4混匀冰上静置5 min。13000×g 4℃离心20min。用6 mL P5平衡层析柱,安装滤纸,用去离子水润湿。将离心好的上清液通过滤纸加入层析柱中,加入16 mL P6洗层析柱两次,加入14 mL P7回收层析柱中的DNA,在回收液中加入10 mL异丙醇,混匀后离心14000×g,4℃,30 min,弃上清,加入4 mL 70%乙醇清洗沉淀两次,在沉淀风干至基本干燥后加入去离子水溶解沉淀,nanodrop测定浓度,-20℃保存。LB-培养基:称取5 g NaCl、5 g TRYPTONE和2.5 g Yeast Extract溶于500 mL去离子水中,高压灭菌后,4℃储存待用。LB平板(氨苄抗性):称取5 g NaCl、5 g TRYPTONE和2.5 g YeastExtract溶于500 mL去离子水中,高压灭菌后,降温后加入500 µL 100 mg/mL氨苄,混匀倒入平板中,冷却后置于4℃储存待用。
2.2 细胞质粒转染以及RNA干扰
(1)HEK293T细胞转染(6孔板)
转染mix配制:在1.5 mL离心管A中加入100 µL Opti-MEM,2 µg质粒,2 µLLipofectamine PLUS;在1.5 mL离心管B中加入100 µL Opti-MEM,6 µL LipofectamineLTX Reagent。将离心管中的液体分别吹打混匀,将离心管B中的液体加入离心管A中,吹打混匀,静置5 min。将6孔板中的培养基换液1.8 mL,再将转染mix加入到孔板中,5 h后换液。
(2)A549细胞转染(6孔板)
转染mix配制:在1.5 mL离心管中加入200 µL Opti-MEM,2 µg质粒,6 µL FugENEHD转染试剂,吹打混匀,静置15 min。将6孔板中的培养基换液1.8 mL,再将转染mix加入到孔板中,5 h后换液。
(3)RNA干扰(12孔板)
干扰mix配制:在1.5 mL离心管A中加入50 µL Opti-MEM,1 µL 20 µM的干扰试剂或者NC对照;在1.5 mL离心管B中加入50 µL Opti-MEM,3 µL RNAiMax Reagent。将离心管中的液体分别吹打混匀,将离心管A中的液体加入离心管B中,吹打混匀,静置15 min。将12孔板中的培养基换液350 µL,再将干扰mix加入到孔板中,5 h后换液。干扰24-48 h后使用。RNA干扰序列信息(锐博生物)见下表1。
表1 RNA干扰序列信息
2.3 细胞总RNA提取、反转录以及实时荧光定量PCR
(1)细胞总RNA提取
使用飞捷fast 200快速提取RNA试剂盒。在12孔板中每孔加入500 µL RA2,将裂解液吹打后加入吸附柱中,10000×g 1 min离心。500 µL Wash buffer离心清洗吸附柱两次。使用40 µL Elution Buffer将RNA回收。Nanodrop测定浓度,-80℃保存备用。
(2)反转录
使用ReverTra Ace qPCR RT Master Mix(去除基因组DNA):RNA 1 µg,Nuclease-Free water补至12 µL。反应条件:65℃,5 min(打开RNA的二级结构);加入4 µL的4× DNAMaster Mix(已加入过gDNA remover)。反应条件:37℃ 5 min(去除基因组DNA);再加入4 µL的5× RT Master MixⅡ。反应条件:37℃ 15 min;50℃ 5 min;98℃ 5 min;4℃ ∞。
(3)实时荧光PCR
cDNA模板 4 µL,SYBR Green Master Mix (2×) 10 µL,Primers F/R mix (10 µM) 0.4 µL,Nuclease-Free water 5.6 µL。反应条件:98℃ 1 min;(98℃ 10 s;56℃ 20s;72℃ 30 s)×40个循环;95℃ 1 min;65℃ 1 min;95℃ 15 s;40℃ 30 s。qPCR所用引物见下表2。
表2 qPCR所用引物
2.4 免疫印迹
在12孔板每个孔中弃掉培养基,用PBS清洗细胞两次,加入120 µL 1×RIPA裂解液,在冰上刮裂细胞,转移到1.5 mL EP管中,13600 rpm离心20 min。将100 µL上清转移到新的EP管中,使用BCA法测定蛋白浓度。加入25 µL 5×loading,100℃,10 min使蛋白变性,-20℃保存待用。使 BioSci New Flash Protein anyKD PAGE-快速蛋白凝胶试剂盒配制SDS-PAGE胶:每块胶加入5 mL分离胶液mix(2.5 mL分离胶液A+2.5 mL分离胶液B+50 µL10% APS)和2 mL浓缩胶液mix(1 mL浓缩胶液A+1 mL浓缩胶液B+20 µL 10% APS),插上梳子,待胶凝固后使用。依据蛋白浓度定量结果,取等量蛋白(10 µg左右)进行聚丙烯酰胺凝胶电泳分离蛋白,80 V,90 min。电泳结束后使用1×转膜液将蛋白从胶上转移到NC膜上(320 mA,60 min)。使用5%的milk封闭NC膜1 h,一抗(1:1000)孵育2 h(室温或4℃过夜),1×TBST洗膜三次(10 min/次),二抗孵育(1:2000)孵育1 h,1×TBST洗膜三次(10 min/次)后,使用Immobilon Western Chemiluminescent HRP Substrate孵育膜显影成像。
2.5 基因敲除细胞系的构建
将构建好的2个sgRNA的质粒以及cas9质粒同时转染细胞,48 h后加5 µg/mL的Puromycin和3 µg/mL Blasticidin筛选一周。0.5个细胞/孔稀释至96孔板中,待长出单克隆后进行基因组鉴定和qPCR验证。sgRNA序列信息见下表3,基因组鉴定敲除的引物见下表4。
表3 sgRNA序列信息
表4 基因组鉴定敲除的引物
2.6 RNA体外转录以及RNA生物素化
(1)RNA体外转录
使用A549细胞的反转录cDNA产物,扩增SCARNA2。再使用含T7启动子的引物扩增T7+SCARNA2的线性化片段作为模板。pMV-165thiM(含T7启动子)由六合华大合成,PCR扩增出线性化模板。使用T7 RiboMAX Express Large Scale RNA Production System试剂盒,进行体外转录,反应体系为:RiboMAX Express T7 2×buffer 10 µL,线性DNA模板 1 µg,Enzyme Mix,T7 Express 2µL,RNase Inhibitor 1 µL,Nuclease-Free water补至20 µL。反应条件:37℃ 30 min。使用RNA Clean&Concentrator-5去除DNA模板并回收RNA。使用Qsep1毛细管电泳或者尿素PAGE胶检测回收到的RNA长度是否正确。
(2)体外转录生物素化的RNA(生物素化在U碱基上)
反应体系为:10×T7 RNA polymerase buffer 2 µL,线性DNA模板 1 µg,T7 RNApolymerase 2 µL,Biotin RNA Labeling Mix 2 µL,RNase Inhibitor 1 µL,Nuclease-Free water补至20 µL。反应条件:37℃ 2 h。使用RNA Clean&Concentrator-5去除DNA模板并回收RNA。
表5 相关引物信息
2.7 RNA pull down
将1 µg生物素化的RNA与过表达STAT1的293T细胞的胞核组分的裂解液于室温风车上共孵育1 h,加入用A液和B液各清洗两次的Dynabeads M-280 Streptavidin beads,4℃风车2 h以上。用NT2 buffer清洗beads四次后加入loading,100℃ 10 min制备样品,-20℃保存,后续进行免疫印迹实验。
2.8 数据统计分析
所有实验均重复三次以上,两组间的差异比较采用非配对双尾T检验的统计分析方法。P<0.05时认为两组数据间有统计学差异。*为P<0.05,**为P<0.01,***为P<0.001。
3、实验结果
3.1 lncRNA SCARNA2可以与STAT1结合
为了验证lncRNA SCARNA2与STAT1的结合,本实施例在293T细胞中过表达了STAT1,将体外转录后的SCARNA2用生物素标记进行了RNA pull down实验。将生物素化的SCARNA2与过表达STAT1的293T细胞的裂解液共孵育,再使用链霉亲和素偶联的磁珠进对biotin-SCARNA2进行富集,免疫印迹检测p-STAT1和STAT1。
结果表明SCARNA2可以结合STAT1,并随着IFNα的处理SCARNA2结合了更多的STAT1,如图1所示。实验结果同样表明SCARNA2可以结合磷酸化的STAT1,而在IFNα处理后只有在STAT1的701位点被磷酸化后STAT1才能入核发挥功能,这也证明了SCARNA2可以在核内与STAT1发生相互作用。
3.2 干扰SCARNA2的表达水平可显著抑制IFNα效应
定制合成了3条针对SCARNA2的反义寡核苷酸(Antisense oligonucleotide,ASO)序列,混合转入细胞中,使得反义寡核苷酸与SCARNA2特异性结合并招募RNase H对SCARNA2-DNA杂交链进行降解,从而降低SCARNA2的表达水平。首先,本实施例在A549细胞中对SCARNA2进行了干扰,发现SCARNA2 表达水平降低了50%,如图2A所示。对比对照组,发现在IFNα处理8 h后干扰SCARNA2的表达水平后可以抑制包括ISG15、IFIT1、MX1在内的ISG的mRNA水平,如图2B所示。由此可见SCARNA2可以促进IFNα的效应。
同时本实施例在人肝癌细胞HepG2细胞系中同样对SCARNA2进行了干扰,发现ASO转入HepG2细胞后SCARNA2的表达水平降低至了对照组的38%,干扰效果明显,如图2C所示。与在A549细胞系中观察到的现象一致,在IFNα处理8 h后干扰SCARNA2的表达可以显著抑制包括RSAD2、IFIT1、MX2在内的多种ISG的mRNA水平,如图2D所示。
本实施例分别在两种细胞系中验证了SCARNA2可以促进IFNα处理后ISG的转录,即可以促进IFNα的效应。
3.3 敲除SCARNA2可显著抑制干扰素效应
为了进一步验证SCARNA2对干扰素效应的影响,本实施例使用CRISPR-cas9体系构建敲除SCARNA2基因的A549细胞系。由于SCARNA2编码于基因间,且存在独立的转录单元,敲除SCARNA2基因并不会影响其他基因的表达。因此本实施例在SCARNA2基因全长的上游38bp处开始和下游32 bp处开始作为敲除位点,构建敲除细胞系。
结果和上述干扰SCARNA2表达水平的结果一致,在敲除SCARNA2后可以显著抑制IFNα处理后的ISG的mRNA水平,如图3所示。
此外,本实施例还使用100 U/mL的IFNγ来处理SCARNA2敲除的细胞系。结果显示,发现敲除SCARNA2也可以显著抑制IFNγ处理后诱导的CXCL10、CXCL11和IL18BP的mRNA表达,如图4所示。
Ⅰ型干扰素在与干扰素受体结合后激活JAK-STAT信号通路,JAK1和TYK2磷酸化可以促进STAT1和STAT2的磷酸化,随后磷酸化的STAT1和STAT2形成异二聚体并与IRF9一起组成ISFG3结构元件入核促进ISRE元件进行转录。而Ⅱ型干扰素(IFNγ)结合干扰素受体激活JAK1和JAK2后促进STAT1磷酸化形成同源二聚体进而入核促进GAS元件转录。因此我们的结果证实SCARNA2可以通过与STAT1结合,具有广泛的促进干扰素效应的调控功能。
实施例2 过表达SCARNA2能够显著抑制VSV、EMCV病毒的复制
1、实验方法
1.1 基因敲除细胞系的构建
同实施例1中2.6部分中所示。
1.2 细胞病毒感染
实验中使用的水泡性口炎病毒(vesicular stomatitis virus, VSV)和脑心肌炎病毒(encephalomyocarditis virus, EMCV)由本实验室扩增和保存。
(1)病毒的扩增和滴度检测
VSV病毒使用HEK293T细胞扩增检测,脑心肌炎病毒使用BHK-21细胞检测。扩增:按贴壁细胞培养方法培养扩增细胞并传代至T175瓶中,待细胞长至90%密度后换液20 mL,取早期的病毒毒株1 μL加入到培养瓶中混匀,16-24 h后观察细胞状态,细胞基本死亡且还未完全漂起时,将培养瓶放入-80℃,反复冻融三次后,在超净台中把培养基全部转移至50 mL离心管中,4℃,4000 rpm离心30 min,取上清即为病毒。滴度(TCID50)测定:将细胞铺至96孔板中,第二天将病毒或者病毒感染细胞后的上清按10倍倍比稀释。然后每个稀释梯度各取100 μL加入一列96孔板的8个复孔中。三天后观察细胞死亡情况,记录一列8个复孔的发生病变的死亡孔数以及前后两个的稀释倍数,按照以下公式来计算病毒滴度。其中,间距=(大于50%的病变率-50%)/(大于50%的病变率-小于50%的病变率);TCID50=10-(大于50%病变率的稀释倍数+间距)。
(2)病毒感染
将细胞系按照1×105密度铺至24孔板中,培养贴壁后分别加入VSV(MOI=1)、EMCV(MOI=1)感染1 h和10 h,随后收取细胞进行后续检测。
1.3 A549细胞系过表达SCARNA2
将细胞系按照5×104密度铺至24孔板中,培养贴壁后进行细胞转染。转染mix配制:在1.5 mL离心管中加入50 µL Opti-MEM,0.5 µg pSIF1-H1-SCARNA2载体,1.5 µLFuGENE HD转染试剂,吹打混匀,静置15 min。将24孔板中的培养基换液0.45 mL,再将转染mix加入到孔板中,5 h后换液。继续培养24 h后进行后续病毒感染等实验。
1.4 细胞总RNA提取、反转录以及实时荧光定量PCR
同实施例1中2.3部分中所示。qPCR所用引物见下表6。
表6 qPCR所用引物
2、实验结果
前期我们发现了SCARNA2可以显著促进干扰素效应,而干扰素信号下游产生的干扰素刺激基因(Interferon-stimulated genes,ISGs)编码的蛋白质已被报道通过多种机制包括抑制病毒转录、翻译和复制,病毒核酸的降解和细胞脂质代谢的改变来抑制病原体,因此我们进一步探究了lncRNA SCARNA2在机体抗病毒过程中的作用。
我们使用CRISPR-cas9体系构建敲除SCARNA2基因的A549细胞系。由于SCARNA2编码于基因间,且存在独立的转录单元,敲除SCARNA2基因并不会影响其他基因的表达。因此我们在SCARNA2基因全长的上游38bp处开始和下游32bp处开始作为敲除位点,构建敲除细胞系。细胞基因组水平上(如图5A所示)以及转录水平上(如图5B所示)均鉴定出SCARNA2成功敲除。
随后我们使用水泡性口炎病毒和脑心肌炎病毒分别感染SCARNA2敲除的细胞系,结果表明,敲除SCARNA2可以抑制ISG的mRNA的表达以及促进病毒的mRNA水平的表达(如图6)。
我们还在A549细胞系中过表达lncRNA SCARNA2,与对照组相比,过表达SCARNA2可以在VSV和EMCV感染后促进ISG的表达水平从而抑制病毒mRNA水平的表达、抑制病毒的复制(如图7)。
以上结果表明,过表达lncRNA SCARNA2能够显著抑制病毒mRNA水平的表达、抑制病毒的复制。
Claims (4)
1.过表达lncRNA SCARNA2的试剂在制备抗病毒药物中的应用,其特征在于,所述病毒为水泡性口炎病毒或脑心肌炎病毒;
所述试剂包括过表达lncRNA SCARNA2的载体、携带lncRNA SCARNA2的纳米颗粒、包裹lncRNA SCARNA2的蛋白微球、包裹lncRNA SCARNA2的脂质体或其任意组合;
所述载体包括DNA质粒载体、慢病毒载体、逆转录病毒载体、痘病毒载体、单纯疱疹病毒载体、腺病毒载体、腺相关病毒载体、结合DNA质粒的脂质体、结合DNA质粒的分子耦联体和/或结合DNA质粒的多聚物。
2.根据权利要求1所述的应用,其特征在于,所述lncRNA SCARNA2在细胞中的过表达能够显著促进ISG的表达。
3.根据权利要求1所述的应用,其特征在于,所述lncRNA SCARNA2在细胞中的过表达能够显著抑制病毒mRNA水平的表达、抑制病毒的复制。
4.根据权利要求1所述的应用,其特征在于,所述抗病毒药物为治疗和/或预防病毒感染疾病的药物。
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