CN106119385A - 一种诊断2型糖尿病的miRNA分子标志物hsa‑miR‑149‑3p及其应用 - Google Patents
一种诊断2型糖尿病的miRNA分子标志物hsa‑miR‑149‑3p及其应用 Download PDFInfo
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Abstract
本发明属于生物医药领域，涉及一种诊断2型糖尿病的miRNA分子标志物及其应用。步骤如下：血液总RNA的抽提及cDNA的制备；荧光定量PCR检测hsa‑miR‑149‑3p水平；miRNA分子标志物的评估。本发明通过大样本检测，发现新的外周血miRNA标志物hsa‑miR‑149‑3p在2型糖尿病患者中明显升高，且其分子水平与受检者血糖水平呈显著正相关。该检测方法操作简便，对受检者的创伤性小，且灵敏度高，特异性强，可用于早期和不同阶段2型糖尿病的诊断。在此基础上，本发明开发了检测血液hsa‑miR‑149‑3p的引物、试剂盒和检测方法，具有明显的临床应用价值。
Description
技术领域
本发明属于生物医药领域，涉及一种诊断2型糖尿病的miRNA分子标志物及其应用。
背景技术
糖尿病是一种以高血糖为主要特征的慢性代谢性疾病，持续性的高血糖引起的并发症会严重损害患者的生命健康。国际糖尿病联盟最新数据显示，2015年全球糖尿病成人患病人数已将近4亿人，而我国患病比例占据1/4左右，给社会和家庭带来日益沉重的负担。糖尿病按照发病机制主要分为1型和2型糖尿病。2型糖尿病也称为成人发病型糖尿病，占糖尿病比例的94％左右。2型糖尿病尚不能完全治愈，且在早期无明显的临床症状，病人常在体检或出现典型的症状才被确诊。如果在糖尿病前期能够及时发现并通过改变生活方式或药物干预即可防止并发症的出现。目前2型糖尿病的临床诊断指标主要包括：空腹血糖(FBG)、葡萄糖耐量试验(OGTT)、糖化血红蛋白(GHb)等，但这些指标多用于罹患糖尿病的诊断及分型，无法进行糖尿病的早期诊断。
microRNA(miRNA)是一类高度保守的非编码小RNA，长度约为18-25个核苷酸。miRNA广泛存在于原核和真核生物体内，对细胞的增殖、分化、凋亡，胚胎的发育，器官的形成，内分泌的调控，疾病的发生、发展都起着重要的调节作用。miRNA通过靶向一个或多个基因的3’非编码区域，降解其mRNA或抑制其翻译的进行，从而达到对基因表达的调控作用。miRNA与肿瘤、心脑血管疾病、内分泌疾病等诸多疾病的发生密切相关。目前已有大量的研究表明miRNA从胰岛发育、胰岛细胞的凋亡、胰岛素的生成、分泌以及胰岛素作用等各个阶段参与糖尿病的发病，但研究手段多局限于动物模型或患病组织，且需要通过创伤性的方式获取。
血液中存在着上千种miRNA，这些小分子RNA不仅含量丰富、性质稳定，便于检测，且与特异性疾病存在着显著的关联性。血液中的miRNA由组织分泌而得，分析血液中的miRNA可以间接反映组织中的miRNA水平。因此通过无创性的手段，快速、灵敏、特异性的定量检测方法，筛选2型糖尿病人血液中特异性表达或异常表达的循环miRNA作为分子标志物，研制糖尿病早期的辅助诊断试剂盒，用于监测2型糖尿病易感人群、评估罹患风险并采取有效的干预措施，对于遏制当前爆发性的糖尿病发病趋势具有显著的社会价值。
发明内容
本发明公开了一种用于诊断2型糖尿病的miRNA分子标志物hsa-miR-149-3p，提供了在制备2型糖尿病辅助诊断试剂盒中的应用。
为解决上述问题，本发明采用以下技术方法：
一种诊断2型糖尿病的miRNA分子标志物hsa-miR-149-3p的应用，步骤如下：
(1)血液总RNA的抽提及cDNA的制备；
(2)荧光定量PCR检测hsa-miR-149-3p水平；
(3)miRNA分子标志物的评估。
所述miRNA分子标志物hsa-miR-149-3p，其miRNA序列如SEQ ID NO：1所示，DNA序列如SEQ ID NO：2所示。
所述步骤(1)中cDNA的制备所用的反转录引物序列如SEQ ID NO：3所示。
所述步骤(2)实时荧光定量PCR检测根据染料法检测、探针法检测。
所述步骤(2)中荧光定量PCR的正向引物如SEQ ID NO：4所示、反向引物如SEQ IDNO：5所示。
诊断2型糖尿病的miRNA分子标志物hsa-miR-149-3p的正向引物和反向引物，在制备2型糖尿病辅助诊断试剂盒中的应用。
本发明的有益效果在于：
第一：本发明通过引入外源性miRNA作为参照基因，通过miRNA荧光定量PCR检测外周血中hsa-miR-149-3p水平；相对于传统的使用内源性血清miRNA作为参照基因进行检测的方法，避免了个体间内参miRNA差异造成的数据偏差，显著提高了检测结果的稳定性。
第二：本发明通过大样本检测，发现新的外周血miRNA标志物hsa-miR-149-3p在2型糖尿病患者中明显升高，且其分子水平与受检者血糖水平呈显著正相关；该检测方法操作简便，对受检者的创伤性小，且灵敏度高，特异性强，可用于早期和不同阶段2型糖尿病的诊断。在此基础上，本发明开发了检测血液hsa-miR-149-3p的引物、试剂盒和检测方法，具有明显的临床应用价值。
附图说明
图1为2型糖尿病病例组和健康对照组中hsa-miRNA-149-3p的表达水平示意图。
图2为检测样本中hsa-miRNA-149-3p水平与空腹血糖水平的相关性分析示意图。
具体实施方式
以下通过实施例进一步描述本发明，其中包括使用材料及具体来源。但应当理解的是，这些只是示例性的，而非限制本发明。与如下试剂、仪器的类型及型号，或性质或功能相似或相同的材料均可以用于本发明的实施。除非另有所指，本发明中用到的试剂可以是任何合适的市售试剂。
下述示例中的方法如无特殊说明均为普通方法。
主要材料：
1.临床样品的采集
发明人于2015年开始至今从河南大学附属淮河医院收集了大量的2型糖尿病人和正常体检人群的外周血样品。整个收集及后续实验过程符合医学伦理道德要求并严格遵循病例资料的保密原则。研究样品的采样、分装、保存条件一致。通过对病历资料的整理，发明人选择了25例符合下列标准的样品作为实时荧光定量PCR检测的实验样品。
空腹血液血糖在3.9-6.1mmol/L之间的健康人群定义为健康对照组；
空腹血液血糖大于或等于7.0mmol/L，连续两次重复测定结果相近，由内分泌医生确诊为2型糖尿病，且未经任何药物治疗的人群定义为病例组。
2.血液中总RNA的抽提
每200μL新鲜的血液样品，加入1μL，1μM的miRNA检测外参(Tiangen)，混匀后再加入600μL Tri reagent，漩涡震荡充分裂解血细胞，室温下静置5分钟后，加入1/10倍Trireagen体积的的BCP溶液，涡旋混匀15秒后室温静置10分钟。4℃，13400g室温离心15分钟；将上清液转移至新的1.5mL离心管，加入等倍上清体积的异丙醇，轻轻颠倒混匀数次后，-80℃条件下静置1小时，4℃，13400g离心1小时后，吸除上清，加入500μL无RNA酶水新鲜配制的75％乙醇溶液，轻吹悬浮清洗RNA，4℃，13400g离心5分钟沉淀RNA。吸除上清后，置于室温通风处晾干，干燥5分钟左右。加入适量无核酸酶水并置于55℃水浴10分钟，待充分溶解后测定OD260和OD280吸收值。一般认为A260/A280在1.8-2.1之间可以初步判定总RNA质量较好。
3.荧光定量PCR检测hsa-miR-149-3p水平
取2μg总RNA作为模板，使用针对miRNA的cDNA第一链合成试剂盒(BioTeke)对miRNA进行加Poly(A)尾反应，反应结束后配制反转录体系：
37℃反应60分钟，反转录成cDNA。将cDNA稀释至4ng/μL作为荧光定量PCR反应的模板。使用针对hsa-miR-149-3p、外参基因的正向引物，通用反向引物及2×SYBR Green qPCRMixture，在ABI 7500荧光定量PCR仪上进行扩增。
miRNA的反转录引物如SEQ ID NO：3所示、正向引物如SEQ ID NO：4所示和通用反向引物如SEQ ID NO：5所示。
本发明提供的检测血液miRNA的引物组合基于poly(A)聚合酶加尾法设计而得。在一些具体的实施方式中，所述的实时荧光定量PCR检测hsa-miRNA-149-3p的引物也可以根据茎环方法设计而得，并不局限于其设计原理。反应体系如下：
外参基因体系为：
hsa-miR-149-3p体系为：
PCR条件为：50℃20秒；95℃10分钟；95℃1分钟；60℃1分钟，重复40个循环；测得样品miR-149-3p扩增的CT值，以外参基因扩增的CT值进行标准化校正。
4.数据处理与分析
两组血液样品miRNA表达量的比值使用2-ΔΔCT法进行计算，其中ΔΔCT＝[CT1(miRNA)-CT1(外参)]-[CT2(miRNA)-CT2(外参)]，CT miRNA是样品miR-149-3p 扩增的CT值，CT外参是样品外参基因扩增的CT值，CT1是病例组或健康对照组样品扩增的CT值，CT2是一例健康对照组样品扩增的CT值。具体结果如下表所示：
表1.荧光定量PCR检测健对照组和病例组血液中hsa-miR-149-3p的表达水平
| 样品编号 | 2型糖尿病 | hsa-miR-149-3p相对表达水平 |
| 1 | 0 | 1.324 |
| 2 | 0 | 0.814 |
| 3 | 0 | 1.389 |
| 4 | 0 | 1.104 |
| 5 | 0 | 0.595 |
| 6 | 0 | 0.463 |
| 7 | 0 | 1.520 |
| 8 | 0 | 0.818 |
| 9 | 0 | 0.973 |
| 10 | 1 | 2.469 |
| 11 | 1 | 2.265 |
| 12 | 1 | 1.027 |
| 13 | 1 | 1.737 |
| 14 | 1 | 1.812 |
| 15 | 1 | 1.533 |
| 16 | 1 | 1.557 |
| 17 | 1 | 1.416 |
| 18 | 1 | 2.574 |
| 19 | 1 | 1.178 |
| 20 | 1 | 1.224 |
| 21 | 1 | 2.915 |
| 22 | 1 | 1.480 |
| 23 | 1 | 1.750 |
| 24 | 1 | 1.292 |
表1中，“0”表示健康人群，“1”表示2型糖尿病患者。
经SPSS软件进行统计分析，hsa-miR-149-3p在2型糖尿病病例组和健康对照组中的表达水平具有显著差异(P＝0.002)，且hsa-miR-149-3p水平与空腹血糖水平呈明显正相关(R＝0.62，P＝0.001)，结果如图1所示。P<0.05认为具有统计学意义。
由此可以确定，hsa-miR-149-3p在2型糖尿病患者的血液中明显升高，可以作为2型糖尿 病的检测分子标志物。
Claims (6)
1.一种诊断2型糖尿病的miRNA分子标志物hsa-miR-149-3p的应用，其特征在于：步骤如下：
（1）血液总RNA的抽提及cDNA的制备；
（2）荧光定量PCR检测hsa-miR-149-3p水平；
（3）miRNA分子标志物的评估。
2.如权利要求1所述的诊断2型糖尿病的miRNA分子标志物hsa-miR-149-3p，其特征在于：所述miRNA分子标志物hsa-miR-149-3p，其miRNA序列如SEQ ID NO：1所示，DNA序列如SEQ ID NO：2所示。
3.如权利要求1所述的诊断2型糖尿病的miRNA分子标志物hsa-miR-149-3p的应用，其特征在于：所述步骤（1）中cDNA的制备所用的反转录引物序列如SEQ ID NO：3所示。
4.如权利要求1所述的诊断2型糖尿病的miRNA分子标志物hsa-miR-149-3p的应用，其特征在于：所述步骤（2）实时荧光定量PCR检测根据染料法检测、探针法检测。
5.如权利要求1所述的诊断2型糖尿病的miRNA分子标志物hsa-miR-149-3p的应用，其特征在于：所述步骤（2）中荧光定量PCR的正向引物如SEQ ID NO：4所示、反向引物如SEQ IDNO：5所示。
6.如权利要求5所述的诊断2型糖尿病的miRNA分子标志物hsa-miR-149-3p的正向引物和反向引物，在制备2型糖尿病辅助诊断试剂盒中的应用。
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| PCT/CN2017/081994 WO2018028249A1 (zh) | 2016-08-11 | 2017-04-26 | 一种miRNA及其在治疗代谢性疾病中的应用 |
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| WO2018028249A1 (zh) * | 2016-08-11 | 2018-02-15 | 河南大学 | 一种miRNA及其在治疗代谢性疾病中的应用 |
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| CN114634977A (zh) * | 2020-12-15 | 2022-06-17 | 南京大学 | 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 |
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| CN107375317A (zh) * | 2017-06-28 | 2017-11-24 | 河南大学 | miR‑149‑3p及其拟似物在制备防治肥胖、动脉粥样硬化、高血脂的药物中的应用 |
| CN107375317B (zh) * | 2017-06-28 | 2020-09-04 | 河南大学 | miR-149-3p及其拟似物在制备防治肥胖、动脉粥样硬化、高血脂的药物中的应用 |
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| CN114634977B (zh) * | 2020-12-15 | 2024-04-09 | 南京大学 | 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 |
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