CN115992141B - 一种炎症相关疾病生物标志物miR-25802簇及其应用 - Google Patents
一种炎症相关疾病生物标志物miR-25802簇及其应用 Download PDFInfo
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Abstract
本发明属于生物检测技术领域,具体涉及一种炎症相关疾病生物标记物miR‑25802簇及其应用。本发明miR‑25802簇包括下述Ⅰ)~Ⅳ)任意一种:Ⅰ)miR‑25802,所述miR‑25802包括如SEQ ID NO.2所示的核苷酸序列;Ⅱ)Ⅰ)中所述miR‑25802经修饰后的衍生物;Ⅲ)长度为18~26nt,并且功能与Ⅰ)中所述miR‑25802相同或基本相同的微小RNA;Ⅳ)Ⅲ)中所述微小RNA修饰后的衍生物,可以作为检测AD的生物标志物,能够诱导小胶质细胞激活,促进炎症反应;反之,下调miR‑25802表达水平,能够抑制小胶质细胞促炎表型表达,抑制炎症反应,从抑制炎症反应的角度发挥防治AD的作用。
Description
技术领域
本发明属于生物检测技术领域,具体涉及一种炎症相关疾病生物标志物miR-25802簇及其应用。
背景技术
炎症是机体对内外源性刺激的免疫应答反应的统称,特点是免疫细胞激活、细胞因子和趋化因子水平升高、活性氧释放增加。炎症作为关键的病理机制广泛地影响多种慢性疾病的病理进展、加剧炎症性病理损伤、推动病情恶化。炎症机理在癌症、心血管疾病、代谢性疾病、痴呆等多种慢性疾病中发挥着重要的作用,特别是持续性的慢性炎症造成不可逆的组织损伤和器官功能障碍。包括阿尔茨海默病在内的多种神经退行性疾病都存在明显炎症过程,如帕金森病、肌萎缩性脊髓侧索硬化症、亨廷顿氏舞蹈病等。目前抗炎药物分为甾体类和非甾体类抗炎药物,主要起退热、镇痛、抗炎、抗风湿等作用,在临床上广泛应用于骨关节炎、类风湿性关节炎和多种发热和各种疼痛症状的缓解。但目前药物发挥长效抗炎作用需要长期服用药物,不能从根本上有效抑制炎症发生,并且可能伴有心血管,胃肠道等药物副作用。因此需要迫切寻找特异性更佳、治疗效果更显著的抗炎靶标。
阿尔茨海默病(Alzheimer’s disease,AD)是一种起病隐匿、与年龄高度相关的进行性神经退行性疾病。临床特征表现为学习和记忆下降的认知功能衰退,主要的病理机制为淀粉样蛋白聚集形成的细胞外老年斑沉积与tau蛋白过度磷酸化形成的细胞内神经元纤维缠结。由于病理机制复杂且不明确,缺乏可靠的生物标记物和有效的药物作用靶标,使得AD的诊断和治疗面临严峻的挑战。目前AD的诊断多以神经心理学测试为主,辅以体液病理标记物检查,诊断方法灵敏性缺乏、特异性和准确度欠佳、适应性不良。但是临床使用或正在研究阶段的具有抗AD药物的疗效有限,不能延缓或治愈疾病进展。因此寻求可靠的AD诊断生物标记物和药物干预靶标,是防治AD亟待解决的科学问题。
微小核糖核酸(microRNA,miRNA)是一类重要的内源性分子,其表达具有显著的组织特异性和时序性,调控关键基因表达水平,影响疾病进展。家族性AD与PSEN1,PSEN2,APP等基因突变密切相关,因而可以通过基因型鉴定对疾病进行早期诊断及干预,但是针对发病率为95%的散发性AD,目前尚无相关基因的报道。此外,以AD为代表的炎症类相关疾病缺乏有效治疗靶标和试剂,且目前有关非编码基因的免疫调控机理研究仍处于初始阶段。基于miRNA多靶向性的特点,miRNA介导表观遗传学调控机制有望通过调节复杂交互的炎症信号通路网络,从上游基因层面干预炎症过程。因此发现AD新型基因类生物标记物、从基因层面发现调控炎症过程的新靶标,为治愈AD及炎症导致的其他慢性疾病具有重要的意义。
发明内容
本发明的目的在于提供一种炎症相关疾病生物标志物miR-25802簇及其应用,设计早期检测试剂盒,有效诊断和/或治疗炎症相关疾病、判断疾病转归及改善患者的生活质量。
本发明提供了一种炎症相关疾病生物标志物miR-25802簇,所述miR-25802簇包括下述Ⅰ)~(Ⅳ)任意一种:
Ⅰ)miR-25802,所述miR-25802包括如SEQ ID NO.2所示的核苷酸序列;
Ⅱ)Ⅰ)中所述miR-25802经修饰后的衍生物;
Ⅲ)长度为18~26nt,并且功能与Ⅰ)中所述miR-25802相同或基本相同的微小RNA;
Ⅳ)Ⅲ)中所述微小RNA修饰后的衍生物。
优选的,所述miR-25802的前体为mir-25802,所述mir-25802包括如SEQ IDNO.1所示的核苷酸序列。
本发明还提供了上述技术方案所述的miR-25802簇在下述a1)~a6)中的一种或多种中的应用:
a1)制备炎症相关疾病人群筛选的试剂盒;
a2)制备炎症相关疾病人群诊断的试剂盒;
a3)制备炎症相关疾病人群治疗状况监测的试剂盒;
a4)制备炎症相关疾病人群预后监测的试剂盒;
a5)制备筛查与炎症相关疾病相关靶标的试剂盒;
a6)制备治疗炎症相关疾病的药物中的应用。
优选的,所述炎症相关疾病包括阿尔茨海默病。
优选的,所述应用包括b1)~b8)中的一种或多种:
b1)促进小胶质细胞活化的药物;
b2)促进小胶质细胞促炎细胞表型的药物;
b3)促进促炎细胞因子释放的药物;所述促炎细胞因子包括TNF-α和/或IL-6;
b4)抑制抗炎细胞因子释放的药物;所述抗炎细胞因子包括IL-4和/或IL-10;
b5)促进小胶质细胞中NF-κB信号通路,促进神经炎症反应的药物;
b6)促进小胶质细胞M1分子标记物的表达的药物;所述小胶质细胞M1分子标记物包括iNOS;
b7)抑制小胶质细胞M2分子标记物的表达的药物;所述小胶质细胞M2分子标记物包括ARG1;
b8)降低KLF4表达水平的药物。
本发明还提供了一种治疗炎症相关疾病的药物,所述药物的有效成分包括抑制miR-25802表达或敲低miR-25802表达的物质。
优选的,所述物质包括化学小分子药物、核酸药物和抗体药物。
本发明还提供了一种检测上述技术方案所述miR-25802簇的引物组,所述引物组包括逆转录引物、上游引物和下游引物;
所述逆转录引物包括如SEQ ID NO.3所示的核苷酸序列;
所述上游引物包括如SEQ ID NO.4所示的核苷酸序列;
所述下游引物包括如SEQ ID NO.5所示的核苷酸序列。
本发明还提供了上述技术方案所述的引物组在制备下述b1)~b4)中的一种或多种试剂盒中的应用:
b1)炎症相关疾病人群筛选的试剂盒;
b2)炎症相关疾病人群诊断的试剂盒;
b3)炎症相关疾病人群治疗状况监测的试剂盒;
b4)炎症相关疾病人群预后监测的试剂盒。
本发明还提供了一种炎症相关疾病筛查试剂盒,所述试剂盒包括上述技术方案所述的引物组;
优选的,所述炎症相关疾病包括阿尔茨海默病。
本发明提供的生物标志物miR-25802簇,包括miR-25802,所述miR-25802包括如SEQ ID NO.2所示的核苷酸序列。本发明通过AD模式细胞、AD模式动物、临床血液样本检测发现,miR-25802簇的微小RNA在阿尔茨海默病中表达显著升高,所述miR-25802簇可以作为检测AD的生物标志物。
另外,本发明使用酶联免疫吸附测定、蛋白免疫印迹、双荧光素酶报告实验、基因功能增益和敲除实验,对miR-25802的功能进行深入系统研究,发现所述miR-25802能诱导小胶质细胞活化,诱导促炎细胞表型;miR-25802表达下调则诱导小胶质细胞呈现为抗炎表型。miR-25802正向调控小胶质细胞NF-kB炎症信号通路,诱导小胶质细胞活化和表型转换,促进炎症反应。因此,敲除或敲低miR-25802的物质,能够抑制小胶质细胞介导的固有免疫反应,改善AD炎症病理进程,有效防治AD。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍。
图1为miRNA高通量测序检测APP/PS1小鼠大脑皮层中miR-25802簇的表达水平的热图;
图2-1为qRT-PCR检测铜离子处理后的不同时间点APPswe细胞中miR-25802的表达水平(AD神经细胞模型);
图2-2为qRT-PCR检测LPS/IFN-γ处理小胶质细胞中miR-25802的表达水平(神经炎症细胞模型);
图2-3为qRT-PCR检测miR-25802在APP/PS1小鼠和WT野生型对照小鼠中的表达水平的结果(动物模型皮层);
图2-4为qRT-PCR检测miR-25802在APP/PS1小鼠和WT野生型对照小鼠中的表达水平的结果(动物模型海马脑组织);
图2-5为qRT-PCR在AD患者血浆和同龄健康志愿者(HAV)中检测miR-25802的表达水平;
图2-6为ROC曲线分析miR-25802在APP/PS1小鼠中的诊断预测价值;
图3-1为qRT-PCR检测miR-25802上调/下调的静息(未激活)状态小胶质细胞促炎M1表型分子标记物水平;
图3-2为qRT-PCR检测miR-25802上调/下调的静息(未激活)状态小胶质细胞抗炎M2表型分子标记物水平;
图3-3为ELISA检测miR-25802上调/下调的静息(未激活)状态小胶质细胞分泌促炎细胞因子TNF-α的水平;
图3-4为ELISA检测miR-25802上调/下调的静息(未激活)状态小胶质细胞分泌促炎细胞因子IL-6的水平;
图3-5为ELISA检测miR-25802上调/下调的静息(未激活)状态小胶质细胞分泌抗炎细胞因子TGF-β的水平;
图4-1为qRT-PCR检测miR-25802上调/下调的炎症(激活,促炎表型)状态小胶质细胞促炎M1表型分子标记物水平;
图4-2为qRT-PCR检测miR-25802上调/下调的炎症(激活,促炎表型)状态小胶质细胞促炎M2表型分子标记物水平;
图4-3为ELISA检测miR-25802上调/下调的炎症(激活,促炎表型)状态小胶质细胞分泌促炎细胞因子TNF-α的水平;
图4-4为ELISA检测miR-25802上调/下调的炎症(激活,促炎表型)状态小胶质细胞分泌促炎细胞因子IL-6的水平;
图4-5为ELISA检测miR-25802上调/下调的炎症(激活,促炎表型)状态小胶质细胞分泌抗炎细胞因子TGF-β的水平;
图5-1为miR-25802所调控的通路富集分析结果;
图5-2和图5-3为利用蛋白印迹Western Blot技术检测小胶质细胞内KLF4蛋白表达水平的结果;
图6-1为利用蛋白印迹Western Blot技术检测miR-25802上调/下调的静息(未激活)状态小胶质细胞NF-κB炎症信号通路相关蛋白表达水平;
图6-2为利用蛋白印迹WesternBlot技术定量检测miR-25802上调/下调的静息(未激活)状态小胶质细胞NF-κB炎症信号通路p65,IKKα&β磷酸化蛋白相对表达水平;
图6-3为利用蛋白印迹WesternBlot技术定量检测miR-25802上调/下调的静息(未激活)状态小胶质细胞NF-κB炎症信号通路IKBα蛋白相对表达水平;
图6-4为利用蛋白印迹Western Blot技术检测miR-25802上调/下调的炎症(激活,促炎表型)状态小胶质细胞NF-κB炎症信号通路相关蛋白表达水平;
图6-5为利用蛋白印迹WesternBlot技术定量检测miR-25802上调/下调的炎症(激活,促炎表型)状态小胶质细胞NF-κB炎症信号通路p65,IKKα&β磷酸化蛋白相对表达水平;
图6-6为利用蛋白印迹WesternBlot技术定量检测miR-25802上调/下调的炎症(激活,促炎表型)状态小胶质细胞NF-κB炎症信号通路IKBα蛋白相对表达水平。
具体实施方式
本发明提供了一种炎症相关疾病生物标志物miR-25802簇,包括miR-25802,所述miR-25802包括如SEQ ID NO.2所示的核苷酸序列,具体为:5’-UCACGGAUACAGCCUCCUUUGGGA-3’。
本发明所述miR-25802簇包括但不局限于miR-25802,与miR-25802序列相似的基因,均属于本发明的保护范围,例如miR-25802经修饰后产生的衍生物,或者长度为18~26nt、功能与miR-25802相同或基本相同的微小RNA,或者长度为18~26nt、功能与miR-25802相同或基本相同的微小RNA经修饰的衍生物均可作为炎症类相关疾病生物标志物,不能仅将miR-25802理解为本发明的全部保护范围。本发明所述炎症类相关疾病优选包括阿尔茨海默病(AD)。
在本发明中,所述miR-25802的前体为mir-25802,所述mir-25802优选包括如SEQID NO.1所示的核苷酸序列,具体为:5’-UCACGGAUACAGCCUCCUUUGGGAUCCUGCUCUGUUCCCAUGAGACU GUAUCUGCCUGUGUCCA-3’。
本发明所述miR-25802为mir-25802的成熟体,具体优选由mir-25802的5’臂端加工而成。本发明对所述加工的方式没有严格要求,常规操作即可。
本发明以稳定转染APP/PS1基因的1、3、6、9月龄双转基因小鼠和野生型小鼠为实验对象,利用基于桥式PCR与边合成边测序结合的测序技术,进行“高通量、高准确率、低成本”的高通量基因组学表达谱二代测序,利用Trizol的方法提取小鼠脑组织RNA并进行分离、构建测序基因文库,挖掘出具有明确特征变化并具有全新序列的miR-25802,miR-25802在不同月龄APP/PS1小鼠脑组织中表达上调。并且采用qRT-PCR技术进行逆转录和实时荧光定量检测miR-25802在AD模式细胞、AD模式动物、AD患者血清中表达上调,miR-25802簇与AD存在疾病关联性,可以作为AD诊断生物标志物。
本发明还提供了上述技术方案所述的miR-25802簇下述a1)~a6)中的一种或多种中的应用:
a1)制备炎症相关疾病人群筛选的试剂盒;
a2)制备炎症相关疾病人群诊断的试剂盒;
a3)制备炎症相关疾病人群治疗状况监测的试剂盒;
a4)制备炎症相关疾病人群预后监测的试剂盒;
a5)制备筛查与炎症相关疾病相关靶标的试剂盒;
a6)制备治疗炎症相关疾病的药物中的应用。
在本发明中,所述炎症相关疾病优选包括阿尔茨海默病。本发明所述药物优选包括b1)~b8)中的一种或多种:b1)促进小胶质细胞活化的药物;b2)促进小胶质细胞促炎细胞表型的药物;b3)促进促炎细胞因子释放的药物;b4)抑制抗炎细胞因子释放的药物;b5)促进小胶质细胞中NF-κB信号通路,促进神经炎症反应的药物;b6)促进小胶质细胞M1分子标记物的表达的药物;b7)抑制小胶质细胞M2分子标记物的表达的药物;b8)降低KLF4表达水平的药物。本发明所述促炎细胞因子优选包括TNF-α和/或IL-6;所述抗炎细胞因子优选包括IL-4和/或IL-10;所述小胶质细胞M1分子标记物优选包括包括iNOS;所述小胶质细胞M2分子标记物优选包括ARG1。
本发明以所述的miR-25802簇作为检测靶点,通过测量样本miR-25802簇的表达情况,能够筛选、诊断炎症相关疾病,并且对炎症相关疾病人群治疗后的状况监测,丰富阿尔茨海默病诊断标记物。
本发明还提供了一种治疗炎症相关疾病的药物,所述药物的有效成分包括抑制miR-25802表达或敲低miR-25802表达的物质。在本发明中,所述物质优选包括化学小分子药物、核酸药物和抗体药物中的一种或多种。
本发明通过敲低miR-25802簇表达,能够降低AD炎症病理进程,有效防治包括AD在内的炎症相关疾病。本发明对所述敲除或敲低miR-25802簇表达的物质的种类没有严格要求,任何敲除或敲低miR-25802簇表达的物质均属于本发明的保护范围,例如miR-25802簇的核酸模拟物、miR-25802簇的抑制剂、核酸药物、小分子化合物和抗体药物。
本发明还提供了检测上述技术方案所述miR-25802簇的引物组,所述引物包括逆转录引物、上游引物和下游引物;
所述逆转录引物包括如SEQ ID NO.3所示的核苷酸序列;
所述上游引物包括如SEQ ID NO.4所示的核苷酸序列;
所述下游引物包括如SEQ ID NO.5所示的核苷酸序列。
本发明利用所述引物组中的逆转录引物能够反转录所述miR-25802簇后利用正向引物和反向引物进行扩增,能够特异性检测miR-25802的表达量,诊断阿尔茨海默病,预测形成阿尔茨海默病的风险,或预测阿尔茨海默病治疗后的的结果。
鉴于本发明所述引物组的优势作用,所述引物组在制备下述b1)~b4)中的一种或多种试剂盒中的应用均属于本发明的保护范围:b1)炎症相关疾病人群筛选的试剂盒;b2)炎症相关疾病人群诊断的试剂盒;b3)炎症相关疾病人群治疗状况监测的试剂盒;b4)炎症相关疾病人群预后监测的试剂盒。
本发明还提供了一种炎症相关疾病筛查试剂盒,所述试剂盒包括上述技术方案所述的引物。在本发明中,所述炎症相关疾病包括阿尔茨海默病。
本发明在发现所述miR-25802簇能够作为阿尔茨海默病标志物的基础上,还可作为分子治疗靶点开发治疗炎症相关疾病的药物。对miR-25802簇的微小RNA的功能进行深入系统研究,发现所述miR-25802簇能够诱导小胶质细胞激活,促进NF-κB炎症信号通路,调控小胶质细胞炎症相关分子标记物表达,促进炎症反应;miR-25802簇的微小RNA表达下调则促进小胶质细胞呈现为抗炎表型、抑制炎症反应。因此,敲除或敲低所述miR-25802簇的表达能够降低炎症病理进程,有效防治阿尔茨海默病。本发明发现miR-25802簇和阿尔茨海默病二者的关系,提供了一种发挥抗炎作用的潜在新靶标,解决了现有技术在基因水平上阿尔茨海默病诊断标记物缺乏问题,有助于解决现有技术中缺乏阿尔茨海默病在内的炎症治疗有效靶标的现状。
为了进一步说明本发明,下面结合附图和实施例对本发明提供的一种阿尔兹海默症生物标志物miR-25802簇及其应用的技术方案进行详细地描述,但不能将它们理解为对本发明保护范围的限定。
在本发明具体实施例中,如无特殊说明,涉及的步骤均为常规步骤,使用的试剂均可常规购买得到或者按照产品说明书自行配制。
实施例1
miRNA高通量测序技术检测AD病理进程中差异表达的微小RNA
以APP/PS1小鼠(记为APP/PS1 mice,购自至善(北京)健康医学研究院)和野生小鼠(记为WT mice,购买自至善(北京)健康医学研究院)为实验材料,利用过量吸入乙醚的方法处死小鼠,分别取1、3、6和9月龄APP/PS1小鼠和野生小鼠的脑组织,分离大脑皮层和海马,即刻放入液氮中,过夜后转至负八十度冰箱保存。利用基于桥式PCR与边合成边测序结合的测序技术,进行“高通量、高准确率、低成本”的高通量基因组学表达谱二代测序,利用Trizol的方法提取小鼠皮层和海马总RNA并进行分离、构建测序基因文库,利用Illu minaHiSeq 2500对构建的样本基因文库进行单端测序,使用FastQC评估测序原始数据质量,通过miRDeep2软件进行miRNA与参考基因组比对、miRNA二级结构分析、miRNA差异表达分析,发现在相同年龄段的APP/PS1小鼠和野生小鼠的海马和皮层中存在差异表达的非编码RNA(如图1所示,结果以均值±SEM(n=3)计),记为miR-25802,测定其核苷酸序列如SEQ IDNO.2所示,具体为,5’-UCACGGAUACAGCCUCCUUUGGGA-3’,其中,miR-25802为成熟体,合成miR-25802的前体mir-25802的核苷酸序列如SEQ ID NO.1所示,具体为,5’-UCACGGAUACAGCCUCCUUUGGGAUCCUGCUCUGUUCCCAUG AGACUGUAUCUGCCUGUGUCCA-3’。miR-25802的逆转录引物序列如SEQ ID NO.3所示,具体为:5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCCCAA-3’;定量PCR(qPCR)检测miR-25802的正向引物如SEQ ID NO.4所示,具体为:5’-CGTCACGGATACAGCCTCCT-3’;定量PCR(qPCR)检测miR-25802的反向引物:SEQ IDNO.5:5‘-AGTGCAGGGTCCG AGGTATT-3’。以上步骤委托生工生物工程(上海)股份有限公司进行。
实施例2
miR-25802簇的微小RNA在阿尔茨海默病(AD)模式细胞中的表达变化
(1)采用细胞培养技术、脂质体瞬时转染、抗生素加压筛选、有限稀释法获得单克隆株,同时利用WesternBlot或ELISA进行相关蛋白检测,构建稳定转染人鼠嵌合型APP基因的人神经母细胞瘤细胞(APPswe细胞),具体参照文献(Wang,C.Y.,et al.(2011).HuperzineA activates Wnt/β-catenin signaling and enhances thenonamyloidogenic pathway in anAlzheimertransgenic mouse model.Neuropsychopharmacology.36(5),1073–1089.)。
(2)将步骤(1)APPswe细胞培养在含10%FBS(胎牛血清)的DMEM培养基中,5%二氧化碳,37℃培养。使用1μg/ml嘌呤霉素维持稳转株细胞表型性状。在细胞汇合度为80%时,使用300μM铜离子处理,使用铜离子诱导处理APPswe细胞后,铜离子与APP、Aβ形成螯合物,加重Aβ的产生与沉积,诱导氧化应激反应和神经细胞的凋亡。因此,利用铜离子处理后的APPswe细胞可以用来模拟AD神经细胞的病理状态和药物作用机制的研究。
随机抽取铜离子处理后的不同时间点APPswe细胞,使用Trizol法提取细胞总RNA(康为生物试剂盒,CW0581)。随后使用茎环法进行逆转录反应(诺维赞南京,MIR-101),采用实时荧光定量聚合酶链反应(诺维赞南京,MQ-101)qRT-PCR技术定量检测APPswe细胞中miR-25802的表达水平,操作按照试剂商说明书进行。其中逆转录引物序列如SEQ ID NO.3所示,实时荧光定量检测的正向引物序列如SEQ ID NO.4所示,实时荧光定量检测的反向引物序列如SEQ ID NO.5所示。
检测结果如图2-1示,其中结果以均值±SEM(n=3)计,*表示与不添加铜离子的0h相比,P<0.05,**表示与0h相比,P<0.01。
根据图2-1可以看出,铜离子刺激诱导的细胞损伤随时间加重,miR-25802的表达量随之增加,这反映了在miR-25802随AD病理进程中表达上调。
(3)小鼠小胶质细胞EOC20细胞购于ATCC,在5%CO2和37摄氏度条件下,在含10%胎牛血清,20%LADMAC条件培养基的DMEM培养基中生长。将EOC20细胞以1×105个/mL接种于六孔板内,同时加入终浓度为100ng/mL LPS和1ng/mL IFN-γ,24h后使用Trizol法提取细胞总RNA(康为生物试剂盒,CW0581),逆转录(诺维赞南京,R323)并使用实时荧光定量聚合酶链反应(诺维赞南京,Q711),以Actb基因为内参,测定TNF-α,IL-6促炎分子mRNA相对表达水平。在此模型中促炎分子表达水平水平明显上调,说明LPS/IFN-γ共同诱导小鼠小胶质EOC20细胞激活并呈现M1促炎细胞表型,由此构建得到体外炎症细胞模型,记为LPS/IFN-γ;未作为对照,记为Control。
采用qRT-PCR技术进行逆转录和实时荧光定量检测神经炎症细胞模型中miR-25802的表达水平,其中逆转录引物序列如SEQ ID NO.3所示,实时荧光定量检测的正向引物序列如SEQ ID NO.4所示,实时荧光定量检测的反向引物序列如SEQ ID NO.5所示。
检测结果如图2-2所示,其中结果以均值±SEM(n=3)计,*表示与Control组相比,P<0.05。
根据图2-2可以看出,miR-25802在炎症细胞模型中的表达量显著增加。
实施例3
miR-25802簇的微小RNA在阿尔茨海默病(AD)模式动物中的表达变化
以1、3、6和9月龄的APP/PS1双转基因小鼠为实验组(记为APP/PS1 mice,购买自至善(北京)健康医学研究院),1、3、6和9月龄的野生对照小鼠为对照组(记为WT mice,购买自至善(北京)健康医学研究院),使用麻醉方法处死小鼠,在冰上快速分离1、3、6、9月龄APP/PS1双转基因小鼠和野生型对照小鼠的皮层和海马脑组织,经液氮冷冻后,随后利用Trizol方法分别提取小鼠皮层、海马脑组织总mRNA,利用紫外分光光度法测定总RNA的浓度和纯度,利用qRT-PCR技术检测miR-25802在AD病理进程中的表达变化,结果如图2-3和2-4所示,其中图2-3为小鼠皮层检测结果,图2-4为海马脑组织检测结果。C和D所示结果以均值±SEM(n=3)计,*表示与WT mice相比,APP/PS1 mice的P<0.05。
根据图2-3和图2-4可以看出,在动物模型皮层和海马脑组织中,与同月龄对照组小鼠(WT小鼠)相比,miR-25802的表达水平在1、3、6、9月龄显著增加。
实施例4
miR-25802簇的微小RNA在阿尔茨海默病(AD)患者血清中的表达变化
收集11名AD患者的血清和11名正常同龄人(HAV)的血清,以此为实验材料,提取患者和正常同龄人总RNA,利用紫外分光光度法进行RNA浓度和纯度验证,利用qRT-PCR技术检测miR-25802在AD患者血清中的含量。使用ROC曲线分析差异性表达的miR-25802作为诊断指标区分AD患者和健康人中的能力,结果如图2-5和图2-6所示,其中图2-5为qRT-PCR技术检测结果,结果以均值±SEM(n=11)计,**表示与HAV相比,AD患者的P<0.01;图2-6为ROC曲线分析结果,其中ROC曲线下面积为AUC=0.920(CI:0.800-1.00,P<0.01),灵敏度87.5%,特异性81.8%。
根据图2-5和图2-6可以看出,AD患者血液中miR-25802的相对表达量显著升高,ROC曲线检测的灵敏度和特异性均较高,以miR-25802差异性的相对表达作为诊断方法能够有效区分患者和健康人,准确度高。
实施例5
miR-25802簇的微小RNA表达失调对静息状态小胶质细胞表型和炎症反应的影响
(1)基于miRNA模拟物(mimics)和miRNA抑制剂(inhibitor),采用脂质体瞬时转染技术构建miRNA过表达或敲除的细胞模型,具体的:
将EOC20小鼠小胶质细胞平均分为4组,依次记为NCM、NCI、miR-25802mimics和miR-25802inhibitor;
NCM组利用脂质体瞬时转染50nM miRNA无关序列阴性对照(negative control,NCM:SEQ ID NO.6,5’-UUGUACUACACAAAAGUACUG-3’);
NCI组利用脂质体瞬时转染50nM miRNA无关序列阴性对照(negative control,NCI:SEQ ID NO.7,5’-CAGUACUUUUGUGUAGUACAA-3’);
miR-25802mimics组利用脂质体瞬时转染50nM novel miR-25802mimics(SEQ IDNO.2,5’-UCACGGAUACAGCCUCCUUUGGGA-3’);
miR-25802inhibitor组利用脂质体瞬时转染50nM novel miR-25802inhibitor(SEQ ID NO.8,5’-UCCCAAAGGAGGCUGUAUCCGUGA-3’)。
各处理组处理后的细胞在37℃孵育,24h后检查mRNA表达水平,分泌型细胞因子检测在孵育48h后进行。
(2)步骤(1)结束后利用qRT-PCR、ELISA技术检测小胶质细胞炎症相关细胞表型标记物,结果如图3-1~图3-5所示,其中图3-1表示qRT-PCR检测miR-25802上调/下调的静息(未激活)状态小胶质细胞促炎M1表型分子标记物水平;图3-2表示qRT-PCR检测miR-25802上调/下调的静息(未激活)状态小胶质细胞抗炎M2表型分子标记物水平;图3-3表示ELISA检测miR-25802上调/下调的静息(未激活)状态小胶质细胞分泌促炎细胞因子TNF-α的水平;图3-4表示ELISA检测miR-25802上调/下调的静息(未激活)状态小胶质细胞分泌促炎细胞因子IL-6的水平;图3-5表示ELISA检测miR-25802上调/下调的静息(未激活)状态小胶质细胞分泌抗炎细胞因子TGF-β的水平,图3-1~图3-5结果以均值±SEM(n=4)计,*表示与NCM相比,P<0.05,**表示与NCM相比,P<0.01,#表示与NCI相比,P<0.05,###表示与NCI相比,P<0.001。
根据图3-1~图3-5可以看出,miR-25802过表达诱导静息小胶质细胞激活并转换为促炎表型,促进分泌促炎细胞因子。
实施例6
miR-25802簇的微小RNA表达失调对炎症状态小胶质细胞表型和炎症反应的影响
按照实施例2步骤(3)构建炎症细胞模型,采用脂质体共转染miR-25802mimics和miR-25802inhibitor方法上调或者下调胶质细胞中miR-25802的表达水平,具体的:
将构建的炎症细胞模型平均分为4组,依次记为NCM、NCI、miR-25802mimics和miR-25802inhibitor;
NCM组利用脂质体瞬时转染50nM miRNA无关序列阴性对照(negative control,NCM:SEQ ID NO.6,5’-UUGUACUACACAAAAGUACUG-3’);
NCI组利用脂质体瞬时转染50nM miRNA无关序列阴性对照(negative control,NCI:SEQ ID NO.7,5’-CAGUACUUUUGUGUAGUACAA-3’);
miR-25802mimics组利用脂质体瞬时转染50nM novel miR-25802mimics(SEQ IDNO.2,5’-UCACGGAUACAGCCUCCUUUGGGA-3’);
miR-25802inhibitor组利用脂质体瞬时转染50nM novel miR-25802inhibitor(SEQ ID NO.8,5’-UCCCAAAGGAGGCUGUAUCCGUGA-3’)。
转染24h或48h后,分别选用qRT-PCR、ELISA技术检测,结果如图4-1~图4-5所示,其中图4-1表示qRT-PCR检测miR-25802上调/下调的炎症(激活,促炎表型)状态小胶质细胞促炎M1表型分子标记物水平;图4-2表示qRT-PCR检测miR-25802上调/下调的炎症(激活,促炎表型)状态小胶质细胞促炎M2表型分子标记物水平;图4-3表示ELISA检测miR-25802上调/下调的炎症(激活,促炎表型)状态小胶质细胞分泌促炎细胞因子TNF-α的水平;图4-4表示ELISA检测miR-25802上调/下调的炎症(激活,促炎表型)状态小胶质细胞分泌促炎细胞因子IL-6的水平;图4-5表示ELISA检测miR-25802上调/下调的炎症(激活,促炎表型)状态小胶质细胞分泌抗炎细胞因子TGF-β的水平,图4-1~图4-5结果以均值±SEM(n=4)计,*表示与NCM相比,P<0.05,#表示与NCI相比,P<0.05。
根据图4-1~图4-5可以看出,miR-25802表达下调可促进小胶质细胞向抗炎表型转换,抑制促炎细胞因子释放,促进抗炎分子标记物表达,缓解炎症反应。
实施例7
miR-25802簇的微小RNA靶基因预测
(1)利用生物信息学软件miRDB、miRanda预测miR-25802的潜在结合靶标,使用Metascape在线软件对miR-25802预测结合基因进行KEGG通路富集分析,结果如图5-1所示。根据图5-1可以看出,miR-25802的靶基因富集于阿尔茨海默病、免疫反应等炎症相关通路。
实施例8
miR-25802簇的微小RNA在翻译水平特异性调控KLF4的表达
基于miRNAmimics/inhibitor,利用脂质体瞬时转染技术,建立miR-25802过表达或敲低的小胶质细胞模型,
具体的:将小胶质细胞平均分为4组,依次记为NCM、NCI、miR-25802mimics和miR-25802inhibitor;
NCM组利用脂质体瞬时转染50nM miRNA无关序列阴性对照(negative control,NCM:SEQ ID NO.6,5’-UUGUACUACACAAAAGUACUG-3’);
NCI组利用脂质体瞬时转染50nM miRNA无关序列阴性对照(negative control,NCI:SEQ ID NO.7,5’-CAGUACUUUUGUGUAGUACAA-3’);
miR-25802mimics组利用脂质体瞬时转染50nM novel miR-25802mimics(SEQ IDNO.2,5’-UCACGGAUACAGCCUCCUUUGGGA-3’);
miR-25802inhibitor组利用脂质体瞬时转染50nM novel miR-25802inhibitor(SEQ ID NO.8,5’-UCCCAAAGGAGGCUGUAUCCGUGA-3’)。
转染48h后,通过RIPA裂解法、超声提取细胞总蛋白质,使用BCA法测定可见光吸收度进行定量。使用二硫苏糖醇还原蛋白并煮沸变性,利用蛋白印迹Western Blot技术检测细胞内KLF4蛋白表达水平,结果如图5-2和图5-3,其中结果以均值±SEM(n=4)计,*表示与NCM相比,P<0.05,#表示与NCI相比,P<0.05。
根据图5-2和图5-3可以看出,miR-25802表达上调可以在翻译水平负向调控特异性靶标KLF4的表达,降低KLF4的蛋白表达量;miR-25802表达下调可以促进KLF4的高表达。
实施例9
miR-25802簇的微小RNA调控NF-κB炎症信号通路
按照实施例6的步骤通过脂质体转染技术构建miR-25802过表达或敲低的炎症细胞模型,通过Western Blot技术检测NF-κB信号通路相关分子表达水平。结果如图6-1~图6-6所示,其中图6-1表示利用蛋白印迹Western Blot技术检测miR-25802上调/下调的静息(未激活)状态小胶质细胞NF-κB炎症信号通路相关蛋白表达水平;图6-2表示利用蛋白印迹WesternBlot技术定量检测miR-25802上调/下调的静息(未激活)状态小胶质细胞NF-κB炎症信号通路p65,IKKα&β磷酸化蛋白相对表达水平;图6-3表示利用蛋白印迹Western Blot技术定量检测miR-25802上调/下调的静息(未激活)状态小胶质细胞NF-κB炎症信号通路IKBα蛋白相对表达水平;图6-4表示利用蛋白印迹WesternBlot技术检测miR-25802上调/下调的炎症(激活,促炎表型)状态小胶质细胞NF-κB炎症信号通路相关蛋白表达水平;图6-5表示利用蛋白印迹WesternBlot技术定量检测miR-25802上调/下调的炎症(激活,促炎表型)状态小胶质细胞NF-κB炎症信号通路p65,IKKα&β磷酸化蛋白相对表达水平;图6-6表示利用蛋白印迹Western Blot技术定量检测miR-25802上调/下调的炎症(激活,促炎表型)状态小胶质细胞NF-κB炎症信号通路IKBα蛋白相对表达水平,图6-1~图6-6结果以均值±SEM(n=4)计,*表示与NCM相比,P<0.05,**表示与NCM相比,P<0.01,***表示与NCM相比,P<0.001,#表示与NCI相比,P<0.05。
根据图6-1~图6-6可以看出,miR-25802过表达上调静息胶质细胞中NF-κB信号通路相关分子表达水平,抑制miR-25802下调促炎表型胶质细胞中NF-κB信号通路相关分子表达水平。
根据上述实施例,本发明提供的miR-25802在阿尔茨海默病病理进程中表达显著升高,基于血清表达水平的ROC曲线显示miR-25802具有良好的诊断效应,可以作为检测AD的生物标志物,并负向调控KLF4基因的表达。miR-25802过表达诱导并促进小胶质细胞转换为促炎表型,上调炎症因子水平,促进炎症反应。miR-25802过表达上调NF-κB炎症信号通路活性,反之miR-25802敲低下调NF-κB炎症信号通路活性,促进胶质细胞炎症表型转换,能够有效防治AD。
尽管上述实施例对本发明做出了详尽的描述,但它仅仅是本发明一部分实施例,而不是全部实施例,人们还可以根据本实施例在不经创造性前提下获得其他实施例,这些实施例都属于本发明保护范围。
Claims (3)
1.抑制miR-25802表达的inhibitor在制备治疗阿尔茨海默病的药物中的应用;
所述miR-25802的核苷酸序列如SEQ ID NO .2所示;
所述inhibitor的核苷酸序列如SEQ ID NO .8所示。
2.根据权利要求1所述的应用,其特征在于,所述miR-25802的前体为mir-25802,所述mir-25802的核苷酸序列如SEQ ID NO .1所示。
3.根据权利要求1或2所述的应用,其特征在于,用于扩增所述miR-25802的引物组包括逆转录引物、上游引物和下游引物;
所述逆转录引物的核苷酸序列如SEQ ID NO .3所示;
所述上游引物的核苷酸序列如SEQ ID NO .4所示;
所述下游引物的核苷酸序列如SEQ ID NO .5所示。
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