CN114107478A - 一种用于帕金森早期诊断的生物标志物及其应用 - Google Patents
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Abstract
本发明公开了一种用于帕金森早期诊断的microRNA生物标志物及其应用,所述生物标志物为miR‑101‑3p,所述生物标志物miR‑101‑3p在帕金森患者外周血中表达显著降低。本发明还公开了一种用于帕金森早期诊断的microRNA生物标志物的筛选方法。利用本发明的生物标志物miR‑101‑3p早期诊断帕金森具有高灵敏度、高特异度且无创的优势,有利于帕金森的早发现、早治疗,具有良好的临床使用和推广价值。
Description
技术领域
本发明涉及生物医药领域,具体涉及一种用于帕金森早期诊断的生物标志物及其应用。
背景技术
帕金森病(PD)是第二常见的神经退行性疾病,其特征是多巴胺能神经元死亡和黑质(SN)中α-突触核蛋白(α-Syn)的异常积聚和聚集,大多数帕金森病患者可能有多因素病因,由环境和遗传因素共同作用所致,包括但不限于有毒化学品接触、头部损伤、生活方式因素、基因突变、线粒体功能障碍等。目前,帕金森病无法完全治愈,现有的临床治疗策略对帕金森病发病早期的治疗效果比发病后期要好,因此,帕金森病的早期诊断至关重要。
近来有研究表明,miRNAs参与了神经退行性疾病如帕金森病的发生和发展,例如已有研究证实miR-7、miR-153、miR-34b和miR-34c直接参与抑制编码SNCA的过程,而miR-7能够抑制α-Syn的基因表达,从而减少应激造成的神经元和多巴胺细胞的凋亡,使PD的发生率降低;miR-205、let-7、miR-184及其靶标也是多巴胺能神经元致病性LRRK2的关键介质;还有一些miRNAs例如miR-128-3p通过激活Wnt/β-catenin通路减少PD的患病概率,miR-216a可通过降解Bax抑制MPP+诱导的神经细胞凋亡,对神经细胞起到保护作用,从而降低PD患病风险,同时也是MPP+引起的PD神经毒性的有效治疗靶标;此外,还有研究发现,PD患者血清中miR-150表达下降,唾液中miR-153和miR-223表达下调,脑脊液中miR-1和miR-19b-3p表达显著降低,miR-153、miR-409-3p、miR-10a-5p、let-7g-3p、miR-136-3p和miR-433表达显著升高,这些都预示其可作为诊断标志物的可能性。
然而,目前的尚未有研究证实外周血中的miR-101-3p作为PD诊断标志物的可行性,寻找和鉴定与PD发病有关的miRNA能够为PD的临床诊断和治疗提供基础,有助于PD的早期诊断。
发明内容
基于此,有必要提供了一种能够用于帕金森早期诊断的外周血microRNA生物标志物。
为实现上述目的,本发明具体技术方案如下:
1.筛选出与帕金森显著相关的microRNA生物标志物miR-101-3p;
2.miR-101-3p表达量在帕金森患者中的应用;
3.ROC曲线验证。
本发明提供了一种用于帕金森早期诊断的生物标志物,所述生物标志物为miR-101-3p。
优选的,所述生物标志物为外周血中microRNA。
优选的,相较于正常人,所述生物标志物miR-101-3p在帕金森患者外周血中表达显著降低。
进一步地,还提供了所述的生物标志物的检测试剂在制备诊断帕金森的试剂盒中的应用。
优选的,所述试剂盒包括检测miR-101-3p表达量的试剂。
优选的,所述检测miR-101-3p表达量的试剂为对miR-101-3p具有检测特异性的PCR引物。
优选的,所述引物序列如下:
U6 F human:GCTCGCTTCGGCAGCACA
U6 R human:GAACGCTTCACGAATTTGCGTG
hsa-miR-101-3p-逆:
GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACTTCAGTT
hsa-miR-101-3p-F:CGGTACAGTACTGTGATAA
miR通用-R:CAGTGCGTGTCGTGGAGT。
优选的,所述试剂盒检测的样本为外周血。
进一步地,还提供了一种用于帕金森早期诊断的生物标志物的筛选方法,其特征在于,包括以下步骤:
(1)细胞系及细胞培养;
(2)RNA的提取;
(3)miRNA芯片实验。
基于上述技术方案,本发明具有以下有益效果:
本发明提供的miR-101-3p在PD患者外周血中的表达量显著低于正常人,外周血中miR-101-3p能够作为PD的诊断标志物,通过对外周血中的miR-101-3p进行特异性检测能够达到辅助PD早期诊断和治疗的目的。具有高灵敏度、高特异度且无创的优势,有利于帕金森的早发现、早治疗,具有良好的临床使用和推广价值。
附图说明
图1A.miRDB数据库、miRWalk数据库、starBase数据库、KM-Plotter数据库中三个或四个数据库的靶mRNA与GSE100054下调基因有交集的基因(即方框中基因);B.miRDB数据库、miRWalk数据库、starBase数据库、KM-Plotter数据库中三个或四个数据库的靶mRNA与GSE100054上调基因有交集的基因(即方框中基因)。
图2A.表达上调的miRNA-mRNA调控网络;B.表达下调的miRNA-mRNA调控网络。
图3 mRNA-DAVID功能富集分析。
图4 mRNA-Metascape功能富集分析。
图5 mRNA在神经系统相关疾病中的评分情况。
图6 mRNA的蛋白质-蛋白质相互作用分析。
图7进一步锁定的miRNA-mRNA网络图。
图8 qRT-PCR溶解曲线为单峰,说明引物特异性良好。
图9 qRT-PCR扩增曲线。
图10 miR-101-3p在PD患者外周血中表达显著下调。与正常人外周血(左)相比,PD患者外周血(右)中miR-101-3p的表达水平显著降低。所有数据均展示为平均值±SD。*代表指定组之间p值<0.05。
图11 qPCR样本琼脂糖凝胶电泳图。琼脂糖凝胶电泳检测为单一条带,PCR扩增特异性较好。
图12关于miR-101-3p的ROC曲线。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
下述实施例所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所有的材料、试剂等,如无特殊说明,均可从商业途径获得。
实施例1
1、细胞系及细胞培养:
细胞株PC12均取自大鼠肾上腺嗜铬细胞瘤细胞系,PC12细胞在DME M高糖培养液中培养,细胞在37℃、5%CO2湿化培养箱中培养,并根据细胞需要定期更换培养液。
所述PC12细胞株来源于褐家鼠(Rattus norvegicus)肾上腺嗜铬细胞瘤,它与神经细胞在发生学上均起源于神经脊,常规条件下培养的PC12细胞为一种儿茶酚胺细胞。应用神经生长因子(nerve growth factor,NGF)处理后,PC12细胞向神经元样细胞分化,神经突起的数量和长度明显增加,生理生化功能更接近于儿茶酚胺神经元,模拟PD患者细胞模型。目前,PC12细胞模型已成为抗PD药物体外基础研究理想的细胞模型。
2、RNA的提取:
a)收集细胞至适当离心管中,1000rpm,8min或5500rpm,1min离心;
b)弃上清,弹匀管底的细胞沉淀,加入1ml PBS,转移至1.5ml RNAase-free EP管中,5500rpm离心1min;
c)吸弃上清,弹匀沉淀以充分打匀细胞团,用1ml RNAase-free枪尖加入1mlTRIzon Reagent,立即涡旋震荡15s使其充分混匀,室温静置10min后转移至-80℃冰箱冻存;
d)打开4℃低温离心机预冷,样本从冰箱中取出充分融化,用RNAase-free枪尖加200μl氯仿,使用涡旋振荡器剧烈震荡混匀15s,室温放置5min;
e)4℃,12000rpm离心15min,在这过程中准备新的1.5ml进口EP管,加入500μl异丙醇,放4℃冰箱预冷;
f)用200μl移液枪小心分次取上层水相,加入到预冷的500μl异丙醇中,上下颠倒混匀,室温放置10min;
g)4℃,12000rpm离心10min,在这过程中配制75%乙醇,4℃冰箱预冷;
h)弃上清,每个EP管加入75%乙醇1ml,上下颠倒混匀;
i)4℃,12000rpm离心5min,小心吸弃上清,不要吸到沉淀,室温放置2-5min,使乙醇挥发,用12μl RNAase-free水溶解沉淀,取2μl测浓度和纯度。
3、miRNA芯片实验:
提取PC12细胞RNA样本进行mRNA芯片实验(mRNA选自GSE100054数据库),提取PC12细胞RNA样本进行miRNA芯片实验(miRNA选自GSE16658)。mRNA和miRNA芯片实验由安捷伦科技有限公司采用Agilent基因表达谱芯片完成。
该基因芯片实验检测PC12RNA,经检测后发现RNA质量完好,随后进行标记杂交实验,杂交芯片经过归一化处理以及差异表达分析得到PC12细胞基因表达谱,分别得到1505个表达上调、1302个表达下调的mRNA和77个表达上调、112个表达下调的miRNA,随后分别筛选出P<0.05且差异倍数前十的表达上调和下调的miRNA各十个,再分别选取miRDB数据库、miRWalk数据库、starBase数据库、KM-Plotter数据库中三个或四个数据库的靶mRNA与PC12细胞中下调基因有交集的基因和与PC12中上调基因有交集的基因,如图1所示,上调miRNA以miR-508-5p为例,下调miRNA以miR-550a-3p为例;
由此构建如图2所示的miRNA-mRNA调控网络;
随后对调控网络中所有mRNA做功能富集分析和Metascape富集分析,功能富集分析结果见图3,Metascape富集分析结果见图4。取功能富集到满足P<0.05的所有项和Metascape富集得到的前20项,从功能富集分析和Metascape富集分析的结果中锁定5个与PD相关的项,通过阅读文献排除adaptive immune response一项,如表1所示:
表1
再把剩余4项中相关mRNA做文献检索,把能查到有文献的10个mRNA作为后续研究基因在神经系统相关疾病中进行CTD评分,如图5所示,以CTD的Parkinson Disease评分>20为选定界限,图5A-G中的7个mRNA评分均大于20,图5H-J中的3个mRNA评分均小于20,故剔除评分小于20的3个mRNA;
随后,将上述7个CTD的Parkinson Disease评分>20的mRNA利用STRING数据库进行蛋白质-蛋白质相互作用分析,如图6所示,图中粗细表示实验验证相关性,细线表示无实验验证,粗线表示有实验验证,线段越粗互作关系验证越强;框中有4个mRNA有直接的互作关系,从而得到可进一步验证的如图7所示miRNA-mRNA网络图,进而最终锁定miR-101-3p。
实施例2
1、RNA的提取
PD患者外周血来源:PD患者外周血全部由中国人民解放军总医院老年医学科提供,均为首诊PD患者,患者尚未接受过任何包括手术等治疗,诊断明确,病理清楚。
正常对照外周血来源:正常对照外周血全部由中国人民解放军总医院体检中心提供,均为健康人群体检获得。
分别提取PD患者外周血RNA和正常对照外周血RNA。
2、引物设计与验证
使用引物和探针设计软件Primer 5.0设计反转录引物和qPCR上游引物,下游引物使用通用引物,目的片段长度约60bp,退火温度设置为60-64℃,引物序列见表2;
表2
引物验证使用与定量PCR相同的条件,以cDNA混合样本为模板,结果溶解曲线均为单一峰,琼脂糖凝胶电泳检测为单一条带,PCR扩增特异性较好。
3、qRT-PCR
将上述步骤中提取的RNA反转录为cDNA,反转录体系为20μl,根据实验目的和所用反转录试剂盒说明书设定反转录条件,本实施例中所用反转录试剂盒品牌为天根,货号:KR118-02;所得cDNA若长期保存,放-80℃冰箱,短期使用可放4℃或-20℃保存。
将反转录为cDNA的样品稀释适当倍数,计算本次实验所需八连排孔数,配置所需如表3所示20μl反应体系。
表3
组成成分 | 使用量 | 终浓度 |
2×SuperReal PreMix Plus | 10μl | 1× |
正向引物 | 0.6μl | 0.3μM* |
反向引物 | 0.6μl | 0.3μM* |
cDNA模板 | 50ng-2μg | -ng-pg |
50×ROX Reference Dye<sup>Δ</sup> | 0.4 | 1× |
RNase-free ddH<sub>2</sub>O | 至20μl | - |
打开ABI 7500 Real Time PCR仪预热,将上述液体全部加入八连排PCR管后,用新的手套将盖子盖好,做好标记,用手指轻轻弹匀,短暂离心,注意不能产生气泡,随后把PCR管放入ABI 7500 Real Time PCR仪中,根据基因选择荧光颜色和参数如表4。
表4
反应结束后,用仪器对应软件分析溶解曲线和扩增曲线,溶解曲线见图8,扩增曲线见图9,最后使用2-ΔΔCt法计算qPCR结果,结果数据见表5。
表5.各样本中miR-101-3p的相对表达量
通过Graph Pad对表5中的数据进行处理,结果如图10所示。
由表5中的数据以及图10可以看出,相较于正常人外周血中的miR-101-3p表达量,在PD患者外周血中miR-101-3p的表达量显著下调。
4、qPCR样本跑胶验证基因的表达
将qPCR样本进行琼脂糖凝胶电泳检测,检测结果见图11,琼脂糖凝胶电泳结果显示样本基因表达情况稳定。
实施例3
利用GSE16658 Non-coding RNA芯片数据,绘制健康对照和帕金森病患者关于miR-101-3p的ROC曲线,其中包含健康对照13例和帕金森病患者19例,采用的样本为外周血单核细胞。根据结果显示,miR-101-3p表达量区分健康对照和帕金森病患者的ROC曲线下面积为0.8462(95%CI:0.6994-0.9929,P=0.001043),相对应的灵敏度和特异度分别是0.842和0.769。AUC在0.7-0.9区间内,代表有一定准确性,结果如图12所示。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (9)
1.一种用于帕金森早期诊断的生物标志物,其特征在于,所述生物标志物为miR-101-3p。
2.如权利要求1所述的生物标志物,其特征在于,所述生物标志物为外周血中microRNA。
3.如权利要求1所述的生物标志物,其特征在于,相较于正常人,所述生物标志物miR-101-3p在帕金森患者外周血中表达显著降低。
4.如权利要求1-3任意一项所述的生物标志物的检测试剂在制备诊断帕金森的试剂盒中的应用。
5.如权利要求4所述的应用,其特征在于,所述试剂盒包括检测miR-101-3p表达量的试剂。
6.如权利要求4或5所述的应用,其特征在于,所述检测miR-101-3p表达量的试剂为对miR-101-3p具有检测特异性的PCR引物。
7.如权利要求6所述的应用,其特征在于,所述引物序列如下:
U6 F human:GCTCGCTTCGGCAGCACA
U6 R human:GAACGCTTCACGAATTTGCGTG
hsa-miR-101-3p-逆:
GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATAC
GACTTCAGTT
hsa-miR-101-3p-F:CGGTACAGTACTGTGATAA
miR通用-R:CAGTGCGTGTCGTGGAGT。
8.如权利要求4或5所述的应用,其特征在于,所述试剂盒检测的样本为外周血。
9.一种如权利要求1-3任意一项所述的用于帕金森早期诊断的生物标志物的筛选方法,其特征在于,包括以下步骤:
(1)细胞系及细胞培养;
(2)RNA的提取;
(3)miRNA芯片实验。
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