CN109504766B - miRNA标志物miRNA-345-3p的应用 - Google Patents
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Abstract
miRNA标志物miRNA‑345‑3p的应用。具体涉及miRNA‑345‑3p在制备抑制基质金属蛋白酶9（MMP‑9）、TNF‑α、IL‑1β和IL‑6中至少一种炎症因子表达制剂中的应用。在本发明中，筛选并验证了miR‑345‑3p对巨噬细胞活力的影响，调节炎症因子的表达作用。发现转染miRNA模拟物（mimics）可以抑制LPS诱导的巨噬细胞细胞活力的降低，抑制LPS诱导的炎症因子TNF‑α、IL‑1β、IL‑6的表达升高，也抑制基质金属蛋白酶9（MMP‑9）的表达升高，为临床诊断和治疗感染性骨不连提供新靶标，新途径。
Description
技术领域
本发明属于分子生物学诊断治疗领域，具体涉及miRNA标志物miRNA-345-3p的应用。
背景技术
感染性骨不连临床上并不少见，其特征是骨不连的断端存在骨性感染。必须兼有骨的感染和骨不连两个因素同时存在，大多由污染严重的开放性骨折造成；也有是闭合性骨折术后感染、慢性骨髓炎引起。感染导致骨折断端和软组织的坏死，局部血运重建时间延长，造成骨折愈合延迟。若是感染严重，骨折端吸收明显致断端的缺损，造成骨不连。感染性骨不连患者一般病程较长，经过多次手术治疗不愈，由于多耐药菌导致抗生素疗效差。一旦诊断为感染性骨不连，即需清除病灶，使用抗生素骨水泥控制局部感染，二期行植骨手术。若是感染严重者会造成骨缺损，甚至需要截肢，大大降低了患者生活质量，加重了经济及心理负担。所以对于此类患者需要早期诊断，制定治疗方案。诊断感染性骨不连需要依靠患者骨折病史；局部皮肤情况、窦道、流脓情况，查体有无异常活动；影像学检查有无骨折不愈合；实验室检查如血沉、C反应蛋白、降钙素原、病原学检查等明确有无感染以及感染的程度等以及最终的组织病理学检查。但是在临床中，往往诊断困难，该类感染多由低毒力致病菌如凝固酶阴性葡萄球菌等引起，其症状往往不典型甚至缺如，感染指标的敏感性及特异性不高，微生物培养阳性率低，且受多种因素影响，包括抗生素的使用、生物膜形成等。组织病理学检查虽然可以确诊，但是其诊断往往在手术干预之后。所以在临床上，针对血沉、CRP等感染指标甚至多次微生物学培养均阴性的骨不连患者，缺乏一种更具优势的术前诊断指标来诊断感染。
miRNA是一类约18-25(20-24)个核苷酸的非编码RNA，通过触发RNA诱导的沉默复合物(RISC)来调节转录后水平的基因表达。它通过调控靶基因(包括细胞增殖，分化和凋亡以及组织发育)从而调节多种生物学和病理学过程。Ruth E.Drury等人利用miRNA在宿主对病原体反应中的作用，提出其在预防，诊断和治疗感染方面可能的临床应用。在骨不连中，一旦诊断为感染性，先控制局部感染，再治疗骨不连是常用的治疗策略；但仍缺乏感染性骨不连骨组织的相关miRNA表达分析。
已有研究表明，基质金属蛋白酶9(MMP-9)对于炎症修复以及骨重塑具有重要的调节作用，它能调节巨噬细胞的骨膜内定位，从而促进膜内骨化。但是在感染时，嗜中性粒细胞、巨噬细胞、淋巴细胞等炎症细胞聚集，引起炎症反应，促进TNF-α、IL-6、IL-1β等炎症介质释放进一步加重炎症反应，这些炎症介质能促进炎症细胞(如巨噬细胞及T细胞等)表达MMP-9。过表达的MMP-9可降解细胞外基质，破坏基膜，不利于骨折愈合。
发明内容
解决的技术问题：为解决上述存在的问题，本发明提供一种miRNA标志物miRNA-345-3p的应用。将感染性骨不连患者的骨组织与对照组即闭合性骨折患者的骨组织进行比较，检测出差异的miRNAs表达谱，进一步通过实时定量PCR进行验证，结合生物信息学分析结果，从中筛选出目标miRNA。进一步制备该miRNA mimics和miRNA inhibitor，研究其对巨噬细胞细胞活力，MMP-9的表达以及炎症介质的释放的影响，为临床诊断和治疗感染性骨不连提供新的标志物。
技术方案：miRNA-345-3p在制备抑制基质金属蛋白酶9(MMP-9)、TNF-α、IL-1β和IL-6中至少一种炎症因子表达制剂中的应用。
上述miRNA标志物miRNA-345-3p为如SEQ ID NO.1所示的miRNA-345-3p mimics。
上述miRNA-345-3p mimics的反义链如SEQ ID NO.2所示
如SEQ ID NO.1所示的miRNA-345-3p mimics在制备诊断感染性骨不连试剂盒中的应用。
如SEQ ID NO.1所示的miRNA-345-3p mimics在制备治疗感染性骨不连药物中的应用。
有益效果：(1)目前对于感染性骨不连诊断和治疗相关的miRNAs少有报道，而在本发明中，我们筛选并验证了miRNA-345-3p在感染性骨不连的骨组织中差异表达，并进一步验证了miRNA-345-3p mimics可以抑制LPS诱导的巨噬细胞细胞活力的降低抑制炎症介质TNF-α、IL-6、IL-1β的表达，从而抑制炎症反应，促进骨折愈合，为临床诊断和治疗感染性骨不连提供新靶标。(2)MMP-9的过表达是炎症反应加重使骨折不愈合的一个重要环节，本发明得出了miRNA-345-3p mimics可以明显降低MMP-9的表达。(3)miRNA-345-3p的模拟物(miRNA-345-3p mimics)通过提高miRNA-345-3p的水平，抑制巨噬细胞活力以及MMP-9的表达，抑制炎症介质的释放，可见作用于巨噬细胞的miRNA-345-3p能作为治疗感染性骨不连的靶标。
附图说明
图1为感染性骨不连患者的骨组织与闭合性骨折患者的骨组织样本芯片分析的聚类图，每一行代表一种miRNA，每一列代表一个样本，I1、I2、I3、I4、I5为感染性骨不连的骨组织样本，C1、C2、C3、C4为闭合性骨折患者的骨组织样本。该图顶部为样本的聚类树，左侧为miRNA的聚类树。顶部的色标用来衡量miRNA的相对表达水平：深色代表高表达，浅色代表低表达；
图2为进一步通过实时定量PCR验证微阵列分析的结果。*代表P＜0.05，**代表P＜0.01；
图3为miRNA-345-3p mimics以及inhibitors对LPS诱导的巨噬细胞的细胞活动的影响；其中，左图为miRNA-345-3p mimics对LPS诱导的巨噬细胞的细胞活动的影响；右图为miRNA-345-3p mimics inhibitors对LPS诱导的巨噬细胞的细胞活动的影响；
图4为miRNA-345-3p mimics以及inhibitors对LPS诱导的巨噬细胞MMP-9蛋白表达的影响；其中，左图为miRNA-345-3p mimics对LPS诱导的巨噬细胞MMP-9蛋白表达的影响；右图为miRNA-345-3p mimics inhibitors对LPS诱导的巨噬细胞MMP-9蛋白表达的影响；
图5为miRNA-345-3p mimics以及inhibitors对LPS诱导的巨噬细胞炎症介质TNF-α、IL-6、IL-1β释放的影响，其中A为miRNA-345-3p mimics对LPS诱导的巨噬细胞炎症介质TNF-α释放的影响，B为miRNA-345-3p mimics inhibitors对LPS诱导的巨噬细胞炎症介质TNF-α释放的影响；其中C为miRNA-345-3p mimics对LPS诱导的巨噬细胞炎症介质IL-1β释放的影响，D为miRNA-345-3p mimics inhibitors对LPS诱导的巨噬细胞炎症介质IL-1β释放的影响；其中E为miRNA-345-3p mimics对LPS诱导的巨噬细胞炎症介质IL-6释放的影响，F为miRNA-345-3p mimics inhibitors对LPS诱导的巨噬细胞炎症介质IL-6释放的影响。
具体实施方式
下面的实施例可使本专业技术人员更全面地理解本发明，但不以任何方式限制本发明。
实施例1差异表达microRNAs的筛选
一、芯片分析筛选差异表达的microRNA
方法：获取感染性骨不连患者与闭合性骨折患者的骨组织，分别提取RNA，利用miRNA芯片分析筛选出差异表达的miRNA。
(1)获取标本：在手术过程中提取感染性骨不连断端的骨片以及闭合性骨折游离的骨碎片，用含0.9wt.％NaCl水溶液冲洗2次，去除血液和骨髓污染，取出后30分钟内液氮保存，分别进行miRNA芯片检测；
(2)样品RNA提取：使用TRIzol法提取RNA，并用RNasey Mini Kit(QIAGEN)纯化。使用NanoDrop ND-1000测量纯化后的RNA浓度，使用变性琼脂糖凝胶电泳检测RNA完整性。
(3)RNA标记与芯片杂交：RNA标记和杂交根据Exiqon提供的方法进行。
1)抽提的RNA通过质检后，使用miRCURYTM Array Power Labeling kit(Cat#208032-A,Exiqon)对miRNA进行标记。具体步骤如下：
a.1微克的RNA加水至2μL加1μL的CIP buffer and CIP酶(Exiqon)。混合后置于37℃下30min。
b.将样品置于95℃下5min终止反应。加入3μL的labeling buffer，1.5μL的fluorescent label(Hy3TM)，2.0μL的DMSO，2.0μL的labeling enzyme。在16℃下反应1h。
c.将样品置于65℃下15min终止反应。
2)标记完成后，将样品与miRCURYTM LNA Array(v.19.0)(Exiqon)芯片杂交，根据Exiqon的实验方法进行。
a.25μL的样品与25μL的杂交缓冲液混合，95℃下变性2min，然后置于冰上2min。
b.与芯片在56℃下杂交16-20h，杂交系统为Nimblegen Systems,Inc.,Madison,WI,USA。
c.杂交完成后，使用Wash buffer kit(Exiqon)清洗芯片。
3)使用Axon GenePix 4000B芯片扫描仪扫描芯片。
(4)芯片数据分析：使用GenePix Pro 6.0读取芯片扫描图像，并提取探针的信号值。相同的探针取中值合并。保留在所有样品中均≥30.0的探针，对全部芯片进行中值标准化，筛选差异表达探针。使用Fold change和P-value筛选两组样品间(有重复)的差异表达miRNA。使用Fold change筛选两个样品间(没有重复)的差异表达miRNAs。选取Fold change＞1.5且P值＜0.05的miRNA作为差异表达的miRNA。最后，对差异表达miRNAs进行聚类并绘制聚类图。
二、实时定量PCR验证差异表达的miRNA
方法：从芯片数据分析结果中选择6种miRNA(hsa-miR-3929、hsa-miR-345-3p、hsa-miR-649、hsa-miR-29b-3p、hsa-miR-498、hsa-miR-328-5p)进行验证。进行实时定量PCR的样本来自进行芯片分析的样本。
(1)cDNA的合成：
1)制备RT混合反应液：
RT特异引物(1μM)
2)在PCR扩增仪(Gene Amp PCR System 9700(Applied Biosystems))进行RT反应：
| 16℃ | 30min； |
| 42℃ | 42min； |
| 85℃ | 5min |
3)反应结束后，将其放在冰上待用或-20℃保存。
(2)进行Realtime PCR反应
1)将所有cDNA样品分别配制Realtime PCR反应体系。体系配制如下：
| 2×Master Mix | 5μL |
| 10μM的PCR特异正向引物 | 0.5μL |
| 10μM的PCR特异反向引物 | 0.5μL |
| 加水至总体积为 | 8μL |
10μM的PCR特异引物F和10μM的PCR特异引物R：
F＝Forward primer；R＝Reverse primer；GSP＝Gene-specific primer.
轻弹管底将溶液混合，5000rpm短暂离心。
2)加样
a.将8μL混合液加到384-PCR板对应的每个孔中。
b.再加入对应的2μL cDNA。
c.小心粘上Sealing Film封口膜，并短暂离心混合。
c.在设置PCR程序前将准备好的PCR板放在冰上。
3)将上述384-PCR板置于Realtime PCR仪(QuantStudio5 Real-time PCR System(Applied Biosystems))上进行PCR反应。
U6&所有的指标均按以下程序进行：
95℃，10min；40个PCR循环(95℃，10秒；60℃，60秒(收集荧光))。
为了建立PCR产物的熔解曲线，扩增反应结束后，按(95℃，10秒；60℃，60秒；95℃，15秒)；并从60℃缓慢加热到95℃(仪器自动进行-Ramp Rate为0.075℃/秒)。
4)各样品的目的miRNA和内参(U6)分别进行Realtime PCR反应。数据采用2-△△CT法进行分析。
三、生物信息学分析：
为了鉴定差异表达的miRNA的潜在靶标，我们基于两种靶基因数据库(即targetScan，miDRB)预测了经PCR验证后差异表达的miRNA的靶基因。将均在两种数据库中都确定靶基因作为差异表达的miRNA的靶基因。接下来将预测的靶基因进行基因本体(GO)和KEGG pathway分析探究其基因功能以及富集途径。P值小于0.05。
结果：经PCR验证后，与闭合性骨折相比，感染性骨不连患者的骨组织miR-345-3p表达下调。根据基因表达差异以及靶基因预测的相关结果，我们筛选出miR-345-3p作为研究对象。
实施例2获得巨噬细胞并研究miRNA-345-3p的表达对巨噬细胞代谢活力(MTT)的影响
一、获得骨髓来源的巨噬细胞(BMDM)
方法：使用L929细胞培养上清诱导巨噬细胞分化。
(1)分离小鼠骨髓：取8-12周雄性C57BL/6小鼠麻醉后脱臼处死，75vt.％乙醇浸泡3-5min，于超净台中取出股骨与胫骨，剪去干骺端。用无菌磷酸缓冲盐溶液(phosphate-buffered saline，PBS)冲洗骨髓腔，直至变白。收集冲洗液离心后弃上清，红细胞裂解液裂解红细胞，离心弃上清。
(2)诱导分化巨噬细胞：用DMEM完全培养基(含10wt.％FBS)于37℃、5vt.％的CO2孵箱中培养L929细胞3-5天，收集细胞上清。将L929细胞上清与含20wt.％FBS的DMEM完全培养基按1:4比例充分混匀，配制成BMDM全培液，重悬骨髓细胞。置于37℃、5％的CO2孵箱中培养7天，获得成熟的BMDM。
二、研究miRNA-345-3p的表达对巨噬细胞代谢活力(MTT)的影响
方法：分三组，设立对照组，转染miR-345-3p mimics(序列为：5’-GCCCUGAACGAGGGGUCUGGAG-3’)以及miR-345-3p inhibitors(序列为：5’-CUCCAGACCCCUCGUUCAGGGC-3’)，加入细菌脂多糖(LPS)刺激巨噬细胞，分别测定巨噬细胞代谢活力。
(1)将细胞接种于培养板(1*106/mL)。实验组加入LPS 10ng/mL刺激骨髓来源的巨噬细胞(BMDM)，对照组加入同等体积的生理盐水，继续置于37℃、5vt.％的CO2孵箱中继续孵育6h；
(2)每孔加入5mg/mL MTT液继续培养4h后，终止培养，弃去孔内培养液。每孔加入150μL；DMSO终止反应，用振荡仪振荡10min后，酶标仪检测492nm处吸光度(A)。
结果：LPS刺激BMDM后，巨噬细胞的活力明显下降，差异有统计学意义(P＜0.05)。与miRNA-345-3p inhibitors相比，转染miRNA-345-3p mimics可以提高巨噬细胞的细胞活力，差异有统计学意义(P＜0.05)。
实施例3测定巨噬细胞MMP-9以及TNF-α、IL-1β、IL-6的表达水平，研究miR-345-3p对免疫调节的影响
一、测定巨噬细胞MMP-9的表达
方法：提取细胞蛋白，明胶酶谱法检测巨噬细胞MMP-9蛋白的表达。
(1)提取细胞蛋白：分别取对照组、miRNA-345-3p mimics组、LPS组以及LPS+miRNA-345-3p mimics组细胞培养上清液，4℃下12000r/min离心15min，取上清，100℃变性3-5min，-20℃保存。
(2)明胶酶谱法检测MMP-9蛋白的表达：
a.电泳：制备8％的分离胶，称取适量的明胶后，加入去离子水，隔热水加热，直到明胶溶解，并依次加入30wt.％丙烯酰胺、10wt.％SDS、Tris-HCl(pH 8.8)、10wt.％APS和TEMED，去离子水压胶。待分离胶完全凝固后，制备4％的浓缩胶，依次加入去离子水、Tris-HCl(pH 6.8)、10wt.％SDS、10wt.％APS和TEMED，插入10孔齿梳。待胶凝固后，装板，加入电泳液，轻轻拔梳子，微量上样器上样，每个样品上样20μL，随后电压80V，时间130min，电泳。
b.脱色与孵育：电泳结束，刮去浓缩胶，小心将分离胶整张移入大小合适的方盒中，在每张胶上做好标记，加入适量洗脱液(Zymogram Renaturing Buffer)在摇床上充分洗脱(130-140r/min)，洗脱四次，每次洗脱摇晃15min，同时将配好的孵育液(ZymogramDeveloping buffer)置于恒温空气摇床(37℃)中预热。待洗脱完毕，小心的将胶用去离子水冲干净，转移入恒温摇床中，加入预温的孵育液，在恒温空气摇床中孵育48小时。
c.染色：将孵育完毕的胶用去离子水冲洗干净后，加入1wt.％考马斯亮蓝染色液轻晃染色40min，然后洗净染色液，加入脱色液脱色四次，每次20min。在显色仪中成像分析。使用Quantity One 4.6.5统计灰度值，取平均值，并计算SEM值，通过GraphPad Prism进行组间t检验，以P<0.05为差异有统计学意义。
二、测定巨噬细胞TNF-α、IL-1β、IL-6的表达水平
方法：ELISA法检测炎症介质TNF-α、IL-1β、IL-6的表达水平。
(1)分别收集对照组、miRNA-345-3p mimics组、LPS组以及LPS+miRNA-345-3pmimics组BMDM的上清，10000r/min离心4min，取上清。
(2)根据试剂盒说明书(Quantikine ELISA,R&D Systems,Minneapolis,MN,USA)，测炎症介质TNF-α、IL-1β、IL-6的表达，具体步骤为：
a.每孔加入50μL分析稀释液及50μL标准品，对照品或样品；用封板胶纸封住反应孔，并在室温下孵育2小时，洗板5次。
b.每孔加入100μL结合物；用封板胶纸封住反应孔，在室温下孵育2小时，洗板5次。
c.每孔加入100μL底物溶液；在室温下避光孵育30分钟。
d.每孔加入100μL终止液；在30分钟内读取OD450值。
结果：LPS刺激后的骨髓来源的巨噬细胞(BMDM)的MMP-9蛋白表达上升，差异有统计学意义(P＜0.05)。转染miRNA-345-3p mimics后，MMP-9的表达量明显下降，差异有统计学意义(P＜0.05)。转染miRNA-345-3p inhibitors后，MMP-9的表达量改变与转染前相比差异没有统计学意义。同时，炎症介质的表达与MMP-9蛋白表达的趋势相同，且转染miRNA-345-3p mimics能够明显减少炎症介质的表达，差异有统计学意义(P＜0.05)。
综上所述，miR-345-3p在感染性骨不连患者的骨组织中表达有明显差异，LPS刺激骨髓来源的巨噬细胞(BMDM)后，巨噬细胞的细胞活力明显下降，其释放MMP-9蛋白以及炎症因子TNF-α、IL-1β、IL-6明显升高。当转染miRNA-345-3p mimics后，MMP-9蛋白、TNF-α、IL-1β、IL-6的表达均能较前下降，说明miRNA-345-3p mimics能够抑制巨噬细胞的功能，减少炎症相关蛋白以及细胞因子释放，从而抑制炎症反应，控制感染，促进骨折的愈合，为临床诊断和治疗感染性骨不连提供新靶标和新途径。
序列表
<110> 胡军
<120> miRNA 标志物miRNA-345-3p的应用
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<170> SIPOSequenceListing 1.0
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<213> 人工序列(Artificial Sequence)
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Claims (1)
1.扩增miRNA-345-3p的引物在制备区分闭合性骨折与感染性骨不连试剂盒中的应用。
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| 炎症对骨折愈合过程中成骨细胞分化的影响及分子机制;李文锋;《中国博士学位论文全文数据库医药卫生科技辑》;20101215(第12期);第E066-33页 * |
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