CN114634977B - 与2型糖尿病胰岛β细胞受损表型相关的血清/血浆miRNA组合标志物及其应用 - Google Patents
与2型糖尿病胰岛β细胞受损表型相关的血清/血浆miRNA组合标志物及其应用 Download PDFInfo
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Abstract
本发明公开了一种与2型糖尿病胰岛β细胞受损表型相关的血清/血浆miRNA组合标志物,包括miR‑4455和/或miR‑4530。本发明还提供了用于胰岛β细胞受损型2型糖尿病检测的荧光定量检测引物组合,包括miR‑4455和miR‑4530的逆转录引物、正向引物和反向引物。以及上述组合标志物和引物组合在制备胰岛β细胞受损型2型糖尿病诊断试剂盒中的应用。本发明公开的标志物能够在早期对胰岛β细胞受损型2型糖尿病进行分型识别诊断,准确地将其与胰岛β细胞正常型2型糖尿病区分开,仅需提供血样即可进行,显著提高了临床应用的可行性和简易性,为特定表型2型糖尿病的早期预防、诊断以及个体化精准治疗方案提供科学依据和指导。
Description
技术领域
本发明属于生物医学技术领域,涉及一种基于血清/血浆miRNA的生物标志物用于2型糖尿病β细胞受损分型诊断的方法。
背景技术
2型糖尿病(T2DM)作为威胁民众健康最主要的慢性疾病之一,是由复杂的遗传-环境因素相互作用引起的多器官代谢紊乱疾病,以慢性高血糖及胰岛素完全或部分分泌不足引起的糖、脂质和蛋白质代谢紊乱为主要特征,常常对多个器官造成不利的影响,包括视网膜病变、肾病、神经病变和心脑血管疾病等糖尿病并发症,严重危及患者生命。根据国际糖尿病联合会(IDF)的报道,全球有3.87亿人患有糖尿病,预计到2035年将增加到5.92亿人。中国糖尿病患病率在过去几十年间也急剧增长,从1980年的0.67%飙升至2010年的11.6%,患病人数达到1.164亿,而且这一数字仍在迅速增长,可见T2DM的防控迫在眉睫。
但是中国70%的糖尿病患者并不知晓自身的疾病状态,仅有26%的患者接受治疗,而在他们当中只仅40%的人群血糖控制良好。这很大程度上归因于糖尿病的复杂病因以及患者在疾病表型、治疗应答和预后恢复等方面有较大的个体差异。因此,及时准确地判定患者的病因和发病进程将大大有助于改善糖尿病的防控形势。
目前T2DM的临床诊断普遍基于美国糖尿病协会(ADA)指南:空腹血糖水平(FPG)≥7.1mmol/L,口服75g葡萄糖耐量试验(OGTT)2小时后血糖水平≥11.1mmol/L,糖化血红蛋白水平≥6.5%。上述诊断指标,联合生活方式(吸烟、饮酒、体育活动、饮食结构),物理因素(年龄、性别、血压、BMI、腰围)和遗传因素(种族、家族遗传)等非侵入性自我评估筛查工具,可显著提高糖调节障碍或T2DM的早期诊断率。临床研究显示综合使用一些生化指标如脂质代谢产物(高密度脂蛋白胆固醇、甘油三酯),C反应蛋白、肝酶,白细胞计数等可提高T2DM的预测准确性。但是,这些代谢标志物还不能特异性地将T2DM和其他代谢疾病区分开来,也无法有效判定T2DM的具体分型。其中,胰岛β细胞通过分泌胰岛素在葡萄糖稳态中发挥核心作用。当胰岛β细胞功能受损甚至凋亡时,胰岛素分泌减少可直接导致T2DM的发生发展,这说明胰岛β细胞功能受损是T2DM的关键发病基础,及时识别β细胞功能受损并进行有效干预就有希望实现这类T2DM的逆转。因此,开发可用于判别胰岛β细胞受损型T2DM的精准防诊治生物标志物,实现以病理生理为基础的糖尿病个体化诊疗仍然是关键需求。
MicroRNA(miRNA)是一类由21-23个核苷酸组成的单链非编码RNA,其广泛调控细胞增殖分化,组织重塑及器官代谢等生物学事件,miRNA的表达变化能灵敏反映多种生理或病理状态,展现出重要的临床应用价值。血清/血浆中循环miRNA作为新型生物标志物,具有创伤小、稳定性高、特异性和灵敏性好、易于检测等优势,是对传统生物标志物的重要补充。作为完美契合精准医学要求的新型疾病标志物和指纹图谱,循环miRNA有望为T2DM的分子分型、个体化治疗和预后判断等方面提供新的判断依据,从而改善目前疾病筛查和检测的精度和准确性。例如,利用血清/血浆miRNA对胰腺癌进行早期诊断已取得突破性进展,相关诊断试剂盒已经获批上市。。然而,血清/血浆miRNA表达谱在用于糖尿病早期分型诊断方面的研究较少,基于胰岛β细胞功能受损的特定表型T2DM患者的miRNA表达谱特征有待进一步研究。
因此,成功研发胰岛β细胞功能受损特定表型的糖尿病精准预测的miRNA标志物,将有利于开创糖尿病特定表型的个体化治疗的全新局面。
发明内容
技术问题:本发明所要解决的第一个技术问题是提供一种用于特定表型2型糖尿病早期分型诊断的血清/血浆miRNA标志物。
本发明还要解决的技术问题是提供上述miRNA标志物的引物。
本发明还要解决的技术问题是提供了上述miRNA标志物及其引物在制备特定表型2型糖尿病诊断试剂盒中的应用。
技术方案:为了解决上述技术问题,本发明提供的一种与胰岛β细胞受损型2型糖尿病相关的血清/血浆miRNA标志物,所述标志物为miR-4530和miR-4455中的一种或两种的组合:
miR-4530的miRNA序列为:CCCAGCAGGACGGGAGCG
miR-4455的miRNA序列为:AGGGUGUGUGUGUUUUU。
上述的与胰岛β细胞受损型2型糖尿病相关的血清/血浆miRNA标志物的引物。这些单独的引物可以通过商业渠道购买获得,也可以在了解这些miRNA序列之后由技术人员自行设计合成,根据已知miRNA序列设计合成引物的方法为本领域技术人员熟知的方法。
各miRNA的逆转录引物、正向引物和反向引物的序列如下:
上述的血清/血浆miRNA标志物在制备胰岛β细胞受损型2型糖尿病诊断试剂盒中的应用。
上述的血清/血浆miRNA标志物的引物在制备胰岛β细胞受损型2型糖尿病诊断试剂盒中的应用。
本发明成功研发了基于胰岛β细胞功能受损病理基础的特定表型2型糖尿病的血清/血浆miRNA表达谱,为胰岛β细胞功能受损表型2型糖尿病精准预测、早期诊断及预后提供了新的判断依据,从而改善了目前疾病筛查和检测的精确性,具有巨大的临床应用价值和社会经济效益。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细说明。
图1是本发明的实验方案设计流程图;
图2为复筛阶段(A)miR-3648、(B)miR-4455和(C)miR-4530在胰岛β细胞受损型2型糖尿病(ISD-T2DM)和正常型2型糖尿病(T2DM)对照组中表达存在明显差异对比图;
图3为大样本验证阶段(A)miR-4455和(B)miR-4530在胰岛β细胞受损型2型糖尿病(ISD-T2DM)和正常型2型糖尿病(T2DM)对照组中表达存在明显差异对比图;
图4是miR-4455和miR-4530作为诊断标志物时对胰岛β细胞受损型2型糖尿病(ISD-T2DM)和正常型2型糖尿病(T2DM)对照组进行聚类分析的结果图;
图5是(A)miR-4455、(B)miR-4530以及(C)组合miRNAs用于胰岛β细胞受损型2型糖尿病(ISD-T2DM)诊断的ROC分析图。
具体实施方式
实施例1、临床样本的收集和临床资料的整理
2017年11月到2019年7月间获取南京市鼓楼医院提供的T2DM患者的血清样本,满足以下三条标准之一者入组:有糖尿病症状,并且随机血糖≥11.1mmol/L;或者空腹血糖≥7.1mmol/L;或者口服葡萄糖耐量实验(OGTT)时2小时血糖≥11.1mmol/L,同时系统收集了完整的临床检验资料。
共计收集成人胰岛β细胞受损型2型糖尿病样本80例,胰岛β细胞正常型2型糖尿病对照组样本240例;两组样本间年龄无显著差异(p>0.05)、BMI值和HOMA-IR值均在正常范围内,空腹C肽值存在显著差异(p<0.001)。
C肽由胰腺β细胞分泌产生,是胰岛素原的中间部分。一分子胰岛素原经蛋白酶切后裂解产生一分子胰岛素和一分子C肽,即导致等摩尔量的胰岛素和C肽的释放。因此,C肽水平常用作评价β细胞功能的指标。本发明采用空腹C肽值作为分型标准,将采集临床样本分成两组:
β细胞功能受损表型:(18≤BMI<26;空腹C肽<200pmol/l;HOMA-IR<2)
β细胞功能正常表型:(18≤BMI<26;空腹C肽≥200pmol/l;HOMA-IR<2)
实施例2、Agilent miRNA Array芯片测序初筛
(1)测序样本的筛选:本发明分别选取胰岛β细胞受损型和正常型2型糖尿病患者血清样本各12例,抽提总RNA后,每3个样本混合为一个样本进行Aligent miRNA芯片测序。
(2)用于Agilent miRNA芯片测序的血清RNA提取:
①采用Trizol法提取total RNA,然后用分光光度计定量,用琼脂糖凝胶电泳Agilent 2100等方法检测其完整性。
②Total RNA过柱纯化,对total RNA使用mirVanaTM miRNA Isolation Kit(AM1561)进行纯化。
(3)Agilent miRNA Array芯片测序:取200ng纯化后total RNA进行实验。使用Agilent公司的miRNA Complete Labeling and Hyb Kit,主要步骤包括:
①在一个新的无RNase的Ep管中,加入100ng total RNA,体积2μL。
②配制CIP Master Mix。在每份100ng total RNA中,加入2μL CIP Master Mix,轻轻吹吸混匀,37℃温育30min。这一去磷酸化过程,在碱性磷酸酶(CIP)的作用下去除RNA5’端磷酸基团。
表1 CIP Master Mix配制表
③在每个样品管中加入2.8μL 100%DMSO,混匀。100℃作用10min,以去除磷酸酶活性。然后转到冰浴。
④配制Ligation Master Mix,在上一步磷酸酶处理后的RNA样品中加入4.5μLLigation Master Mix,轻轻吹吸混匀,16℃孵育2h。这一标记反应过程,在T4 RNA ligase作用下将Cyanine 3-pCp连接到RNA 3’端。
表2 Ligation Master Mix配制表
⑤将标记反应产物在真空浓缩仪中浓缩抽干,设定温度45℃,浓缩时间约3h。
⑥配制杂交体系。其中标记并浓缩后的RNA产物,加水调至17μL,加入其他成分,轻轻吹吸混匀,100℃加热5min。然后转到冰浴。
表3杂交体系配制表
⑦组装好杂交装置,将45μL杂交液加样至杂交盖片上,安放miRNA芯片,旋紧杂交装置。
⑧将杂交装置放在Agilent公司杂交炉中过夜杂交(约16h,20rpm)。
⑨杂交结束后,取出芯片,先在42℃左右含0.2%SDS,2×SSC的洗液I中洗5min,而后在室温的0.2×SSC的洗液II中洗5min,玻片甩干后即可用于扫描。
⑩使用Agilent芯片扫描仪(G2565CA)对清洗后的芯片进行扫描,得到杂交图片。
(4)数据提取及分析:使用Agilent Feature Extraction(v10.7)软件对杂交图片进行分析并提取数据。然后使用Agilent GeneSpring软件对数据进行归一化和差异分析。利用CLUSTER 3.0软件对数据进行Log2转化,采用平均连锁的聚类方法分析组间差异。根据测序结果,我们选择与胰岛β细胞正常型2型糖尿病对照组(NISD-T2DM)相比,胰岛β细胞受损型2型糖尿病组(ISD-T2DM)血清miRNA拷贝数大于30且变化1.5倍以上,P值小于0.05,作为初步筛查的候选miRNAs,见表4。
表4 ISD-T2DM与NISD-T2DM患者中表达差异显著的6种候选miRNAs
实施例3、胰岛β细胞受损型T2DM血清候选miRNA的qRT-PCR验证
(1)样本的筛选:本发明通过复筛和验证两个阶段对初筛得到的候选miRNAs在患者个体中进行检测,前者在测序样本(12例ISD-T2DM和12例NISD-T2DM)中复检,后者在新的更大样本中(68例ISD-T2DM和228例NISD-T2DM)验证,均采用实时荧光定量PCR(qRT-PCR)方法。
备注说明:每个样本均单独进行如下测定。
(2)苯酚氯仿抽提法提取血清总RNA:
①取300μL血清加入2.0mL无酶管,加入300μL DEPC水稀释,涡旋混匀。
②混匀毕。加入200μL PH=4.7-5.5水饱和酚并剧烈震荡,室温静置2min,再加入200μL氯仿并剧烈震荡,室温静置5min后20℃,16000g,离心15min。
③小心吸取上清(约600μL),加入0.1倍体积的PH=5.3,3M醋酸钠溶液,再加入2倍体积-20℃预冷的异丙醇,涡旋混匀后-20℃静置2h,之后4℃,16000g,离心20min。
④弃上清,留沉淀,加入1mL 75%乙醇DEPC水溶液,轻柔颠倒数次洗涤沉淀,洗涤毕4℃,16000g,离心20min。
⑤弃上清,留沉淀,在室温下晾干约10min后,加入50μL DEPC水溶解沉淀,待完全溶解后放入-80℃低温冰箱留存待用。
⑥Nanodrop测定RNA样品浓度。
⑦把每管RNA都稀释成50ng/μL,-80℃保存,备用。
(3)检测用引物序列
1)miRNA茎环结构逆转录引物:
hsa-miR-4455
5’-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAAAAACAC-3’
hsa-miR-18b-3p
5’-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGCCAGAAG-3’
hsa-miR-671-5p
5’-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCTCCAGCC-3’
hsa-miR-3648
5’-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCCCTCGGC-3’
hsa-miR-4530
5’-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCGCTCCCG-3’
hsa-miR-1539
5’-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGGGCATCT-3’
2)PCR正向引物分别为:
hsa-miR-4455
5’-ACACTCCAGCTGGGAGGGTGTGTGT-3’
hsa-miR-18b-3p
5’-ACACTCCAGCTGGGTGCCCTAAATGCCCCT-3’
hsa-miR-671-5p
5’-ACACTCCAGCTGGGAGGAAGCCCTGGAGGGG-3’
hsa-miR-3648
5’-ACACTCCAGCTGGGAGCCGCGGGGATCGC-3’
hsa-miR-4530
5’-ACACTCCAGCTGGGCCCAGCAGGACG-3’
hsa-miR-1539
5’-ACACTCCAGCTGGGTCCTGCGCGTCCCAG-3’
3)miRNA通用PCR反向引物:
hsa-miR-4455 5’-TGGTGTCGTGGAGTCG-3’
hsa-miR-18b-3p 5’-TGGTGTCGTGGAGTCG-3’
hsa-miR-671-5p 5’-TGGTGTCGTGGAGTCG-3’
hsa-miR-3648 5’-TGGTGTCGTGGAGTCG-3’
hsa-miR-4530 5’-TGGTGTCGTGGAGTCG-3’
hsa-miR-1539 5’-TGGTGTCGTGGAGTCG-3’
(4)血清中miRNA表达量的检测方法
采用qRT-PCR(SYBR Green I染料法)方法检测血清中miRNA表达量。
①实验准备:
从-80℃冰箱中取出提取好的血清miRNA样品,冰上溶解。同时将引物取出冰上放置。
②逆转录反应合成cDNA(RT-PCR):
1)10μL逆转录反应体系(RT-PCR)
2)按照上述逆转录反应体系配方配制8μL母液,含有DEPC水、5x AMV buffer、2.5mM dNTP Mixture、miRNA特异性的RT-Primer、AMV逆转录酶,混合均匀。
3)向8μL母液加入2μL RNA,混合均匀。
4)将10μL的混合物放入PCR仪进行逆转录,反应参数设置为:
16℃,15min;42℃,1h;85℃,5min,得cDNA。
备注:无模板阴性对照cDNA制备,仅需用DEPC水代替血清RNA,其余的反应体系、反应条件与样本完全相同。本发明我们对每种miRNA设计了各自的特异性茎环结构逆转录引物,单独进行如上操作,单管合成cDNA。
③qRT-PCR:
将逆转录产物(cDNA)进行PCR扩增,反应体系如下:
1)20μL实时定量反应体系(Q-PCR)
2)将miRNA特异性的正向引物、反向引物及DEPC水按照1:1:3的比例混合,配制Q-PCR引物MIX。正向引物和反向引物混合后的浓度各为10μM。
3)按照前述逆转录反应体系配方配制59μL母液,含有DEPC水、10xPCR buffer、2.5mM dNTP Mixture、Taq酶、miRNA特异性的Q-PCR-Primer MIX、Evagreen,混合均匀。
4)向59μL母液加入3μL cDNA,混合均匀。
5)按照每孔19.5μL,每个样本设置三个复孔,加入96孔板,贴膜压实,上机SteponePlus型荧光定量PCR仪运行,荧光定量流程如下:
95℃,5min预热;PCR循环条件:95℃,15s;60℃,1min;40个循环
备注:阴性对照cDNA与样本同时进行扩增。每个样本的每种miRNA分别设置3个平行孔,以表示结果的可重复性。
(5)qRT-PCR数据处理:
1)用方程-△△Ct表示两组样本血清miRNA的相对表达量,其中△△Ct=(△CTISD-T2DM-△CTlet-7dgi)-(△CTNISD-T2DM-△CTlet-7dgi),本发明采用let-7d、let-7g、let-7i这三种组合miRNA的总表达量作为内参校正数据后,计算血清中miRNA的相对表达量。以上数据,即Ct值均可用荧光定量检测仪的检测软件读出。最后数据用Graphpad8.0进行统计分析,数据表示方法为平均值±标准差(means±SD),组间比较采用T检验,P<0.05表示有统计学差异。聚类分析采用Cluster3.0软件进行。miRNA的特异性和灵敏性采用ROC曲线下面积及选定的最佳截断点(cut off)值进行计算。
2)复筛阶段:将测序初筛得到的6条miRNAs,在测序个体样本中进行qRT-PCR复检。定义miRNA具有显著差异的标准为:①变化倍数大于2.0倍;②对照组与实验组的P值小于0.05;③Ct小于35。结果发现在12例ISD-T2DM患者与12例NISD-T2DM患者对照组中,有3条miRNAs在胰岛β细胞受损型2型糖尿病患者血清中表达明显异于对照组,且结果具有显著统计学意义。这3条miRNAs分别是miR-3648,miR-4530和miR-4455(见图2A-C)。
3)验证阶段:我们进一步在新的更大的样本中检测了上述差异miRNA的表达情况,证实其作为胰岛β细胞受损型2型糖尿病早期分型诊断分子标记物的可能性。结果在68例ISD-T2DM患者与228例NISD-T2DM患者对照组中,发现miR-4455和miR-4530这2条miRNA的表达变化差异显著,与测序初筛和qRT-PCR复检结果一致(见图3A-B)。
4)我们进一步借助CLUSTER3.0进行聚类分析,结果见图4。从图中可以看出,用miR-4530和miR-4455作为诊断标志物,可以将ISD-T2DM与NISD-T2DM对照组明显区分开。
5)为了评估miR-4530和miR-4455在胰岛β细胞受损型2型糖尿病(ISD-T2DM)检测中的诊断能力,通过绘制ROC曲线并计算曲线下面积AUC,来评价每条miRNA的在诊断胰岛β细胞受损型2型糖尿病的敏感性和特异性。结果如图5A-C。
用miR-4530区分胰岛β细胞受损型T2DM患者时,AUC为0.774,最佳临界点的灵敏度为87%,特异度为80%。
用miR-4455区分胰岛β细胞受损型T2DM患者时,AUC为0.999,最佳临界点的灵敏度为99%,特异度为100%。
用miR-4530和miR-4455组合以区分胰岛β细胞受损型T2DM患者时,AUC为0.970,最佳临界点的灵敏度为90%,特异度为100%。综合上述分析结果,说明这2条miRNAs对胰岛β受损型2型糖尿病早期分型诊断具有较好的诊断价值,可以作为特定表型2型糖尿病临床早期诊断的生物标志物。
以上结合附图对本发明的实施方式作出详细说明,但本发明不局限于所描述的实施方式。对本领域的普通技术人员而言,在本发明的原理和技术思想的范围内,对这些实施方式进行多种变化、修改、替换和变形仍落入本发明的保护范围内。
序列表
<110> 南京大学
<120> 与2型糖尿病胰岛β细胞受损表型相关的血清/血浆miRNA组合标志物及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 1
cccagcagga cgggagcg 18
<210> 2
<211> 17
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 2
agggugugug uguuuuu 17
Claims (2)
1.与胰岛β细胞受损型2型糖尿病相关的血清/血浆miRNA标志物的检测引物组合物在制备胰岛β细胞受损型2型糖尿病诊断试剂盒中的应用,其中该引物组合物包括miR-4530和/或miR-4455的检测引物;
各miRNA的逆转录引物、正向引物和反向引物的序列如下:
2.根据权利要求1所述的应用,其特征在于所述的荧光定量检测引物还包括miR-4530和/或miR-4455的检测探针。
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