CN113186224B - 具有抑制乙肝病毒复制活性的microRNA-27a及其应用 - Google Patents
具有抑制乙肝病毒复制活性的microRNA-27a及其应用 Download PDFInfo
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Abstract
本发明公开了一种具有抑制乙肝病毒复制活性的microRNA‑27a及其应用,将miR‑27a的至少一个编码基因插入病毒载体得到重组载体;将重组载体转入宿主细胞包装得到所述重组病毒。所述的重组载体或重组病毒能够应用于制备乙肝相关药物;所述乙肝相关药物包括靶向抑制HBXmRNA的药物、抑制HBV复制和表达的药物、预防和/或治疗肝脏肿瘤的药物。
Description
技术领域
本发明涉及生物技术领域,尤其涉及一种具有HBV复制和表达抑制活性的重组载体及其应用R。
背景技术
乙型肝炎病毒(hepatitis B virus,HBV)感染是一个全球性的公共健康问题,全世界有超过2.5亿乙型肝炎病毒携带者,每年约有78万人死于HBV相关性肝病[1-2]。HBV感染引起免疫介导的肝损伤和癌变,最终导致肝细胞癌[3]。目前尽管存在有效的疫苗和基于干扰素和核苷酸/核苷类似物的治疗策略,但彻底清除患者体内的乙肝病毒仍然是一个巨大挑战[4-5]。因此,深入研究HBV复制和表达的调控机制,寻找新的和有效的抑制HBV感染和复制的靶点,有助于开发更好的治疗策略来实现对HBV的控制。
发明内容
本发明要解决的技术问题是提供一种具有HBV复制和表达抑制活性的重组载体及其应用。
为解决上述技术问题,本发明所采取的技术方案如下。
一种具有HBV复制和表达抑制活性的重组载体,所述重组载体为将miR-27a的至少一个编码基因插入病毒载体得到的重组载体。
作为本发明的一种优选技术方案,所述miR-27a为序列表中的SEQ ID NO:1所示的RNA。
作为本发明的一种优选技术方案,所述病毒载体为腺病毒载体。
作为本发明的一种优选技术方案,所述重组载体为将miR-27a的至少一个编码基因插入到腺病毒载体的多克隆位点间得到的重组载体。
一种具有HBV复制和表达抑制活性的重组病毒,将任一所述的重组载体转入宿主细胞,包装得到所述重组病毒。
作为本发明的一种优选技术方案,所述宿主细胞为人体细胞,优选为人胚肾细胞。
所述的重组载体在制备乙肝相关药物中的应用;所述乙肝相关药物包括抑制乙肝病毒DNA拷贝的药物、靶向抑制HBX mRNA的药物、抑制了HBV复制和表达的药物、抑制乙肝病毒抗原表达的药物、预防和/或治疗肝脏肿瘤的药物、减小肝脏肿瘤数量和/或肿瘤重量的药物。
所述的重组病毒在制备乙肝相关药物中的应用;所述乙肝相关药物包括抑制乙肝病毒DNA拷贝的药物、靶向抑制HBX mRNA的药物、抑制了HBV复制和表达的药物、抑制乙肝病毒抗原表达的药物、预防和/或治疗肝脏肿瘤的药物、减小肝脏肿瘤数量和/或肿瘤重量的药物。
采用上述技术方案所产生的有益效果在于:在本发明的研究中,我们首先检测了miR-27a在HBV稳定表达细胞系HepG2.2.15和对照细胞系HepG2中的表达差异,然后进行生物信息学和功能实验,阐明了miR-27a在调节HBV复制和表达中的作用及潜在的分子机制。我们发现miR-27a在HBV感染的Huh7细胞中表达下调;miR-27a通过直接靶向抑制HBX mRNA进而抑制了HBV复制和表达。上述研究结果提示,miR-27a在HBV-宿主的相互作用中发挥重要作用,miR-27a可作为一种潜在的控制病毒感染的治疗靶点。
附图说明
图1为miR-27a过表达抑制HBV复制和表达图。图中,A.miR-27a mimics及NC转染Huh7细胞,miR-27a相对表达量检测;B~G.miR-27a mimics及NC分别与pHBV1.2组合转染Huh7细胞,HBV复制和表达相关指标检测,HBV DNA(B)、HBV RNA(C)、HBeAg吸光度(D)、HBsAg吸光度(E)、HBeAg浓度(F)、HBsAg浓度(G)。
图2为抑制内源性miR-27a表达促进HBV复制和表达图。图中,A.miR-27ainhibitor及NC转染Huh7细胞,miR-27a相对表达量检测;B~G.miR-27a inhibitor及NC分别与pHBV1.2组合转染Huh7细胞,HBV复制和表达相关指标检测,HBV DNA(B)、HBV RNA(C)、HBeAg吸光度(D)、HBsAg吸光度(E)、HBeAg浓度(F)、HBsAg浓度(G)。
图3为miR-27a直接靶向抑制HBX表达图。图中,A.miR-27a与HBX的结合位点及其突变位点;B.miR-27a mimics/miR-27a inhibitor及其各自的阴性对照(NC)分别与miR-27a结合位点的HBx报告基因载体组合转染Huh7细胞,双荧光素酶报告基因检测相对荧光强度;C.miR-27a mimics及NC分别与miR-27a结合位点突变HBx报告基因载体组合转染Huh7细胞,双荧光素酶报告基因检测相对荧光强度;D和E.miR-27a mimics及NC分别HBx表达载体组合转染Huh7细胞,检测HBx RNA(D)和HBx蛋白(E)。
图4为HBV抑制miR-27a表达图。图中,A.pHBV1.2及其对照(Ctr)转染Huh7细胞,qRT-PCR检测miR-27a相对表达;B.qRT-PCR检测HepG2和HepG2.2.15细胞miR-27a相对表达;C.腺病毒HBx表达载体和空腺病毒感染Huh7细胞,qRT-PCR检测miR-27a相对表达。
具体实施方式
以下实施例详细说明了本发明。本发明所使用的各种原料及各项设备均为常规市售产品,均能够通过市场购买直接获得。
应当理解,当在本申请说明书和所附权利要求书中使用时,术语“包括”指示所描述特征、整体、步骤、操作、元素和/或组件的存在,但并不排除一个或多个其它特征、整体、步骤、操作、元素、组件和/或其集合的存在或添加。
还应当理解,在本申请说明书和所附权利要求书中使用的术语“和/或”是指相关联列出的项中的一个或多个的任何组合以及所有可能组合,并且包括这些组合。
如在本申请说明书和所附权利要求书中所使用的那样,术语“如果”可以依据上下文被解释为“当...时”或“一旦”或“响应于确定”或“响应于检测到”。类似地,短语“如果确定”或“如果检测到[所描述条件或事件]”可以依据上下文被解释为意指“一旦确定”或“响应于确定”或“一旦检测到[所描述条件或事件]”或“响应于检测到[所描述条件或事件]”。
另外,在本申请说明书和所附权利要求书的描述中,术语“第一”、“第二”、“第三”等仅用于区分描述,而不能理解为指示或暗示相对重要性。
在本申请说明书中描述的参考“一个实施例”或“一些实施例”等意味着在本申请的一个或多个实施例中包括结合该实施例描述的特定特征、结构或特点。由此,在本说明书中的不同之处出现的语句“在一个实施例中”、“在一些实施例中”、“在其他一些实施例中”、“在另外一些实施例中”等不是必然都参考相同的实施例,而是意味着“一个或多个但不是所有的实施例”,除非是以其他方式另外特别强调。术语“包括”、“包含”、“具有”及它们的变形都意味着“包括但不限于”,除非是以其他方式另外特别强调。
实施例1、材料和方法
1.1材料腺病毒Ad-HBx、pcDNA3.1-HBx等HBV蛋白表达载体由本研究室留存。miR-27amimics、miR-27a inhibitor及其无关阴性对照(negative control,NC)由GenePharmaCo.Ltd.(中国上海)合成;HBsAg诊断试剂盒、HBeAg检测试剂盒购自北京万泰生物药业股份有限公司;HBsAg测定试剂盒(化学发光法)、HBeAg测定试剂盒(化学发光法)购自北京中同兰博医学检验实验室有限公司;HBV核酸定量检测试剂盒(PCR-荧光探针法)购自凯杰生物工程(深圳)有限公司;GoScript Reverse Transcription System反转录试剂盒、Go TaqqPCR Master Mix定量检测试剂盒购自Promega公司。连接酶、pMD-18T载体等基因克隆及表达相关试剂购自北京全式金生物技术有限公司;HBXIP抗体(H-5)购自圣克鲁斯生物技术有限公司。
1.2方法
1.2.1细胞培养和转染肝癌细胞系HepG2、Huh7由本实验室留存,培养条件为含10%胎牛血清、2mmol/L L-谷氨酰胺、100U/ml青霉素和100mg/mL链霉素的DMEM培养基。HepG2.2.15(由HepG2细胞系稳定转染1.3倍全长HBV获得)由本室留存,培养条件为含380μg/mL G418的RPMI 1640培养基。细胞均在37℃含5%CO2的恒温孵箱中进行培养,并根据细胞生长情况及时换液。
将HBx全长序列克隆pcDNA3.1质粒中,构建重组pcDNA3.1-HBx表达载体;将HBx 3’UTR的基因序列克隆到报告基因质粒pGL3中,构建重组pGL3-HBx 3’UTR;构建HBx 3’UTR靶位点突变报告基因检测载体pGL3-HBx 3’UTR-Mutant。克隆构建、靶位点突变等相关引物见表1。
提前24小时将要转染的细胞用胰酶消化,计数后以铺六孔板(5×106个/孔)或12孔板(5×105个/孔)(转染时每组三个重复),16~18小时后待细胞生长到90%-95%融合度用Lipofectamine 2000进行转染。mRNA和蛋白质检测于72小时后收集细胞,荧光素酶报告基因检测于48小时后收集细胞。
1.2.2 qRT-PCR检测miRNA、mRNA和HBV DNA细胞培养24小时后,首先按照TRIzolReagent(invitrogen)说明书提取细胞内总RNA,检测基因表达水平。根据GoScriptReverse Transcription System反转录试剂盒说明书,取1μg总RNA反转录为cDNA,逆转录反应条件为:16℃退火30min,42℃反应30min,85℃反应5min。然后根据Go Taq qPCRMaster Mix定量检测试剂盒说明书,取2μL反转录的cDNA,分别以U6 snRNA和β-actin为miRNA和mRNA检测内参,检测miR-27a、HBV mRNA表达水平(反转录及定量检测引物见表1)。实时定量反应条件为:95℃,2min;95℃,15s;60℃,60s共40循环;熔解曲线。用2-△△Ct法计算相对表达量[20]。HBV DNA检测按照HBV核酸定量检测试剂盒(PCR-荧光探针法)说明书进行。
表1引物和序列
Table 1Primers and Sequences
1.2.3 ELISA检测HBsAg、HBeAg吸光度及浓度吸光度检测根据HBsAg诊断试剂盒、HBeAg检测试剂盒说明书进行;浓度检测根据HBsAg测定试剂盒(化学发光法)、HBeAg测定试剂盒(化学发光法)说明书进行。
1.2.4 Western blot检测HBx蛋白表达细胞处理:将细胞用PBS清洗,用胰酶消化后收集细胞,加入适量的冰上预冷的裂解液(含蛋白酶抑制剂)置于冰上10~20min裂解细胞,12000g 4℃离心5min去除细胞碎片,取等量样品加5×loading buffer煮沸10min后上样。电泳:以初始电压为60V的电流强度进行稳流电泳,当样品跑到浓缩胶与分离胶交界处时调为80V,当目的蛋白泳动至距胶下缘1cm左右结束。转膜:使用半干法进行转膜,转膜条件为200mA,45min。取出膜并用TBST洗膜液漂洗,在含5%牛奶的封闭液中封闭1小时。将一抗稀释后室温下轻摇孵育1小时,4℃静置过夜。一抗孵育结束后用洗膜液洗5次,每次8min。根据一抗来源选择合适的二抗,1:1000稀释,在室温条件下轻摇一小时。二抗孵育结束后用洗膜液洗5次,每次8min。在暗室中显色。
1.2.5靶基因预测使用ViTa(http://vita.mbc.nctu.edu.tw/)预测miR-27a在HBV上的靶位点
1.2.6双荧光素酶报告基因检测将含有miR-27a结合位点的pGL3-HBx 3’UTR或结合位点突变的pGL3-HBx 3’UTR-M与miR-27a表达质粒及内参质粒(pRL-TK)按照Lipofectamine 2000说明书进行转染,48小时后用PBS清洗细胞,加入Passive LysisBuffer裂解细胞,室温震荡15min。按照说明书分别加入裂解液检测萤火虫荧光素酶反应强度和内参海肾荧光素酶反应强度。RLU1为萤火虫荧光素酶反应强度,RLU2为内参海肾荧光素酶反应强度,相对荧光强度为Ratio=RLU1/RLU2。
1.3统计学处理每个实验至少重复3次,用GraphPad Prism 8软件进行统计分析,正态分布的计量数据用mean±S表示。2组荧光素酶相对活性、mRNA和miRNA表达水平的比较采用成组t检验。P<0.05表明差异具有统计学意义。
实施例2、试验结果
2.1 miR-27a过表达抑制HBV复制和表达探究miR-27a过表达对HBV复制和表达的影响。首先验证miR-27a mimics的表达效果,将miR-27a mimics及NC分别转染Huh7细胞,qRT-PCR检测miR-27a表达水平,结果如图1A所示,miR-27a mimics转染细胞后miR-27a表达水平显著升高。进一步探究miR-27a过表达对HBV复制和表达的影响,将miR-27a mimics及NC分别与pHBV1.2组合转染Huh7细胞,qRT-PCR检测miR-27a过表达对HBV DNA和HBV RNA的影响,结果如图1B(HBV DNA)和图1C(HBV RNA)所示,miR-27a表达抑制HBV DNA和HBV RNA,提示miR-27a抑制HBV的复制和转录;ELISA检测miR-27a过表达对HBeAg和HBsAg的影响,吸光度检测结果如图1D(HBeAg)和图1E(HBsAg)所示,浓度检测结果如图1F(HBeAg)和图1G(HBsAg)所示,结果提示miR-27a过表达抑制HBeAg和HBsAg表达,提示miR-27a抑制HBV蛋白表达。以上研究结果表明,在肝癌细胞系Huh7中,miR-27a过表达抑制了HBV复制和表达。
2.2抑制内源性miR-27a表达能够促进HBV复制和表达进一步探究抑制内源性miR-27a对HBV复制和表达的影响。首先验证miR-27a inhibitor抑制效果,将miR-27ainhibitor及NC转染Huh7细胞,qRT-PCR检测miR-27a表达水平,结果如图2A所示,转染miR-27a inhibitor后miR-27a表达水平显著降低,提示miR-27a inhibitor可有效抑制miR-27a表达。将miR-27a inhibitor及NC分别与pHBV1.2组合转染Huh7细胞,qRT-PCR检测抑制内源性miR-27a对HBV DNA和HBV RNA的影响,结果如图2B(HBV DNA)和图2C(HBV RNA)所示,抑制内源性miR-27a使HBV DNA和HBV RNA表达水平升高,提示抑制内源性miR-27a能够促进HBV的复制和转录;ELISA检测抑制内源性miR-27a对HBeAg和HBsAg的影响,吸光度检测结果如图2D(HBeAg)和图2E(HBsAg)所示,浓度检测结果如图1F(HBeAg)和图1G(HBeAg)所示,结果提示抑制内源性miR-27a促进HBeAg和HBsAg表达,提示miR-27a促进HBV蛋白表达。以上研究结果表明,在肝癌细胞系Huh7中,抑制内源性miR-27a表达促进了HBV复制和表达。
2.3 miR-27a直接靶向HBX以上研究发现miR-27a可抑制HBV的复制与表达,进一步分析miR-27a调控HBV复制和表达的分子机制。推测存在两种可能的机制,第一种可能机制是miR-27a通过直接靶向HBV RNA而抑制HBV复制和表达;第二种可能机制是miR-27a通过靶向HBV复制过程中的调控因子而间接影响HBV复制和表达。通过ViTa在线预测,发现HBx含有miR-27a的靶位点,如图3A所示。
为了探究miR-27a是否可直接靶向HBx RNA,构建含有miR-27a靶位点的HBx mRNA报告基因载体pGL3-HBX 3’UTR。将miR-27a mimics/miR-27a inhibitor及相应的NC分别与pGL3-HBX 3’UTR-Mutant组合转染Huh7细胞,48小时后收集细胞,检测双萤光素酶活性,结果如图3B所示,miR-27a使含有HBx 3’UTR的报告基因活性降低。进一步将HBx上miR-27a的结合位点突变(突变位点如图3A所示),构建含有HBX 3’UTR突变序列的报告基因载体pGL3-HBX 3’UTR-Mutant,将miR-27a mimics及其阴性对照分别与pGL3-HBX 3’UTR-Mutant组合转染Huh7细胞,48h后收集细胞,检测双萤光素酶活性,结果如图3C所示,miR-27a表达对含有突变位点的报告基因活性没有显著影响。以上结果提示miR-27a能够直接靶HBX RNA。进一步构建含有HBx序列的表达载体pcDNA3.1-HBx,探究miR-27a对HBX RNA及蛋白表达的影响,将miR-27a mimics及NC分别与pcDNA3.1组合转染Huh7细胞,72小时后收集细胞,qRT-PCR检测HBX RNA,结果如图3D所示,western blot检测HBX蛋白表达,结果如图3E所示,转染miR-27a能够抑制HBX RNA和蛋白表达。以上结果提示,miR-27a能够直接靶向HBX RNA,从而间接抑制HBV的复
2.4 HBV抑制miR-27a表达HBV感染会导致miRNA表达水平发生显著变化,因此本研究探究了HBV对miR-27a表达的影响。将pHBV1.2及其空载体转染Huh7细胞,72小时后收集细胞,qRT-PCR检测miR-27a表达水平,结果如图4A所示,瞬时转染pHBV1.2使miR-27a的表达水平下降,提示HBV能够抑制miR-27a的表达。进一步,我们比较了稳定表达HBV的HepG2.2.15细胞及其对照细胞HepG2的miR-27a表达水平,结果如图4B所示,与HepG2细胞相比,稳定表达HBV的HepG2.2.15细胞miR-27a表达水平显著降低。由于miR-27a能够直接靶向HBx 3’UTR,我们将表达HBx的腺病毒Ad-HBx感染Huh7细胞,检测HBx的表达对miR-27a的影响,结果如图4C所示,Ad-HBx感染Huh7细胞使miR-27a表达水平下降,提示HBx使miR-27a表达水平降低。
实施例3、应用价值
HBV感染是导致活动性肝炎、肝纤维化、肝硬化和肝癌等肝脏疾病的主要因素[21]。尽管针对HBV感染的诊断和治疗已经取得了很大的进步,但这并不能从根本上消除HBV感染[22]。因此,亟需寻找和开发新的抗HBV靶点和药物。越来越多的证据表明miRNA在HBV感染中发挥重要的调控作用。miR-27a在多种生物学过程中发挥重要作用,包括基因多态性、肿瘤发生、增殖、细胞凋亡、侵袭、迁移和血管生成[23]。在患肝癌的肥胖患者中,miR-27a通过靶向FOXO1促进肝癌细胞的增殖[24]。miR-27a在肝癌组织和肝癌细胞系中表达上调,通过调节PPAR-γ的表达促进肝癌细胞增殖,并作为癌基因在肝癌细胞中发挥重要作用[25]。然而,miR-27a在HBV感染中的潜在作用和分子机制仍未明确。
本研究探索miR-27a在HBV表达和复制中的作用。发现在HBV感染的肝癌细胞Huh7中miR-27a表达水平显著降低。本研究发现,随着miR-27a表达水平的升高,Huh7细胞中HBVRNA、HBV DNA、HBeAg和HBsAg等HBV复制和基因表达的指标显著降低,而抑制内源性miR-27a的表达能够促进HBV的复制和表达,提示miR-27a可能是一种潜在的HBV复制抑制因子。
miRNAs调控HBV的机制主要分为两类:一类是miRNA直接靶向作用于HBV的转录本,如miR-125a-5p,miR-199a-3p,miR-210,miR-1231,其中miR-125a-5p和miR-199a-3p靶位点位于表面蛋白和聚合酶重叠的编码区域,而miR-210的靶位点位于pre-s1编码区域,miR-1231靶向HBV核心蛋白mRNA[12,26]。第二类是通过作用于相关的细胞蛋白影响HBV生命周期,如microRNA-146a靶向flap endonuclease 1[27],miR-802靶向SMARCE1分子[28],miR-200c靶向nuclear factor IA[29],miR-302c-3p靶向BMPR2[30],MiR-185-5p靶向ELK1分子等[31]。我们的研究发现miR-27a可以直接靶向HBX 3’UTR,抑制HBx转录和表达,最终抑制HBV的复制与表达。
研究表明,HBV X蛋白可以引起宿主miRNA的变化。如:HBx促进miR-29a和miR-143,抑制miR-101,miR-122,miR-132,miR-148a,miR-152,let-7和miR-16家族的成员[32]。有研究探索了HBV病毒影响miRNA表达的分子机制。目前主要有两种机制,一种是HBV mRNA通过自身含有的miRNA互补位点与miRNA结合,这样细胞内大量的HBV RNA起到海绵体作用从而抑制其miRNA发挥作用,miR-122和miR-15a[33-34]。另一种是转录因子参与HBV对miRNA的调控,如c-Myc参与HBx对miR-15a/16的抑制作用[35]。我们的研究表明miR-27a可以直接靶向HBX 3’UTR,提示在HBV感染中,HBx的转录产物可能作为“海绵体”与miR-27a结合,从而使miR-27a表达水平降低。
本研究有不足支持,由于缺乏体内研究,可能会削弱miR-27抑制HBV复制和表达的证据。HBV感染导致miR-27a表达水平下降,但是有研究表明在肝癌中miR-27a的表达水平升高,因此,miR-27a在HBV相关的肝癌中的变化及临床意义需要进一步深入研究。
综上所述,我们发现miR-27a在HBV感染的Huh7细胞中表达下调;miR-27a通过直接靶向抑制HBX mRNA进而抑制了HBV复制和表达。上述研究结果提示,miR-27a在HBV-宿主的相互作用中发挥重要作用,miR-27a可作为一种潜在的控制病毒感染的治疗靶点。
以上所述实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围,均应包含在本发明的保护范围之内。
序列表
<110> 中国人民解放军陆军军医大学士官学校
<120> 具有抑制乙肝病毒复制活性的microRNA-27a及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 1
cgccuugaau cggugacacu u 21
Claims (1)
1.miR-27a在体外抑制HBV复制和表达中的应用,所述miR-27a的序列如SEQ ID NO.1所示。
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