CN116396961B - 一种circRNA_BMPR2诊断生物标志物及其应用 - Google Patents
一种circRNA_BMPR2诊断生物标志物及其应用 Download PDFInfo
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Abstract
本发明公开了一种circRNA_BMPR2诊断生物标志物及其应用。其基因序列如序列表SEQ ID NO:1所示。本发明检测了PCOS病人和正常人卵巢颗粒细胞中circRNA_BMPR2的表达情况,发现circRNA_BMPR2表达水平显著升高,表明circRNA_BMPR2可以作为多囊卵巢综合征诊断用生物标志物。
Description
技术领域
本发明属于多囊卵巢综合征诊断技术领域,具体涉及一种circRNA_BMPR2诊断生物标志物及其应用。
背景技术
多囊卵巢综合征(PCOS)是一种常见的内分泌和代谢紊乱,扰乱了育龄女性个体。PCOS的症状涉及严重的生殖功能障碍(包括不孕和妊娠并发症),不平衡的代谢功能(包括胰岛素抵抗、2型糖尿病等)以及心理障碍(主要包括抑郁和焦虑)和其他影响,干扰女性患者的年龄范围从青春期到更年期。PCOS被认为是一种同时具有内分泌功能障碍和代谢功能障碍的疾病,影响近20%的育龄女性患者。PCOS的症状包括雄激素过量(多毛症和/或高雄激素血症)和卵巢功能障碍(寡排卵和/或多囊卵巢形态)。
circRNA是一类新型的、非多聚腺苷酸化的环形非编码RNA,没有5′帽或3′尾巴的共价闭合的RNA环,由pre-mRNA转录本的反向剪接形成。circRNA主要来源于外显子、内含子和基因间区域,广泛分布于动物细胞的染色体上。因其特殊的共价闭合环状结构以及RNA酶逃逸性,使其比之其他线性的ncRNA(如miRNA、lncRNA等)在体内存在更丰富和稳定。目前circRNA在疾病中的研究已在肿瘤、心血管领域中取得了具有诊疗价值的成果,同时一些研究表明PCOS患者卵泡液、颗粒细胞、外泌体中含有极其丰富且具有大量潜在生物信息的circRNA,但具体的相关circRNA在PCOS中的功能机制方面国内外尚未见报道。
发明内容
本发明的目的在于提供一种circRNA_BMPR2诊断生物标志物及其应用。
本发明的有益效果:本发明检测了PCOS病人和正常人卵巢颗粒细胞中circRNA_BMPR2的表达情况,发现circRNA_BMPR2表达水平显著升高,表明circRNA_BMPR2可以作为多囊卵巢综合征诊断用生物标志物。
附图说明
图1为病例信息。
图2为PCOS和非PCOS患者卵泡液中circRNA测序及表达谱分析;
图中,A:circRNA长度,B:circRNA数量,C:circRNA外显子数量。
图3为PCOS和非PCOS患者卵泡液中circRNA表达谱分析;
图中,D:circRNA染色体分布。
图4为PCOS和非PCOS患者卵泡液中circRNA表达谱分析;
图中,E:circRNA差异聚类分析,F:circRNA差异火山图。
图5为qRT-PCR验证。
图6为过表达载体结构图。
图7 circRNA_BMPR2的相对表达。
图8为circRNA_BMPR2对人卵巢颗粒细胞的生物学作用。
图9为流式细胞仪检测结果图。
具体实施方式
为了便于理解本发明,下面将对本发明进行更全面的描述。但是,本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
下述实施例中采用的主要试剂及仪器:TRK-1001总RNA纯化试剂盒(LC Sciences,Houston,USA);Ribo-Zero rRNA去除试剂盒(Epicentre,Madison,WI);cDNA合成试剂盒(美国Thermo Fisher Scientific公司);Superscript II逆转录酶(Invitrogen);1X SYBRGreenPCRMasterMix(美国Thermo Fisher Scientific公司);NanoDrop ND-2000紫外可见光谱仪(美国Thermo Fisher Scientific公司);Agilent Bioanalyzer 2100(AgilentTechnologies,Santa Clara,CA);Applied Biosystems 7900HT快速实时PCR系统(美国Thermo Fisher Scientific公司)。
实施例1circRNA_BMPR2基因的筛选与验证
患者标本选择严格遵守成人的鹿特丹诊断标准,严格的标准要求,既要求不规则周期,又要有LH/FSH比值明显增加。使用超声检查可以看到PCOS患者的“项链征”。排除甲状腺疾病(促甲状腺激素)、库欣综合征、产生雄激素的肿瘤和高泌乳素血症(催乳素),并考虑男性因素进行评估。筛选出3例原发性不孕症(非PCOS组)和3例原发性不孕症伴PCOS组(PCOS组),其配偶诊断为原发性不孕症伴畸形精子症。本研究遵循的程序符合江苏省苏北人民医院人体试验委员会所制定的伦理学标准,得到该委员会批准。
多囊卵巢综合征的诊断标准对PCOS的诊断标准采用2003年的鹿特丹共识(三者中的两个):(a)少排卵和/或无排卵;(b)高雄激素血症的临床和/或生化体征;(c)多囊卵巢。应排除其他病因(先天性肾上腺增生、雄激素分泌肿瘤或库欣综合征)和应用诊断标准的详细记录(并在研究论文中描述),以供未来评估。推荐以下诊断标准:高雄激素血症的临床或生化证据,慢性无排卵,并排除其他已知的疾病(图1)。
卵泡液外泌体样本采集方法:卵泡液的外泌体来自PCOS患者以及在苏北人民医院生殖医疗中心登记的非PCOS不孕成人对照组。所有实验均经医院伦理委员会批准,并在外泌体样本收集前获得所有参与者的知情同意。穿刺前用阴道B型超声(B超)记录卵泡直径至少3次,并取平均值。所有患者均接受了促排卵治疗。当至少一个卵泡直径为≥18mm时注射人绒毛膜促性腺激素(hCG,2000U;丽珠医药集团有限公司,广州珠海,中国)。在hCG触发36小时后,在阴道B超引导下穿刺卵泡,并收集第一管无血清污染的卵泡液。卵泡液是从直径大于15mm的卵泡中收集的。样品在室温下1500g离心15min,上清液于-80℃保存。立即在4℃下以2000g离心30min,获得外泌体。将上清液转移到干净的Amicon Ultra 30K离心装置(Millipore,Bedford,MA)中,在4℃下10000g离心30min。丢弃流动液,将剩余的上清液与0.2体积的总外泌体分离试剂(来自其他体液;Invitrogen,Carlsbad,CA)混合。将合成的混合物在室温下孵育30min,然后在4℃下以10,000g离心30min。弃上清液,将颗粒重新重悬在1.5ml微离心管中的磷酸盐缓冲盐水中,最终体积相当于浓缩外泌体样品溶液的四分之一。这样制备的外泌体样本立即用于总RNA分离,无需进一步处理。
基因测序方法:用TRK-1001总RNA纯化试剂盒(LC Sciences,Houston,USA)按照制造商的说明分离出总RNA。分离的RNA的浓度和完整性分别在NanoDrop ND-2000紫外可见光谱仪(Thermo Fisher Scientific,Waltham,MA)和Agilent Bioanalyzer 2100(AgilentTechnologies,Santa Clara,CA)上测定。为了构建互补DNA(cDNA)文库,首先使用人/小鼠/大鼠Ribo-Zero rRNA去除试剂盒(Epicentre,Madison,WI)按照制造商的说明处理4μg提取的RNA,去除所有核糖体RNA。聚(A)-和聚(A)+RNA随后在二价阳离子存在的情况下在高温下破碎,然后使用第一链cDNA合成试剂盒(Thermo Fisher Scientific)与随机六聚体引物进行逆转录,然后使用dUTP方法构建cDNA文库。RNA文库在Illumina HiSeq.2500平台上同125bp的成对末端读数进行了测序。成对端文库中插入片段的平均大小约为300bp。
环状RNA的检测和注释:对circRNA的识别是基于之前描述的称为发现circ的计算管道,并稍作修改。人类参考基因组hg19(2009年2月,GRCh37)从基因编码项目(http://www.gencodegenes.org/)下载,并用于所有后续的生物信息学分析。Bowtie2(版本2.1.0;Langmead&Salzberg,2012)首次被用来消除所有可以完全映射到一个或多个rRNA序列的原始RNA-seq读数。然后,使用BWA(版本0.7.15-r1140)将剩余的读数与基因组对齐,而不进行修剪。不能连续映射到基因组上的读数被保留下来,其余的被丢弃。接下来,每个保留读数两端的20个核苷酸的锚定序列被独立地重新匹配到基因组上,然后扩展到整个读数。如果一个读数的两个末端锚点连续与基因组反向排列(头尾剪接)对齐,则被认为是一个潜在的circRNA候选基因。相反,锚点与基因组的顺序排列标志着线性剪接事件。此外,我们还采用了以下标准来提高circRNA鉴定的准确性。首先,两个剪接位点之间的距离不得超过100kb,且两侧必须有GU/AG。第二,整个读数的排列不能包含两个以上的错配。第三,必须至少有两个独立的读数来确认头尾剪接。第四,确定的断点必须在锚定对齐的两个核苷酸内。第五,最佳排列的得分必须超过第二最佳排列的得分至少35。对于circRNA注释,假定circRNA中已知内含子是被剪接出来的。如果每个circRNA与各自的特征完全或部分重叠,则其被划分为一个基因结构类别。
基于circRNA在转录及转录后水平具有重要的调控作用,本发明的项目前期以PCOS患者卵泡液中的circRNA作为研究对象,并对PCOS组和非PCOS组育龄女性卵泡液进行circRNA高通量测序分析。根据circRNA长度、数量及外显子数量进行筛选分类。结果发现,circRNAs在除Y染色体外的所有的染色体中大量存在(图2中ABC及图3中D)。其中,两组共检测出412个差异环状circRNAs,其中上调circRNAs为167个,下调circRNAs为245个(图4中EF),为下一步的实验提供了数据支持。
通过定量逆转录聚合酶链反应(qRT-PCR)验证circRNA:为每个假定的circRNA候选基因设计了一对不同的引物,并用于从基因组DNA和cDNA中进行扩增。使用SuperscriptII逆转录酶(Invitrogen)根据制造商的说明进行了第一链cDNA的合成。将由2μg RNA和2μl逆转录酶组成的逆转录混合物依次在42℃下孵育50min,46℃下孵育10min,然后在75℃下孵育15min。随后,RNA模板在37℃下被RNase H消化20min,然后在65℃下酶失活10min。然后将合成的cDNA作为模板,对每个候选circRNA进行实时PCR扩增。每个PCR反应混合物设置为在无核酸酶无菌水中,包含30ng cDNA样本,每个引物5μM,1X SYBR GreenPCRMasterMix。在Applied Biosystems7900HT快速实时PCR系统中进行PCR扩增。此外,在类似的PCR反应中使用聚合引物扩增参考甘油醛3-磷酸脱氢酶基因和线性转录本。Ct值转化为RNA表达的倍数变化。
反拼接结点的读数和线性映射的读数都被结合起来,并因每百万映射的读数(RPM)来量化circRNA的表达水平。所有的qRT-PCR扩增均为3个重复。PCOS组与非PCOS组之间环状RNA表达谱的差异采用t检验进行分析。p<0.05被认为是有统计学意义的差异。
根据circRNA表达差异倍数及p值筛选10个差异circRNAs进行了qRT-PCR验证,90%的circRNAs趋势与高通量测序一致,证实本项目测序结果的可靠性。其中,circRNA_BMPR2在PCOS组卵泡液中显著表达,推测其可能参与了PCOS的发生发展。circRNA_BMPR2位于chr2:203329531-203332412,长度为342bp(图5)。
实施例2过表达载体构建及鉴定
以PCR扩增基因circRNA_BMPR2全长342bp序列(SEQ IDNO:1),以In-Fusion克隆连接到pLC5(图6)。
Alu-circRNA_BMPR2-UnF:CTTTTTTTTTTTTTTTTTGCAGCTT CGCAGAATCAAGAACGGC;
Alu-circRNA_BMPR2-UnR:AAAGTATGGAGTTGTTACCTACTG AGTGGTGTTGTGTCAGGAG。
测序峰图正常,无杂峰或叠带,序列对比吻合,表明circRNA_BMPR2成功插入pLC5中,过表达载体构建成功,实验步骤如下:
载体测序引物pC5-seqF:TGTGAATTTGACCCTTAAGA。
目的片段PCR扩增:95℃5min,38个循环(98℃10s,58℃30s,68℃60s),68℃5min,4℃保存,反应体系如表1所示。
表1PCR扩增反应体系
PCR扩增产物回收:按照琼脂糖凝胶DNA回收试剂盒(Magen)说明书进行凝胶回收,PCR产物-20℃保存。
空载体双酶切:向空载体中加入两种内切酶EcoRI和BamHI进行双酶切。反应体系如表2所示,37℃温育1h。然后灌制1.5%的琼脂糖凝胶进行电泳,回收目的条带。
表2双酶切反应体系
In-Fusion连接:参考HieffPlus One Step Cloning Kit(上海翊圣)说明书配置连接反应液,保持50℃20min,然后进行转化或于-20℃保存。反应体系如表3所示。
表3连接反应体系
转化感受态细胞Trans1 T1,挑取单个菌落接种于LA培养液中,37℃摇床220rpm培养4h后取1μL菌液作为模板进行PCR扩增;然后灌制1.5%琼脂糖凝胶,取5μL PCR产物进行电泳检测。
阳性菌液测序鉴定:将通过菌液PCR检测的阳性菌液进行测序,将得到的结果进行BLAST序列对比分析,确定其是否为目的基因。
无内毒素质粒抽提:按照低内毒素质粒小提试剂盒(Magen)说明书进行低内毒素质粒抽提,-20℃保存。
细胞转染:分Alu-BMPR2、NC(pLC5)和control三组转染293T细胞,48h后收集细胞进行qPCR检测,操作步骤如下:
细胞复苏:
1)将水浴锅预热至37℃;
2)用75%酒精擦拭紫外线照射30min的超净工作台台面;
3)在超净工作台中按次序摆放好消过毒的离心管、吸管、培养瓶等等;
4)取出冻存管,迅速将冻存管投入到已经预热的水浴锅中迅速解冻,并要不断的摇动,使管中的液体迅速融化,在冻存管内还存在一点点没融化的时候,取出;
5)用酒精棉球擦拭冻存管的外壁,再拿入超净台内;
6)制备细胞悬液,将细胞转移至一个15mL离心管内,一滴一滴地加入预热好的培养基,同时一边晃动离心管;加入培养基的量要达到10mL以上;
7)以800rpm离心5min;吸去上清,然后用1mL培养基重悬细胞;
8)将细胞悬液分装入培养皿内,将培养皿放入37℃含5%CO2的培养箱内培养,换液的时间由细胞沉降速度情况而定。
细胞培养:
1)用75%酒精擦拭紫外线照射30min的超净工作台台面;
2)在超净工作台中按次序摆放好消过毒的离心管、吸管、培养瓶等等;
3)取出细胞培养瓶,无菌操作;
4)打开瓶盖,吸掉旧培养液;
5)用PBS洗涤细胞2次;
6)加入适量trypsin-EDTA溶液,轻洗细胞皿底部。放入37℃培养箱3min,于倒立显微镜下观察,当细胞将要分离而呈现圆粒状时,轻拍培养瓶壁使大部分细胞脱落,加入适量含血清之新鲜培养基终止trypsin作用;
7)以吸管上下吸放数次以打散细胞团块,混和均匀后,补足3n(n为传代瓶数)mL培养基,依稀释比例转移至新的培养瓶中;
8)将细胞悬液分装入培养皿内,将培养皿放入37℃含5%CO2的培养箱内培养,换液的时间由细胞生长速度情况而定。
细胞瞬时转染:
1)转染前一天,5×105个细胞接种在6孔板上,2mL完全培养基,转染前细胞汇合达到70-90%;
2)在100μL无血清培养基加入2μg质粒,柔和混匀;
3)混匀lipofectamine试剂,用100μL无血清培养基稀释5μLlipofectamine试剂,轻轻混匀,室温放置5min;
4)将稀释好的质粒和lipofectamine试剂混合,轻柔混匀,室温放置20min,以便形成质粒-lipofectamine复合物;
5)将200μL质粒-lipofectamine复合物加到含800μL无血清培养基的细胞孔中,来回轻柔摇晃细胞培养板;
6)细胞在37℃含5%CO2培养箱中,培养5h后吸去转染培养基,更换完全培养基;
7)转染36h后,荧光显微镜观察转染效率,拍照。
qPCR验证转染后表达效率:提取细胞样品总RNA,逆转录后进行qPCR检测,分别以内参、circRNA_BMPR2特异引物进行qPCR。同时设计测序引物,PCR后回收条带测序验证环化位点接头,实验步骤如下:
RNA提取步骤:
1)细胞加Trizol后,室温放置5min,使其充分裂解;
2)按200ul氯仿/mL Trizol加入氯仿,剧烈振荡15s,室温静置5min;
3)4℃、12,000×g离心15min;
4)吸取上层水相,移至另一离心管中;
5)按0.5mL异丙醇/mL Trizol加入异丙醇混匀,室温放置10min;
6)4℃、12,000×g离心10min,弃上清;
7)按1mL 75%乙醇/mL Trizol加入75%乙醇,温和振荡离心管,悬浮沉淀;
8)4℃、7,500×g离心5min,尽量弃上清;
9)室温晾干8min,加入20μL DEPC水溶解沉淀。
测OD值定量RNA:打开取样臂,用移液枪吸取1μL H2O滴加到检测平台上,调零;放下取样臂,用移液枪吸取1μL样品滴加到检测平台上,按“Measure”键;记录A260/280比值和RNA浓度;用干净的吸水纸擦去上下检测平台上的样品。
逆转录:
配制第一链cDNA合成反应液
按下列条件进行第一链cDNA合成反应
qPCR实验:
qPCR反应体系配制
qPCR反应程序设置
qPCR引物
GAPDH
GAPDH-F:AGAAGGCTGGGGCTCATTTG;
GAPDH-R:GCAGGAGGCATTGCTGATGAT;
扩增片段大小140bp
circRNA_BMPR2
circRNA_BMPR2-F1:CTGACACAACACCACTCACT;
circRNA_BMPR2-R1:GCCATAGCAGGTGCTACCTT;
circRNA_BMPR2的扩增片段大小146bp。
qPCR数据处理,根据2-△△Ct公式计算目标基因进行表达情况,RT-qPCR检测结果如表4和图7所示。
表4circRNA_BMPR2的相对表达差异
相比于对照组,Alu-circRNA_BMPR2组的过表达倍数为460,PCR产物测序峰图正常,无杂峰或叠带,序列与环化位点参考序列对比吻合。以上结果表明circRNA_BMPR2能在真核细胞中成功过表达目的circRNA。说明circRNA_BMPR2参与了PCOS的发生发展。
实施例3circRNA_BMPR2对人卵巢颗粒细胞的生物学作用
将KGN细胞接种到96孔板,每孔密度为4*103个细胞,转染后分别孵育0、24、48、72h和96h。接下来,向每孔中加入10μL细胞计数试剂盒-8(CCK-8)溶液(诺唯赞,南京,中国)4小时。最后,使用酶标仪(BioTek,VT,美国)在450nm处测量吸光度。
结果如图8所示,过表达circRNA_BMPR2促进了KGN细胞的增殖。
实施例4:细胞凋亡试验
细胞凋亡分析是通过使用Annexin V-FITC Apoptosis Detection Kit(Vazyme,南京,中国)和FACScan流式细胞仪(BD Biosciences,美国)进行的。简单地说,KGN细胞在转染后以1×105个细胞/ml的密度重新悬浮在结合缓冲液中。将5μl Annexin-V FITC和10μLPropidium Iodide(PI)加入500μl细胞悬液中,室温下避光孵育10min。随后,用冷的PBS清洗和悬浮染色的细胞,在1h内进行流式细胞仪检测。
结果如图9所示,过表达circRNA_BMPR2抑制了KGN细胞的凋亡。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
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1.一种circRNA_BMPR2诊断生物标志物在制备诊断多囊卵巢综合征的试剂中的应用,其特征在于,所述标志物的基因序列如序列表SEQ ID NO:1所示。
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Patent Citations (1)
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CN115011596A (zh) * | 2022-01-25 | 2022-09-06 | 江苏省苏北人民医院 | 一种环状circRNA_BECN1基因及其应用 |
Non-Patent Citations (5)
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Aberrant Expression of Long Non-coding RNAs in Exosomes in Follicle Fluid From PCOS Patients;Liping Wang 等;Frontiers in Genetics;第11卷;全文 * |
High throughput circRNAs sequencing profile of follicle fluid exosomes of polycystic ovary syndrome patients;Li‐ping Wang 等;J Cell Physiol.;第234卷(第9期);全文 * |
hsa_circ_06837;circRNADb;circRNADb;全文 * |
Identification of Three Potential circRNA Biomarkers of Polycystic Ovary Syndrome by Bioinformatics Analysis and Validation;Pengyu Huang 等;International Journal of General Medicine;第14卷;全文 * |
多囊卵巢综合征与正常卵泡液中环状RNA的差异表达分析;何笑 等;扬州大学学报(农业与生命科学版);第40卷(第2期);全文 * |
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