CN111073973A - 一种用于早期诊断2型糖尿病的microRNA序列及其应用 - Google Patents
一种用于早期诊断2型糖尿病的microRNA序列及其应用 Download PDFInfo
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Abstract
本发明公开了一种用于早期诊断2型糖尿病的microRNA序列及其应用，属于医药技术领域，所述序列为microRNA‑X，该microRNA的序列如SEQ：ID：NO：1所示，设计引物的序列如SEQ：ID：NO：2‑SEQ：ID：NO：3所示。本发明收集562名维吾尔族个体血清,运用Illumina Infinium Global Screening Array‑24 v1.0(GSA)Bead Chip技术进行分析，发现一段与空腹血糖显著正相关microRNA序列。
Description
技术领域
本发明属于医药技术领域，涉及一种用于早期诊断2型糖尿病的microRNA序列及其应用。
背景技术
2型糖尿病(Type 2 diabetes,T2DM)的发病率近年来在全球逐年增长，成为世界第5位死亡主因。据世界卫生组织(WHO)预测，到2025年全世界糖尿病患者将达到3亿人，其中2.3亿人在发展中国家，而中国糖尿病新增人数将占世界糖尿病新增人数的首位。Ning G等的调查结果显示，目前中国成年人T2DM患病率达11.6％，并且在成年人群中有50.1％处于前糖尿病状态。流行病学资料显示，80％的T2DM患者伴有肥胖，肥胖成为IR和T2DM的一个重要的危险因素。最近的研究表明，中国成年人群中，男性肥胖率由1980年的0.33％增加至2015年的5.02％，女性肥胖率由0.9％增加至5.51％。近年文献表明肥胖会引起T2DM，但具体机制尚不十分明确。
microRNA是一类由内源基因编码的长度约为22个核苷酸的非编码单链RNA分子，它们在动植物中参与转录后基因表达调控。最近的文献表明，体内大部分循环外泌体microRNAs来源于脂肪组织并通过血液循环到达靶组织器官，影响其糖、脂代谢关键基因的表达，导致脂肪肝、动脉粥样硬化、癌症以及糖尿病等疾病的发生。近年来，microRNAs在糖脂代谢领域中的作用越来越受到重视，大量报道表明，microRNAs是调控胰岛素敏感性的重要因子，在肥胖机体中表达异常，诱发胰岛素抵抗(Insulin Resistance,IR)。对于治疗肥胖及其相关慢性疾病，microRNAs具有非常大的潜力，可作为诊断和治疗靶点。
发明内容
本发明的目的在于提供一种用于早期诊断2型糖尿病的microRNA序列及其应用。
其具体技术方案为：
一种用于早期诊断2型糖尿病的microRNA序列，所述序列为microRNA-X，该microRNA的序列如SEQ：ID：NO：1所示，设计引物的序列如SEQ：ID：NO：2-SEQ：ID：NO：3所示。
本发明所述用于早期诊断2型糖尿病的microRNA序列在治疗2型糖尿病药物制备过程中的应用。
与现有技术相比，本发明的有益效果：
本发明收集562名维吾尔族个体血清,运用Illumina Infinium GlobalScreening Array-24 v1.0(GSA)Bead Chip技术进行分析，发现一段与空腹血糖显著正相关microRNA序列。随后我们在汉、哈、维三个不同民族的正常、肥胖和糖尿病血清样本中进行了验证，发现该microRNA在肥胖和糖尿病血清样本中显著高表达，并且该microRNA的表达与血糖、血脂相关指标显著正相关；体外培养人肝癌细胞系HepG2细胞我们发现转染了该microRNA模拟物的一组甘油三酯合成增加，葡萄糖利用率和胰岛素敏感性降低，提示该microRNA可能影响了肝细胞内的糖、脂代谢。该microRNA的表达在正常、肥胖和糖尿病三组中呈现逐级递增的趋势，与现有技术相比，该microRNA对早期糖尿病更敏感，对于早期糖尿病的诊断更准确更具有优势。
附图说明
图1 miR-X在正常、肥胖和糖尿病个体血清内的表达水平显著增加A.维吾尔族正常血清样本24例，肥胖血清样本46例，糖尿病血清样本23例,miR-X的表达水平；B.哈萨克族正常血清样本21例，肥胖血清样本48例，糖尿病血清样本10例，miR-X的表达水平；C.汉族正常血清样本21例，肥胖血清样本22例，糖尿病血清样本20例，miR-X的表达水平。秩和检验，*P<0.05，**P<0.01，差异具有统计学意义；
图2A.0.1mmol/L FFA刺激及50nM miR-X-Mimic转染HepG2细胞24h后，400倍镜下油红O染色结果；图2B.570nm处油红O吸光度，t检验，***P<0.001,差异具有统计学意义；B.0.1mmol/L FFA刺激及50nM miR-X-Mimic转染HepG2细胞0h和24h后，细胞内甘油三酯含量，t检验，*P<0.01,与NC组相比，差异具有统计学意义；
图3 A.miR-NC、miR-X-mimic转染24h后HepG2葡萄糖利用率，与NC组相比，秩和检验，**P<0.01,差异具有统计学意义；
图3 B.miR-NC、miR-X-mimic转染22h后加100μg/ml胰岛素刺激HepG2，测定HepG2细胞胰岛素敏感性；
图4本发明的技术路线图。
具体实施方式
下面结合附图和实施例对本发明的技术方案作进一步详细地说明。
实施例1
1.课题组收集562名维吾尔族个体血清,运用Illumina Infinium GlobalScreening Array-24 v1.0(GSA)Bead Chip技术进行分析，发现一段与空腹血糖显著正相关microRNA序列，经过数据库比对确定该序列为microRNA-X，该microRNA的序列为AAAGGUAAUUGUGGUUUCUGC。课题组设计引物如下：
F:5’-AAAGGUAAUUGUGGUUUCUGC-3’
R:5’-AGAAACCACAAUUACCUUUUU-3’
2.人体样本收集：
(1)样本分组及来源：2016年12月-2018年12月，分别在新疆维吾尔自治区喀什地区、伊犁地区以及石河子地区，收集维吾尔族正常个体血清样本24例，肥胖个体血清样本48例，糖尿病个体血清样本24例；哈萨克族正常个体血清样本24例，肥胖个体血清样本48例，糖尿病个体血清样本10例；汉族正常个体血清样本21例，肥胖个体22例，糖尿病个体20例。
(2)样本的纳入和排除标准：正常组：40岁<年龄<50岁；体质指数(BMI)<24kg/m2；空腹血糖<6.1mmol/l；甘油三酯(TG)<1.7mmol/l；总胆固醇(TC)<5.17mmol/l；性别：1：1配对；肥胖组：40岁<年龄<50岁；BMI≥28kg/m2；空腹血糖<6.1mmol/l；性别：1：1配对；T2DM组：38岁<年龄<50岁；空腹血糖≥7.0mmol/l；性别：1：1配对。
(3)血液样本的收集和保存：样本自收集后，室温4000rpm离心5min，吸取上层血清至1.5ml RNase-Free EP管中，置于-80℃冰箱保存。
(4)血液标本中microRNA的提取方法：
1)样品处理：每200μL血清或血浆中加入900μL裂解液MZA,振荡器振荡混匀30sec至完全匀浆，颠倒混匀；
2)室温放置5min，使得核酸蛋白复合物完全分离；
3)加入200μL氯仿，盖好管盖，剧烈振荡15sec，室温放置5min；
4)12000rpm，4℃，离心15min，样品会分为3层：黄色的有机相，白色的中间层和无色的水相，RNA主要在水相中，把水相转移到新管中，进行下一步操作；
5)量取转移液的体积，缓慢加入转移液2倍体积的无水乙醇，混匀，将得到的溶液和沉淀一起转入吸附柱miRelute，室温放置2min，室温12000rpm离心30sec，离心后弃掉流出液，保留吸附柱miRelute；
6)向吸附柱miRelute中加入700μL去蛋白液MRD，室温静置2min，室温12000rpm离心30sec，弃废液；
7)向吸附柱miRelute中加入500μL漂洗液RW，室温静置2min，室温12000rpm离心30sec，弃废液；
8)重复7步骤一次；
9)室温12000rpm离心2min，弃收集管；
10)将吸附柱miRelute转入一个新的RNase-Free 1.5ml离心管中，向吸附膜中心位置加15-30μL RNase-Free ddH2O，室温放置2min，室温12000离心2min。
(5)体外扩增的反应体系和条件：
1)qRT-PCR反应体系如表1所示：
表1 qRT-PCR反应体系
2)实时定量PCR扩增程序如表2所示：
表2实时定量PCR扩增程序
3.在体外培养人肝癌细胞系HepG2的基础上，运用microRNA-X体外mimic模拟物序列(AAAGGUAAUUGUGGUUUCUGC),上调microRNA-X后观察对HepG2细胞糖、脂代谢能力的影响：
(1)人肝癌细胞系HepG2体外培养：细胞由中国科学院上海细胞库购入，DMED培养基，10％FBS(胎牛血清)，青霉素(100单位/mL)和链霉素(100ug/mL)，常规培养于37℃含5％CO2培养箱内。
(2)mimic模拟物序列的合成：模拟物合成序列购自吉玛基因，
F:5’-AAAGGUAAUUGUGGUUUCUGC-3’
R:5’-AGAAACCACAAUUACCUUUUU-3’
(3)细胞糖代谢能力的检测方法：
细胞糖消耗实验：
1)正常培养基培养12h后更换无糖培养基剥夺葡萄糖12h；
2)利用无糖DMEM和lipo 2000将模拟物转染至细胞内；
3)4h后更换高糖DMEM含血清培养基，检测0h和24h培养基中葡萄糖含量。
细胞胰岛素敏感性实验：
1)正常培养基培养12h后更换无血清培养基饥饿处理12h；
2)利用正常DMEM和lipo 2000将模拟物转染至细胞内；
3)4h后更换正常DMEM含血清培养基22h后，加入含高浓度胰岛素和血清的高糖培养基，检测0min，30min，120min培养基内葡萄糖含量。
(4)细胞脂代谢能力的检测方法：
细胞内甘油三酯检测：
1)正常培养基培养12h后更换无血清培养基饥饿处理12h；
2)利用正常DMEM和lipo 2000将模拟物转染至细胞内；
3)4h后更换含0.1mmol/L的游离脂肪酸和血清的正常培养基，检测0h，24h细胞内的甘油三酯含量。
油红O染色鉴定脂滴生成：
1)正常培养基培养12h后更换无血清培养基饥饿处理12h；
2)利用正常DMEM和lipo 2000将模拟物转染至细胞内；
3)4h后更换含0.1mmol/L的游离脂肪酸和血清的正常培养基，培养24h；
4)24h后加入10％甲醛磷酸盐缓冲液至覆盖所有细胞，室温固定一小时。
5)吸弃甲醛磷酸盐缓冲液，再用1×PBS溶液冲洗2-3次。
6)加入油红O工作液，室温或37度环境染色30min左右。
7)吸弃油红O工作液，六孔板每孔使用1-2mL的1×PBS冲洗2-3次，肉眼观察无沉淀及杂质，显微镜下观察脂滴被红染的效果。
4.统计学方法：检测结果数据用均值±标准差(x±s)表示，应用SPSS 25.0软件进行数据统计分析。数据分析采用t检验和秩和检验，多组间比较采用单因素方差分析，数据相关性分析采用Pearson和Spearman相关性分析，P<0.05认为有统计学意义。
实施例2miR-X与肥胖和2型糖尿病密切相关
1.miR-X在肥胖和2型糖尿病个体体内显著高表达
在24例正常，48例肥胖和24例T2DM维吾尔族个体及24例正常，48例肥胖及10例T2DM哈萨克族个体血清中对microRNA-X(AAAGGUAAUUGUGGUUUCUGC)的拷贝数进行验证，结果显示与正常组相比，该序列在肥胖组和T2DM组中的拷贝数显著升高(P<0.05)，提示microRNA-X(AAAGGUAAUUGUGGUUUCUGC)可能与2型糖尿病的发生发展密切相关。
2.miR-X的表达与糖脂代谢显著正相关
SPSS 25.0分析miR-X与糖脂代谢相关指标的相关性，Spearman相关性分析结果显示miR-X与BMI、空腹血糖、TG、TC、LDL和HDL均显著相关。
表3 miR-X与糖脂代谢相关指标的相关性分析
Spearman相关性分析,*P<0.05，**P<0.01，***P<0.001,相关性具有统计学意义
3.miR-X促进了肝脏细胞的脂肪合成能力
将50nm的miR-X-mimic转染至肝脏细胞(HepG2)中24小时同时加入0.1mM的游离脂肪酸(FFA)刺激后，油红O染色鉴定细胞内的脂滴，酶法检测细胞内的甘油三酯(TG)含量，结果提示：转染的miR-X的细胞内脂滴合成增多，甘油三酯含量显著增加。
4.miR-X降低了细胞葡萄糖消耗能力和胰岛素敏感性
将50nm的miR-X-mimic转染至肝脏细胞(HepG2)中24小时检测0h、24h细胞内葡萄糖含量，将50nm的miR-X-mimic转染至肝脏细胞(HepG2)中22小时后同时加入大浓度胰岛素刺激，检测0h、30min和2h的葡萄糖含量，结果提示：转染了miR-X的细胞葡萄糖消耗能力显著降低，胰岛素敏感性显著下降。
以上所述，仅为本发明较佳的具体实施方式，本发明的保护范围不限于此，任何熟悉本技术领域的技术人员在本发明披露的技术范围内，可显而易见地得到的技术方案的简单变化或等效替换均落入本发明的保护范围内。
序列表
<110> 石河子大学
<120> 一种用于早期诊断2型糖尿病的microRNA序列及其应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 1
aaagguaauu gugguuucug c 21
<210> 2
<211> 21
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 2
aaagguaauu gugguuucug c 21
<210> 3
<211> 21
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 3
agaaaccaca auuaccuuuu u 21
Claims (2)
1.一种用于早期诊断2型糖尿病的microRNA序列，其特征在于，所述序列为microRNA-X，该microRNA的序列如SEQ：ID：NO：1所示，设计引物的序列如SEQ：ID：NO：2-SEQ：ID：NO：3所示。
2.一种权利要求1所述的用于早期诊断2型糖尿病的microRNA序列在治疗2型糖尿病药物制备过程中的应用。
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130109628A1 (en) * | 2010-07-15 | 2013-05-02 | Emory University | Microrna compositions and methods related thereto |
| CN103555724A (zh) * | 2013-10-24 | 2014-02-05 | 浙江理工大学 | 2型糖尿病的血清miRNA生物标志物及其应用 |
| CN104955950A (zh) * | 2012-09-26 | 2015-09-30 | 米尔克斯治疗学公司 | 具有改善的脱靶特征谱的寡核苷酸 |
| CN106148537A (zh) * | 2016-08-08 | 2016-11-23 | 南京市妇幼保健院 | 一种与2型糖尿病相关的血清或血浆miRNA标志物及其应用 |
| WO2017136662A1 (en) * | 2016-02-05 | 2017-08-10 | St. Johns University | Circulating serum microrna biomarkers and methods for determining parkinson's disease |
| EP3642366A1 (en) * | 2017-06-19 | 2020-04-29 | St. Johns University | Circulating serum microrna biomarkers and methods for alzheimer's disease diagnosis |
-
2019
- 2019-12-10 CN CN201911256089.7A patent/CN111073973A/zh active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130109628A1 (en) * | 2010-07-15 | 2013-05-02 | Emory University | Microrna compositions and methods related thereto |
| CN104955950A (zh) * | 2012-09-26 | 2015-09-30 | 米尔克斯治疗学公司 | 具有改善的脱靶特征谱的寡核苷酸 |
| CN103555724A (zh) * | 2013-10-24 | 2014-02-05 | 浙江理工大学 | 2型糖尿病的血清miRNA生物标志物及其应用 |
| WO2017136662A1 (en) * | 2016-02-05 | 2017-08-10 | St. Johns University | Circulating serum microrna biomarkers and methods for determining parkinson's disease |
| CN106148537A (zh) * | 2016-08-08 | 2016-11-23 | 南京市妇幼保健院 | 一种与2型糖尿病相关的血清或血浆miRNA标志物及其应用 |
| EP3642366A1 (en) * | 2017-06-19 | 2020-04-29 | St. Johns University | Circulating serum microrna biomarkers and methods for alzheimer's disease diagnosis |
| US20200190588A1 (en) * | 2017-06-19 | 2020-06-18 | St. John's University | Circulating serum microrna biomarkers and methods for alzheimer's disease diagnosis |
Non-Patent Citations (1)
| Title |
|---|
| 徐民桢等, 上海科学技术出版社 * |
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