CN116751866B - 中耳胆脂瘤的标志物及其应用 - Google Patents
中耳胆脂瘤的标志物及其应用 Download PDFInfo
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Abstract
本发明公开了中耳胆脂瘤的标志物及其应用,具体的,所述的标志物为circRNA‑000425或miRNA‑17。本发明提供了一种诊断中耳胆脂瘤的产品。本发明还提供了一种治疗中耳胆脂瘤的药物组合物。
Description
技术领域
本发明属于生物医药领域,具体涉及中耳胆脂瘤的标志物及其应用。
背景技术
中耳胆脂瘤是一种在颞骨内角化过度的鳞状上皮异常堆积并渐进性对周围骨质破坏的炎性疾病,并非真性肿瘤,是耳鼻喉头颈外科的常见疾病。胆脂瘤最常发生的部分是中耳和乳突,或者两者兼而有之,极少数发生在外耳道。胆脂瘤虽属于一种良性病变,但是具有类似肿瘤的恶性表现,主要体现在其异常增殖和侵袭能力对临近组织和器官的破坏,如未及时治疗干预,则会逐渐侵袭周围中耳听力结构以及造成颅骨破坏,易造成听力受损、前庭功能障碍、面瘫、甚至继发颅内感染引起严重颅内并发症,危及生命。目前,手术是主要的治疗方式,但术后的复发率高,患者往往面临多次手术,面对心理、经济双重负担。因此,对于中耳胆脂瘤的非手术治疗方式以及治疗新靶点的研究显得尤为有意义。
发明内容
基于现有技术的不足,本发明的第一目的是提供一种诊断中耳胆脂瘤的产品;本发明的第二目的是提供一种治疗中耳胆脂瘤的药物组合物。
为实现上述目的,本发明采用了如下技术方案:
第一方面,本发明提供了检测样本中生物标志物的表达水平的试剂在制备诊断中耳胆脂瘤的产品中的应用,所述的生物标志物为circRNA-000425或miRNA-17。
在本发明所使用的术语“样本”、“样品”是指获得自或衍生自受试者的组合物,其包含有待根据例如物理,生化,化学和/或生理特点来表征和/或鉴定的细胞和/或其它分子实体。例如,短语“疾病样品”或其变体指得受试者的任何样品,预计或已知其包含待表征的细胞和/或分子实体。样品包括但不限于,组织样品(例如肿瘤组织样品),原代或培养的细胞或细胞系,细胞上清,细胞裂解物,血小板,血清,血浆,玻璃体液,淋巴液,滑液,精液,羊水,乳,全血,血液衍生的细胞,尿液,脑脊髓液,唾液,痰,泪,汗液,粘液,肿瘤裂解物,和组织培养液,组织提取物如匀浆化的组织,肿瘤组织,细胞提取物,及其组合。在本发明的具体实施例中,所述的样本为组织。在一些实施方案中,样本由医学专业人员在分离的实体的指导下从受试者获取,然后提供给所述实体如测试实验室。在一些实施方案中,样本由受试者或受试者的照料者在家中采集,并且提供给从样本中获取生物标志物数据的一方。
进一步,所述的试剂包括采用测序技术、探针杂交技术、基因芯片技术或荧光定量PCR技术检测样本中生物标志物的表达水平的试剂。
进一步,所述的试剂选自:
特异性识别circRNA-000425或miRNA-17基因的寡核苷酸探针;或
特异性扩增circRNA-000425或miRNA-17基因的引物。
进一步,所述的产品包括芯片、试剂盒或核酸膜条。
进一步,所述芯片为基因芯片,所述基因芯片包括用于检测circRNA-000425或miRNA-17基因转录水平的针对circRNA-000425或miRNA-17基因的寡核苷酸探针;所述试剂盒包括基因检测试剂盒,所述基因检测试剂盒包括用于检测circRNA-000425或miRNA-17基因转录水平的试剂、或芯片。
进一步,所述试剂盒包括通过RT-PCR法、qRT-PCR法、生物芯片检测法、DNA印迹法、RNA印迹法检测circRNA-000425或miRNA-17表达水平的试剂。
第二方面,本发明提供了一种治疗中耳胆脂瘤的药物组合物,所述的药物组合物包含circRNA-000425的抑制剂。
本发明所述的药物组合物可以制备成注射剂或口服制剂,包括注射剂、片剂、胶囊剂、丸剂、栓剂、气雾剂、口服液体制剂、颗粒剂、散剂、缓释剂、纳米制剂、糖浆剂、酒剂、酊剂、露剂。所述药物组合物一般制成注射剂,还可以开发成口服制剂,从而提高患者的服药依从性。
本发明所述的药物组合物可采用本领域技术人员熟知的常规制备方法进行制备,如将各有效成分混匀,或者根据各种剂型的常规制备方法,将药效成分与相应辅料混配后制备获得。本发明所述的药物组合物还可与其他能够用于治疗中耳胆脂瘤的治疗剂一起使用。
进一步,所述的抑制剂包括shRNA、小干扰RNA、dsRNA、微小RNA、反义核酸及其构建物。
进一步,所述的药物组合物还包含药学上可接受的载体。
在本发明中,“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和变态反应)的,即具有合理的效益/风险比的物质。
在本发明中,药学上可接受的载体包括但不限于软化水或蒸馏水;盐水溶液;植物油类,如花生油、红花油、橄榄油、棉籽油、玉米油,麻油类,如花生油、红花油、橄榄油、棉籽油、玉米油、麻油、花生油或椰子油;硅油类,包括聚硅氧烷类,如甲基聚硅氧烷、苯基聚硅氧烷和甲基苯基聚硅氧烷;挥发性硅酮类;矿物油类,如液体石蜡、软石蜡或角鲨烷;纤维素衍生物,如甲基纤维素、乙基纤维素、羧甲基纤维素、羧甲基纤维素钠或羟丙基甲基纤维素;低级烷醇类,例如乙醇或异丙醇;低级芳烷醇类;低级聚烷撑二醇类或低级烷撑二醇类,例如聚乙二醇、聚丙二醇、乙二醇、丙二醇、1,3-丁二醇或甘油;脂肪酸酯类,如棕榈酸异丙酯、肉豆蔻酸异丙酯或油酸乙酯;聚乙烯吡咯烷酮;琼脂;黄耆树胶或阿拉伯胶和凡士林。
第三方面,本发明提供了本发明第二方面所述的药物组合物在制备治疗中耳胆脂瘤的药物中的应用。
第四方面,本发明提供了一种促进成纤维细胞增殖的方法,所述的方法包括在成纤维细胞的培养体系中添加circrRNA-000425的促进剂。
进一步,所述的促进剂包括含有circrRNA-000425的核酸序列。
第五方面,本发明提供了一种调控成纤维细胞中circRNA-000425、Tnfsf11或RANKL的表达水平的方法,所述的方法包括将角化上皮细胞来源的外泌体与成纤维细胞共孵育。
第六方面,本发明提供了如下任一项应用:
(1)circRNA-000425在促进破骨细胞前体细胞活化中的应用;
(2)circRNA-000425在抑制成纤维细胞中miRNA-17的表达中的应用;
(3)circRNA-000425在促进成纤维细胞增殖中的应用。
本发明的有益效果:
本发明首次公开了circRNA-000425、miRNA-17与中耳胆脂瘤的相关性,为中耳胆脂瘤的诊断和治疗提供了新方法。
附图说明
图1是原代细胞的培养与鉴定的结果图,其中,图1中A是光镜下观察不同时间的原代胆脂瘤、正常耳后皮肤相关成纤维细胞的结果图,图1中B是光镜下观察不同时间的原代胆脂瘤、正常耳后皮肤相关角化上皮细胞的结果图,图1中C是免疫化学染色利用特异性Vmention、Cytokeratin蛋白鉴定原代胆脂瘤成纤维细胞以及角化上皮细胞的结果图,图1中D是流式细胞术检测Cytokeratin抗体标记的原代胆脂瘤角化上皮细胞的结果图,图1中E是流式细胞术检测Vmention抗体标记的原代胆脂瘤成纤维细胞的结果图;
图2是检测circRNA-000425、miRNA-17、Tnfsf11、RANKL在胆脂瘤组织、原代胆脂瘤成纤维中的表达情况的结果图,其中,图2中A是qPCR检测胆脂瘤中circRNA-00425的结果图,图2中B是qPCR检测胆脂瘤中Tnfsf11的结果图,图2中C是qPCR检测胆脂瘤中miRNA-17的结果图,图2中D是胆脂瘤中RANKL蛋白免疫印迹图,图2中E是Western blotting检测胆脂瘤中RANKL的表达量图,图2中F是qPCR检测原代成纤维细胞circRNA-00425的表达量的结果图,图2中G是qPCR检测原代成纤维细胞Tnfsf11的表达量的结果图,图2中H是qPCR检测原代成纤维细胞miRNA-17的表达量的结果图,图2中I是原代成纤维细胞中RANKL蛋白免疫印迹图,图2中J是Western blotting检测原代成纤维细胞RANKL的表达量的结果图,图2中K是Elisa检测RANKL的分泌量的结果图;
图3是检测circRNA-000425的亚细胞定位的结果图,其中,图3中A是荧光原位杂交技术标记circRNA-000425的结果图,图3中B是circRNA-000425核质分离实验的结果图;
图4是角化上皮细胞外泌体的分离与鉴定的结果图,其中,图4中A是透射电镜图,图4中B是通过Western blotting验证外泌体标志蛋白CD9、TSG101、Calnexin的结果图,图4中C是外泌体大小信息图,图4中D是外泌体粒径大小图;
图5是证明角化上皮细胞通过分泌外泌体的方式来促进成纤维细胞RANKL的表达的结果图,其中,图5中A是荧光显微镜下观察外泌体被摄取的结果图,图5中B是PCR检测外泌体中circRNA-000425的相对表达量的结果图,图5中C是PCR检测成纤维与不同处理组别共孵育后miRNA-17的相对表达量的结果图,图5中D是PCR检测成纤维与不同处理组别共孵育后circRNA-000425的相对表达量的结果图,图5中E是PCR检测对不同处理组别的成纤维细胞中Tnfsf11表达量的结果图,图5中F是RANKL的免疫印迹图,图5中G是Westernblotting检测不同处理组别的RANKL的结果图;
图6是证明circRNA-000425促进破骨细胞前体细胞的分化的结果图,其中,图6中A是PCR检测干扰、过表达质粒的效率的结果图,图6中B是PCR检测c-FOS的相对表达量的结果图,图6中C是PCR检测CtsK的相对表达量的结果图,图6中D是PCR检测MMP-9的相对表达量的结果图,图6中E是PCR检测NFATc1的相对表达量的结果图,图6中F是过表达circRNA-000425后MMP-9、NFATc1、CtsK、c-FOS的免疫印迹图,图6中G是过表达circRNA-000425后CtsK的表达量的结果图,图6中H是过表达circRNA-000425后c-FOS的表达量的结果图,图6中I是过表达circRNA-000425后MMP-9的表达量的结果图,图6中J是过表达circRNA-000425后NFATc1的表达量的结果图;
图7是证明circRNA-000425能够与miRNA-17相结合的结果图,其中,图7中A是双荧光素酶实验检测circRNA-000425与miRNA-17结合情况的结果图,图7中B是荧光原位杂交技术检测circRNA-000425、miRNA-17的结果图;
图8是证明circRNA-000425竞争性结合miRNA-17,促进RANKL表达,促进细胞增殖的结果图,其中,图8中A是qPCR检测转染miRNA-17 mimic、miRNA-17 inhibitor后Tnfsf11的相对表达量的结果图,图8中B是RANKL的免疫印迹图,图8中C是RANKL的相对表达量的结果图,图8中D是qPCR检测过表达、干扰circRNA-000425后miRNA-17的相对表达量的结果图,图8中E是qPCR检测过表达、干扰circRNA-000425后Tnfsf11的相对表达量的结果图,图8中F是过表达、干扰circRNA-000425的RANKL的免疫印迹图,图8中G是Western blotting检测过表达、干扰circRNA-000425检测RANKL的表达量的结果图,图8中H是CK8检测过表达、干扰circRNA-000425后细胞的增殖情况的结果图。
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解,现结合具体实施例对本发明的技术方案进行以下详细说明,应理解这些实例仅用于说明本发明而不用于限制本发明的范围。实施例中,各原始试剂材料均可商购获得,未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件,或按照仪器制造商所建议的条件。
实施例1 circRNA-000425/miRNA-17/RANKL中耳胆脂瘤中的表达与诱导破骨细胞分化和活化的功能的机制研究
一、实验材料
1.实验试剂与耗材
角质形成细胞培养基(KM)购自美国Sciencell公司,TRNzol Universal 总 RNA提取试剂(DP424)购自天根生化科技(北京)有限公司,MiRNA 1st Strand cDNA SynthesisKit(MR101-02)购自南京诺唯赞生物科技有限公司,Human Receptor Activator OfNuclear Factor Kappa B Ligand(RANkL)ELISA Kit购自武汉基因美,CCK-8试剂盒(MA0218-5)购自大连美仑生物技术有限公司,干扰、过表达RNA质粒购自上海和元生物技术股份有限公司。
2.实验样本
2.1 组织标本
于2021年3月到2022年10月,收集在行乳突根治术治疗后切除的胆脂瘤组织以及耳后切口处正常皮肤组织标本15对。一份放入4度RNAstore样本保存液中,并尽快转至液氮中保存用于后续实验分析。一份组织浸泡在预冷的PBS缓冲液中,尽快运输至实验室,用于后续原代细胞的培养。所有临床标本的取材和使用均征得患者及其家属的知情同意。本研究获得山东第一医科大学附属省立医院伦理委员会批准。
2.2 细胞标本:BMMs细胞株来自中心实验室赠予。
二、实验方法
1.免疫荧光(Immunofluorescence,IF)
对分离的胆脂瘤成纤维细胞和角化上皮细胞检测其对应标志物。检测成纤维细胞标志物Vimentin、角化上皮细胞标志物角蛋白。
2.实时荧光定量PCR(Quantitative Real-time PCR,qPCR)
2.1 总RNA的提取
2.2 琼脂糖凝胶电泳
2.3 cDNA的制备
其中,实验方法2.1、2.2、2.3均为本领域的常规方法。
2.3.1 miRNA逆转录
按照MiRNA 1st Strand cDNA Synthesis Kit(MR101-02)说明书进行反转录。
2.3.2 除miRNA外的其他RNA逆转录
参照FastQuant cDNA第一链合成试剂盒(去基因组)(KR116)的说明书(进行逆转录。
2.4 引物设计
以目的基因序列为依据设计特异性扩增引物,所涉及的引物是由北京鼎国昌盛生物科技有限责任公司设计引物序列并合成引物,引物序列如下:
表1 引物序列
2.5 实时荧光定量PCR
(1)制备反应体系
(2)配制体系,混匀混合溶液后,使用实时荧光定量PCR仪进行PCR。按照95℃,15min;95℃,10 s;60℃,30 s的顺序设定程序,共进行40次循环。每个样品至少做3个重复。
(3)数据分析采用2-ΔΔCT法。
3.荧光原位杂交(Fluorescence in situ hybridization,FISH)
(1)消化细胞后,将细胞接种含有细胞爬片的24孔板内,将细胞培养至融合60-70%。
(2)细胞爬片固定:向细胞爬片滴加4%多聚甲醛固定液,固定细胞15-20 min。固定完成后,使用PBS缓冲液,振荡漂洗,3次,每次5 min。
(3)消化:将20 μg/mL的蛋白酶K的工作液滴入,进行3-5 min的消化。纯水冲洗后PBS洗3次,每次5 min。
(4)预杂交:每孔加入适量预杂交液,37℃恒温箱孵育1小时。
(5)杂交:吸弃预杂交液,滴加含有探针hsa-circ-0000425+hsa-mir-17-5p的杂交液,浓度为500nM,40℃杂交过夜。
(6)洗涤:吸弃杂交液,2×SSC洗脱液,37℃漂洗10 min,利用甲酰胺洗涤,洗去非特异杂交体。
(7)DAPI复染细胞核:向爬片滴加适量DAPI染液复染细胞核,避光孵育8min,漂洗后滴加抗荧光淬灭封片剂封片。
(8)镜检拍照:将细胞爬片置于尼康正置荧光显微镜下观察并采集图像。
(9)探针序列:
hsa-circ-0000425:5’-CATGAGTACCTTTGTTGCGATTCTC-3’(SEQ ID NO.12)
4.数据统计以及软件分析
数据分析均采用Graphpad Prism9.0版本,本研究所有实验均重复三次及以上,使用Graphpad Prism9.0版本进行图表制作。组间数据比较采用t检验或者方差分析,数据均采用平均值±标准差表示,P<0.05 时认为具有统计学意义。
三、实验结果
1.胆脂瘤相关成纤维细胞、角化上皮细胞的培养与鉴定
本发明通过组织块法处理正常组织以及中耳胆脂瘤组织分别培养出相关成纤维细胞和角化上皮细胞。如图1中A所示,光镜下观察原代胆脂瘤成纤维细胞呈现细长的梭形,胞体有长短不一的突起,增殖活跃,细胞融合生长后排列紧凑,放射状走形。如图1中B所示,原代胆脂瘤相关角化上皮细胞单层紧密排布,多角形,呈现“铺石路样”、鹅卵石样排列。为了进一步鉴别胆脂瘤相关原代细胞相关表型,免疫荧光方法用来原代细胞的进一步鉴定。如图1中C所示,角化上皮细胞特异标志物Cytokeratin在分离出的原代胆脂瘤相关角化上皮细胞胞质中表达,成纤维细胞特异性标志物Vimentin在原代胆脂瘤成纤维细胞胞质中表达。如图1中D和E所示,而后通过流式细胞术再次验证分离出的胆脂瘤相关原代细胞是原代胆脂瘤成纤维细胞以及原代胆脂瘤相关角化上皮细胞。
2.circRNA-000425在中耳胆脂瘤中高表达
对收集的15对中耳胆脂瘤以及正常外耳道组织,通过qPCR分别检测组织中的circRNA-000425、miRNA-17以及Tnfsf11(编码RANKL)的表达情况。结果如图2中A-C所示,相较于正常组中,胆脂瘤组组织中circRNA-000425、Tnfsf11表达含量明显增加,miRNA-17的表达量明显降低。如图2中D和E所示,Western Blotting结果显示RANKL在胆脂瘤组织表达增加。P<0.05,均具有统计学差异。
接着检测原代胆脂瘤成纤维细胞中的circRNA-000425、miRNA-17、Tnfsf11,结果如图2中F-K所示,circRNA-000425、Tnfsf11的表达量增加,而miRNA-17与两者相反。P<0.05,均具有统计学差异。
3.circRNA-000425的亚细胞定位
circRNA的生物学功能与其亚细胞定位有密切联系,为了更深入的探究circRNA-000425在胆脂瘤中的生物学作用,本发明通过荧光原位杂交技术,证明circRNA-000425主要在胞浆分布,如图3中A所示。接着,如图3中B所示通过核质分离实验原代角化上皮细胞胞质以及胞浆的RNA,以GAPDH作为细胞浆定位标志物,以U6作为细胞核定位标志物,结果也验证了circRNA-000425主要分布在细胞质中。亚细胞定位说明circRNA-000425具有竞争性内源RNA的潜能,可能通过作为miRNA海绵在细胞中发挥生物学效应。
4.角化上皮细胞来源的外泌体的分离与鉴定
为了进一步探究胆脂瘤中过度增殖的角化上皮细胞分泌的外泌体对成纤维细胞的生物学作用,本发明收集原代胆脂瘤角化上皮细胞培养上清液,通过超速离心的方法分离出外泌体,接着对其鉴定是否为外泌体。首先,使用透射电镜对外泌体形态进行观察,记录100nmnm、200nm、500nm的图像如图4中A所示。其次利用纳米颗粒分析技术对提取出的外泌体进行粒径分析,结果如图4中C和D所示,分析显示角化上皮细胞来源的外泌体直径在30-150nm之间,平均直径为82.3nm。然后对外泌体进行总蛋白提取,提取的蛋白用于检测外泌体的特异性标记蛋白阳性蛋白CD9、TSG101和阴性蛋白Calnexin。如图4中B所示,上清液中Calnexin蛋白呈现阴性表达,CD9、TSG101蛋白呈现阳性表达,证明成功从角化上皮细胞细胞培养上清液中成功提取出外泌体。
5.角化上皮细胞通过分泌外泌体的方式来促进成纤维细胞RANKL的表达
为了验证角化上皮细胞来源的外泌体可被转移至胆脂瘤成纤维细胞中,本发明使用PKH26(外泌体标记染料)对外泌体进行染色。将染色后的外泌体与成纤维细胞体外共培养24小时,24小时后在荧光显微镜下观察,如图5中A所示,镜下可以观察到成纤维细胞发出信号,表明被PKH26标记的外泌体被转移至成纤维细胞中。如图5中B所示,利用qPCR检测角化上皮来源的外泌体中circRNA-000425,发现circRNA-000425在外泌体中富集,提示其可以被角化上皮细胞通过外泌体的方式被分泌发挥生物学功能。接下来,对成纤维细胞进行不同处理。分别将成纤维细胞与缓冲液组、角化上皮细胞来源的外泌体组、角化上皮细胞组、角化上皮细胞+GW4869(外泌体抑制剂)组进行共孵育。如图5中C和D所示,通过qPCR检测成纤维细胞circRNA-000425和miRNA-17的表达。与对照组相比,外泌体组、角化上皮细胞组的circRNA-000425的相对表达明显增加,而加入外泌体抑制剂GW4869组则表达量明显降低(P<0.05)。miRNA-17的相对表达量显著增加的是角化上皮细胞+GW4869组,外泌体组、角化上皮细胞组则有不同程度的降低(P<0.05)。然后,将不同处理组与成纤维细胞孵育48h,qPCR检测成纤维细胞中Tnfsf11的表达变化。如图5中E中折线图所示,Tnfsf11的表达水平最高的是孵育36小时后,角化上皮细胞组以及角化上皮细胞外泌体组两组趋势明显,而抑制剂组则与对照组一样表达水平没有明显时间差异。其次如图5中F和G所示,WesternBlotting检测RANKL与qPCR结果相同(P<0.05)。以上结果显示,角化上皮细胞可以通过外泌体的方式影响成纤维细胞中miRNA-17/Tnfsf11级联途径,调控RANKL的分泌。
6.circRNA-000425促进破骨细胞相关标志物表达量增加
首先,本发明验证了构建的circRNA-000425过表达以及沉默载体的效率,如图6中A所示,与各自对照组比较,OV-circRNA-000425过表达有效(P<0.05),sh-2、sh-3干扰效率高,任意选择sh-3进行后续实验。
本发明使用ov-circ对正常原代角化上皮细胞进行不同处理,而后进行正常成纤维细胞、BMMs细胞(破骨细胞前体细胞)共培养,探究circRNA-000425与破骨细胞前体细胞分化的关系。分为:ov-control+成纤维细胞+BMMs组、ov-circ+成纤维细胞+BMMs组。Western Blotting、qPCR检测NFATc1 、c-FOS、CtsK和MMP-9。Western Blotting结果如图6中F-J所示,各组表达量增高的均为过表达circRNA-000425组(P<0.05)。如图6中B-E所示,qPCR结果也相同。这说明circRNA-000425可以促进BMMs的活化,从而促进胆脂瘤的骨质破坏。
实施例2 角化上皮细胞来源Ker-Exo中circRNA-000425作为miRNA海绵在转录后水平调控RANKL表达的细胞内信号
一、实验材料
荧光素酶活性检测试剂盒、CCK8试剂盒购自MedChemExpress公司,干扰、过表达RNA质粒购自上海和元生物技术股份有限公司。
二、实验方法
1.荧光原位杂交(Fluorescence in situ hybridization,FISH)
(1)消化细胞后,将细胞接种含有细胞爬片的24孔板内,将细胞培养至融合60-70%。
(2)细胞爬片固定:向细胞爬片滴加4%多聚甲醛固定液,固定细胞15-20min。固定完成后,使用PBS缓冲液,振荡漂洗,3次,每次5min。
(3)消化:滴加蛋白酶K(20 μg/mL)工作液消化3-5 min。纯水冲洗后PBS洗3次,每次5 min。
(4)预杂交:每孔加入适量预杂交液,37℃恒温箱孵育1小时。
(5)杂交:吸弃预杂交液,滴加含有探针hsa-circ-0000425+hsa-mir-17-5p的杂交液,浓度为500 nM,40℃杂交过夜。
(6)洗涤:吸弃杂交液,2×SSC洗脱液,37℃漂洗10min,若非特异杂交体较多,可以增加甲酰胺洗涤
(7)DAPI复染细胞核:向爬片滴加适量DAPI染液复染细胞核,避光孵育8min,漂洗后滴加抗荧光淬灭封片剂封片。
(8)镜检拍照:将细胞爬片置于尼康正置荧光显微镜下观察并采集图像。
(9)探针序列:
hsa-mir-17-5p:5’-CTACCTGCACTGTAAGCACTTTG-3’(SEQ ID NO.13)
2.实时荧光定量PCR
2.1 总RNA的提取
2.2 琼脂糖凝胶电泳
2.3 cDNA的制备
其中,实验方法2.1、2.2、2.3均为本领域的常规方法。
2.3.1 miRNA逆转录
按照MiRNA 1st Strand cDNA Synthesis Kit(MR101-02)说明书进行反转录。
2.3.2 除miRNA外的其他RNA逆转录
按照FastQuant cDNA第一链合成试剂盒(去基因组)(KR116)说明书进行逆转录。
2.4 引物设计
以目的基因序列为依据设计特异性扩增引物,由北京鼎国昌盛生物科技有限责任公司设计引物序列并合成引物,引物序列如下:
表2 引物序列
3.数据统计以及软件分析
数据分析均采用Graphpad Prism9.0版本,本研究所有实验均重复三次及以上,使用Graphpad Prism9.0版本进行图表制作。组间数据比较采用t检验或者方差分析,数据均采用平均值±标准差表示,P<0.05 时认为具有统计学意义。
三、实验结果
1.circRNA-000425能够与miRNA-17相结合
通过生物信息学网站circBANK(http://www.circbank.cn/),预测miRNA-17与circRNA-000425可能存在结合位点。而前期实验,也提示circRNA-000425可能会与miRNA-17结合进而发挥其生物学效能。本发明通过构建circRNA-000425的突变序列以及野生序列,分别与miRNA-17 mimics、以及 miRNA mimics共转染正常原代成纤维细胞。结果如7中A所示,circRNA-000425 MUT与miRNA-17 mimic共转染组的表达量显著高于circRNA-000425WT与miRNA-17 mimic共转染组,说明circRNA-000425与miRNA-17存在结合位点,二者可以结合。如图7中B所示,利用荧光原位杂交技术双染circRNA-000425、miRNA-17,检测二者在细胞中的定位,探究circRNA-000425其作为miRNA海绵的潜能。实验结果如图所示,circRNA-000425与miRNA-17存在细胞质共定位。
2.circRNA-000425竞争性结合miRNA-17调控下游靶基因表达
为了探究circRNA-000425是否具有竞争性内源RNA,吸附miRNA的作用。首先,利用miRNA-17 mimic、miRNA-17 inhibitor本发明通过qPCR检测miRNA-17对Tnfsf11的调控,WB检测RANKL的表达,结果如图8中A-C所示,证明验证了miRNA-17对于Tnfsf11的负向调控,抑制RANKL的分泌。本发明对正常原代成纤维细胞进行了不同处理。分组情况:control组、ov-control组、ov-circRNA000425组、sh-NC组、sh-circRNA000425组。转染后,检测miRNA-17、Tnfsf11的相对表达量以及RANKL的表达情况。实验结果如图8中D和E所示,过表达circRNA组,Tnfsf11的表达量相比于增加显著。与空白对照组相比,sh-circ组的miRNA-17的表达量增加,WB实验结果如8中F和G所示,与Tnfsf11相同。以上结果,表明circRNA-000425可以负向调控miRNA-17的表达,发挥海绵作用,调节下游靶基因的表达被逆转。
3.circRNA-000425竞争性抑制miRNA-17促进成纤维细胞增殖
为了验证circNA-000425对细胞增殖的影响,本发明利用CCK8实验,对成纤维细胞进行不同处理,转染分组如下:control组、ov-control +miRNA-17 mimic组、ov-circRNA000425组、miRNA-17 mimics组、ov-circ+miRNA-17 mimic组。在450nm初,测定吸光值,吸光值与细胞活性呈现正相关。结果如图8中H所示:与对照组相比,过表达circrRNA-000425组,细胞增殖能力最强,其次为共转染组,miRNA-17 mimic组与control组差异不大。表明circRNA-000425促进成纤维细胞增殖,加速疾病进展。
为了对本发明的技术特征、目的和有益效果有更加清楚的理解,现结合具体实施例对本发明的技术方案进行以下详细说明,应理解这些实例仅用于说明本发明而不用于限制本发明的范围。实施例中,各原始试剂材料均可商购获得,未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件,或按照仪器制造商所建议的条件。
Claims (4)
1.检测样本中生物标志物的表达水平的试剂在制备诊断中耳胆脂瘤的产品中的应用,其特征在于,所述的生物标志物为circRNA-000425。
2.根据权利要求1所述的应用,其特征在于,所述的样本为组织。
3.根据权利要求1所述的应用,其特征在于,所述的试剂包括采用测序技术、探针杂交技术、基因芯片技术或荧光定量PCR技术检测样本中生物标志物的表达水平的试剂。
4.根据权利要求1所述的应用,其特征在于,所述的产品包括芯片、试剂盒或核酸膜条。
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