血瘀质非创伤性股骨头坏死miRNA差异表达筛选及生物信息学分析
2020-08-28周明旺李盛华邓昶付志斌吉星陈彦同胡星荣刘一飞王鹏志赵秋玥
周明旺,李盛华,邓昶,付志斌,吉星,陈彦同,胡星荣,刘一飞,王鹏志,赵秋玥
血瘀质非创伤性股骨头坏死miRNA差异表达筛选及生物信息学分析
周明旺1,李盛华1,邓昶2,付志斌1,吉星1,陈彦同2,胡星荣2,刘一飞2,王鹏志2,赵秋玥2
1.甘肃省中医院,甘肃 兰州 730050;2.甘肃中医药大学中医临床学院,甘肃 兰州 730000
筛选血瘀质、非血瘀质非创伤性股骨头坏死(NONFH)患者与健康人外周血中差异表达的miRNA,建立miRNA表达谱,分析其生物学信息,为血瘀质NONFH的防治提供依据。选取血瘀质、非血瘀质NONFH患者各10例(NONFH组)和健康志愿者8例(正常组),采用RT-PCR检测其血浆miRNA相对表达量,筛选差异表达的miRNA。应用实时荧光定量PCR对差异表达的hsa-miR-365a-3p、hsa-miR-483-3p进行验证。采用TargetScan、microRNAorg、PITA在线数据库对差异miRNA进行靶基因预测,并进行靶基因生物信息学分析。与正常组比较,NONFH组有差异表达的miRNA 50个,其中上调21个,下调29个。与非血瘀质NONFH组比较,血瘀质NONFH组有差异表达的miRNA 33个,其中上调10个,下调23个。经筛选,最终确定血瘀质NONFH差异表达miRNA 5个。实时荧光定量PCR验证结果与miRNA芯片结果相符。3个在线数据库共筛选出交集靶基因11个,仅表达下调miRNA中hsa-miR-365a-3p检出交集靶基因11个,余未检测出交集靶基因。GO分析显示,差异表达miRNA主要与受体结合、蛋白及酶的活性有关。Pathway分析显示,血瘀质NONFH差异表达miRNA靶基因主要富集于轴突导向、Rap1、赖氨酸降解、Fc epsilon RI、甘油磷脂代谢、T细胞受体、血小板活化、成骨细胞分化等多个信号通路。血瘀质NONFH差异表达miRNA可能通过调控Rap1、赖氨酸降解、甘油磷脂代谢、血小板活化、成骨细胞分化、Ras、PI3K-Akt等信号通路参与NONFH发生发展过程。
血瘀质;非创伤性股骨头坏死;miRNA;基因芯片;生物信息学
非创伤性股骨头坏死(non-traumatic osteonecrosis of femoral head,NONFH)主要由血液供应的破坏和凝血纤溶系统紊乱引起,最终导致股骨头塌陷,为骨科难治性疾病[1]。由于发病较隐匿,缺乏早期临床症状,对其早期检测尚存困难,因此阐明其病因病机成为当今研究主要方向。中医药重视辨析体质状态,对NONFH的治疗有积极作用。本课题组前期研究显示,血瘀质是NONFH的高发体质类型之一[2]。miRNA是一类非编码RNA,近年来,越来越多数据表明,其参与破骨细胞的形成、分化、凋亡和吸收[3-4],在NONFH的发病中扮演了极为重要的角色[5]。基于此,本课题组通过miRNA芯片技术筛选出血瘀质NONFH患者血浆中异常表达的miRNA,并对其进行生物信息学分析,探讨血瘀质及相关miRNA异常表达与NONFH发病的相关性,以期为中医药调体干预NONFH提供新的思路和依据。
1 资料与方法
1.1 诊断标准
参照《股骨头坏死临床诊疗规范》[6]、《中药新药临床研究指导原则》[7]、《成人股骨头坏死诊疗标准专家共识(2012年版)》[8]制定诊断标准。
1.2 中医体质判定标准
依据《中医体质分类与判定》[9]制定血瘀体质NONFH相关miRNA组学分析及其靶基因调控网络研究调查表。按照转化分对中医体质类型进行判断,分为平和质、气虚质、阳虚质、阴虚质、痰湿质、湿热质、血瘀质、气郁质、特禀质共9种类型。平和质:转化分≥60分,其他8种体质转化分均小于30分,判断为“是”;转化分≥60分,其他8种体质转化分均小于40分,判断为“基本是”;不满足上述条件者判断为“否”。偏颇体质:转化分≥40分判断为“是”,转化分30~39分判断为“倾向是”,转化分<30分判断为“否”。
1.3 纳入标准
①NONFH组符合上述诊断标准及中医体质判定标准;②年龄18~70岁;③受试者对本研究知情。
1.4 排除标准
①创伤性股骨头坏死者;②强直性脊柱炎、骨肿瘤、髋臼发育不良、类风湿关节炎者;③合并严重内脏病变或严重代谢异常疾病者;④妊娠期妇女;⑤精神疾病或智力障碍者;⑥正在参加其他临床试验者。
1.5 一般资料
选择2016年6月-2017年6月甘肃省中医院骨科经问卷调查判定为血瘀质NONFH患者及非血瘀质NONFH患者各10例作为NONFH组,选取同期于甘肃省中医院体检的健康志愿者8例作为正常组,3组性别(2=0.223,=0.890)、年龄(=0.102,=0.903)比较差异无统计学意义,具有可比性。本研究经甘肃省中医院伦理委员会审查批准(2015-018-01)。10例血瘀质NONFH患者为从前期数据库筛选出的典型单纯血瘀体质,经过反复体质判定,不存在偏颇体质。
1.6 病例调查及样本采集
对调查者进行相关理论知识、调查方法及试验方法培训。因NONFH分布不集中,故指定专职调查者进行病例收集。取患者空腹外周血3 mL,置入装有EDTA抗凝剂的无菌管,混匀,于1 h内室温下3000 r/min离心10 min,分离血浆,收集上层淡黄色血浆,移至2 mL冻存管内,标记患者信息后置于甘肃省中医院中心实验室-80 ℃冰箱保存。
1.7 试验材料
TRIzol试剂(Ambion公司)、Gene Expression Wash Pack、miRNA complete Labeling and Hyb Kit(24×)、miRNA Spike In Kit、Package 20 backings(8 HD arrays per slide)、Package 20 backings(4 arrays per slide)、Chip(Agilent公司,美国),miScript Ⅱ Reverse Transcription Kit(Qiagen公司,德国),QuantiFast®SYBR®Green PCR Kit(Qiagen公司,德国),Micro Bio-Spin 6(Bio-RAD公司,美国),荧光定量PCR仪(Roche公司,瑞士),引物由Agilent公司提供。
1.8 试验方法
1.8.1 RNA提取及质量检测
根据TRIzol试验步骤进行RNA沉淀、清洗、再溶解、沉淀及抽提,得到总RNA。总RNA采用NanoDrop RND-2000微量紫外分光光度计进行检测并经Agilent Bioanalyzer 2100(Agilent Technologies)检测RNA完整性。
1.8.2 Agilent microRNA芯片操作及数据分析
RNA质检合格后,按照流程进行样本的标记、芯片杂交及洗脱。首先,总RNA经过去磷酸化,变性,再进一步用Cyanine-3-CTP标记。标记好的RNA纯化后和芯片杂交,洗脱后利用Agilent Scanner G2505C(Agilent Technologies)扫描得到原始图像。采用Feature Extraction(version10.7.1.1,Agilent Technologies)处理原始图像提取原始数据,利用Genespring(version13.1,Agilent Technologies)进行quantile标准化和后续处理。对标准化后的数据进行过滤,用于比较的每组样本中至少有1组100%标记为Detected的探针留下进行后续分析。利用检验的值和倍数变化值进行差异miRNA筛选,筛选标准为上调或下调倍数变化值≥2.0且≤0.05。
1.8.3 实时荧光定量PCR验证差异表达miRNA
利用miScript Ⅱ Reverse Transcription Kit将待测RNA反转录成cDNA。反转录体系:总RNA 0.5 μg,5×miScript HiSpec Buffer 2 μL,10×Nucleics Mix 1 μL,miScript Reverse Transcriptase Mix 0.5 μL,Nuclease-free H2O加至10 μL。反应程序:37 ℃、60 min,95 ℃、5 min。反转录完毕后加入90 μL Nuclease-free H2O储存在-20 ℃冰箱备用。以此cDNA为模板,用QuantiFast®SYBR®Green PCR Kit进行实时荧光定量PCR。反应体系:2×QuantiFast®SYBR®Green PCR Master Mix 5 μL,10 μmol/L Universal primer 0.2 μL,10 μmol/L microRNA-specific primer 0.2 μL,cDNA 1 μL,Nuclease-free H2O 3.6 μL。PCR程序:95 ℃、5 min,95 ℃、10 s,60 ℃、30 s,40个循环。以2-ΔΔCt值表示miRNA的相对表达水平。重复3次。
1.8.4 差异表达miRNA生物信息学分析
通过TargetScan(http://www.targetscan.org/)、microRNAorg(http://www.microrna.org/microrna/home. do)及PITA(http://www.pita.org.fj/)在线数据库预测差异表达miRNA的靶基因,TargetScan采用保守miRNA家族的保守位点、PITA采用Scaled Score分值不超过-9.73、microRNAorg采用mirSVR分值不超过-0.1的保守miRNA进行预测。选取3个在线数据库预测的交集靶基因,进行信号转导通路(Pathway)富集分析,以判定差异miRNA主要影响的信号通路。
1.9 统计学方法
2 结果
2.1 miRNA差异表达及筛选
与正常组相比,NONFH组有差异表达的miRNA 50个。上调21个,其中hsa-miR-4739、hsa-miR-3610、hsa-miR-134-5p是表达前3位的miRNA,差异倍数(FC)值分别为18.452 68、7.215 677、6.312 345;下调29个,其中hsa-miR-6834-3p、hsa-miR-631、hsa-miR-6892-5p是表达前3位的miRNA,FC值分别为7.900 493、6.327 500、6.299 431。与非血瘀质NONFH组相比,血瘀质NONFH组有差异表达的miRNA 33个。上调10个,其中hsa-miR-6786-5p、hsa-miR-550a-5p、hsa-miR-4788是表达前3位的miRNA,FC值分别为3.598 814、3.477 553、3.325 008;下调23个,其中hsa-let-7b-5p、hsa-miR-25-3p、hsa-miR-16-5p是表达前3位的miRNA,FC值分别为9.518 297、8.887 547、6.615 946。经筛选,最终确定血瘀质NONFH差异表达miRNA 5个,其中表达上调miRNA为has-miR-4270,表达下调为hsa-miR-129-1-3p、hsa-miR-365a-3p、hsa-miR-483-3p、hsa-miR-6731-3p,详见表1。
表1 miRNA差异表达筛选结果
miRNA名称FC值P值差异表达 has-miR-42705.067 789 0.016 611上调 hsa-miR-129-1-3p5.918 068 9.16E-04下调 hsa-miR-365a-3p6.746 917 0.004 935下调 hsa-miR-483-3p9.078 883 0.013 898下调 hsa-miR-6731-3p7.225 763 0.001 938下调
2.2 实时荧光定量PCR验证
实时荧光定量PCR结果显示,与正常组比较,血瘀质NONFH组hsa-miR-365a-3p、hsa-miR-483-3p表达水平明显下调(<0.05),与miRNA芯片检测结果一致。见表2。
表2 实时荧光定量PCR验证结果(2-ΔΔCt)
组别has-miR-365a-3phas-miR-483-3p 正常组 1.000 0 1.000 0 血瘀质NONFH组 0.230 0* 0.240 0*
注:与正常组比较,*<0.05
2.3 差异表达miRNA生物信息学分析结果
采用GeneSpring13.1软件,通过TargetScan、PITA、microRNAorg在线数据库对差异miRNA进行靶基因预测。结果显示,hsa-miR-365a-3p检出交集靶基因11个,余未检测出交集靶基因,结果见图1。
图1 靶基因预测结果
针对差异表达miRNA进行GO分析,共得到111条相关注释描述,包括分子功能(MF)23条、生物学过程(BP)65条和细胞组分(CC)23条。进一步针对分子功能分析,发现差异表达miRNA主要与受体结合、蛋白与酶的活性有关。见图2。
Pathway分析显示,靶基因主要富集于轴突导向、Rap1、赖氨酸降解、Fc epsilon RI、甘油磷脂代谢、T细胞受体、血小板活化、成骨细胞分化等多个信号通路。见表3。
图2 差异表达miRNA GO分析分子功能结果
表3 差异表达miRNA信号通路及靶基因预测
KEGG IDGeneSymbols hsa04360:Axon guidanceSEMA6D;EFNA3 hsa04015:Rap1 signaling pathwayLCP2;EFNA3 hsa00310:Lysine degradationKMT2D hsa04664:Fc epsilon RI signaling pathwayLCP2 hsa00564:Glycerophospholipid metabolismETNK1 hsa04660:T cell receptor signaling pathwayLCP2 hsa04611:Platelet activationLCP2 hsa04380:Osteoclast differentiationLCP2 hsa04650:Natural killer cell mediated cytotoxicityLCP2 hsa04014:Ras signaling pathwayEFNA3 hsa05206:miRNAs in cancerEFNA3 hsa04151:PI3K-Akt signaling pathwayEFNA3 hsa01100:Metabolic pathwaysETNK1
3 讨论
本研究显示,血瘀质NONFH差异表达miRNA共5个,其中表达上调为has-miR-4270,表达下调为hsa-miR-129-1-3p、hsa-miR-365a-3p、hsa-miR-483-3p、hsa-miR-6731-3p。随后对hsa-miR-365a-3p、hsa-miR-483-3p进行实时荧光定量PCR验证,结果与芯片数据高度一致。研究表明,miR-483可抑制胰岛素生长因子1表达[10-12],促进血管内皮细胞凋亡[13]。研究发现,miR-483与急性心室缺血、血管腔形成及成骨分化有关[14-15]。多项研究表明,miR-365与骨代谢、冠状动脉粥样硬化有关[16-18],这可能是NONFH的发生途径。
Pathway分析显示,靶基因主要富集于轴突导向、Rap1、赖氨酸降解、Fc epsilon RI、甘油磷脂代谢、T细胞受体、血小板活化、成骨细胞分化等多个信号通路。其中多条通路参与了骨代谢、脂代谢、血管新生及血小板活化等可能导致NONFH发生的调控。研究表明,Rap1是一氧化氮产生和内皮功能调节的关键物质,而一氧化氮系血管稳态的关键决定因素[19-20]。Rap1是血小板活化、血管新生和血栓形成关键[21]。Ras信号通路被证实与血管内皮细胞增殖及血管形态有关[22-23]。而PI3K/Akt信号通路是细胞内重要的信号通路,是一条从细胞膜到细胞核的快速通路转导系统,其与细胞凋亡、血管再生密切相关,若受到抑制将导致血管缺血[24]。
综上所述,本研究采用miRNA芯片技术研究血瘀质NONFH差异表达miRNA,并对这些miRNA进行生物信息学分析。结果表明,血瘀质NONFH差异表达miRNA可能通过调控Rap1、赖氨酸降解、甘油磷脂代谢、血小板活化、成骨细胞分化、Ras、PI3K-Akt等信号通路参与NONFH的发生发展过程。本研究是对NONFH的miRNA基因表达谱的初步探讨,仅观察了NONFH患者及健康志愿者外周血中miRNA的表达情况。今后需扩大样本进一步针对NONFH高发中医体质类型探讨miRNA与靶基因的关系。
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Screening and Bioinformatics Analysis of Differentially Expressed MicroRNAs in Non-traumatic Osteonecrosis of Femoral Head with Blood Stasis
ZHOU Mingwang1, LI Shenghua1, DENG Chang2, FU Zhibin1, JI Xing1, CHEN Yantong2,HU Xingrong2, LIU Yifei2, WANG Pengzhi2, ZHAO Qiuyue2
To establish the miRNA expression profile and analyze its biological information by screening the miRNA differentially expressed in the peripheral blood of the patients with and without blood stasis and non-traumatic osteonecrosis of femoral head (NONFH); To provide a basis for the prevention and treatment of NONFH with blood stasis.Totally 10 cases of NONFH with out blood stasis (NONFH group), 10 cases of NONFH with blood stasis and 8 cases of healthy volunteers (normal group) were selected. The relative expression of miRNA in plasma was detected by RT-PCR, and the miRNA with different expression was screened. The differential expression of hsa-miR-365a-3p and hsa-miR-483-3p was verified by real-time fluorescence quantitative PCR. Three online databases, TargetScan, microRNAorg, PITA were used to predict the target genes of different miRNAs, and the target genes were analyzed by bioinformatics.Compared with the normal group, 50 miRNAs were differentially expressed, 21 up-regulated and 29 down-regulated in NONFH groups. Compared with the NONFH without blood stasis group, 33 miRNAs were differentially expressed, 10 up-regulated and 23 down-regulated in NONFH with blood stasis group. After screening, five miRNAs with different expression of NONFH with blood stasis group were identified. The results of real-time fluorescent quantitative PCR were consistent with those of miRNA microarray. In the three online databases, 11 cross target genes were screened out; only 11 cross target genes were detected in hsa-miR-365a-3p expression down regulated miRNA; the rest were not detected. GO analysis showed that the differential expression of miRNA was mainly related to receptor binding, protein and enzyme activity. Pathway analysis showed that the differentially expressed miRNA target genes of NONFH with blood stasis were mainly enriched in axon guidance, Rap1, lysine degradation, Fc epsilon RI, glycerophospholipid metabolism, T cell receptor, platelet activation, osteoblast differentiation and other signaling pathways.The different expressions miRNA of NONFH with blood stasis are obtained. These differentially expressed miRNAs may be involved in the development of NONFH by regulating the signal pathways of Rap1, lysine degradation, glycerophospholipid metabolism, platelet activation, osteoblast differentiation, RAS, PI3K-Akt and so on.
blood stasis; non-traumatic osteonecrosis of femoral head; microRNA; gene chip; bioinformatics
R272.968
A
1005-5304(2020)08-0036-05
10.3969/j.issn.1005-5304.201910076
国家自然科学基金(81473712、81560782)
李盛华,E-mail:820512343@qq.com
(2019-10-08)
(2020-01-20;编辑:季巍巍)