CN108866195A - miR-3942-5p作为肿瘤标志物在制备无创型泛癌诊断及预后药物中的应用 - Google Patents
miR-3942-5p作为肿瘤标志物在制备无创型泛癌诊断及预后药物中的应用 Download PDFInfo
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Abstract
本发明通过qPCR检测miR‑3942‑5p结果显示，miR‑3942‑5p在结直肠癌、肝细胞癌、胃癌、宫颈癌及子宫内膜癌组织中较癌旁高表达，在癌症组血清中较健康及良性组均显著增高，且在转移组血清中的水平显著高于未转移组，提示miR‑3942‑5p可作为无创型泛癌诊断及预后肿瘤标志物；功能实验显示，miR‑3942‑5p可促进结肠癌细胞转移及侵袭。此外，本发明将为肿瘤筛查及预后提供新思路，并对阐明肿瘤恶性行为的分子机制具有重要意义。并且可以根据肿瘤标志物miR‑3942‑5p来开发无创型泛癌诊断及预后药物。
Description
技术领域
本发明属生物技术领域，涉及一种用于可作为无创型泛癌诊断及预后标志物的微小非编码RNA(MicroRNA,miRNA)。
背景技术
恶性肿瘤的发生发展是一个多因素、多步骤的生物学过程，以无限增生、浸润和转移为特征。恶性肿瘤中许多相关分子发生改变，包括经典的编码蛋白基因及最近发现的非编码RNA，非编码RNA主要包括小RNA(miRNA)和长链非编码RNA。MiRNA是一类长度约为19～25个核苷酸的非编码RNA片段，广泛存在于动植物中，它通过调节基因表达和蛋白翻译过程参与细胞增殖、分化及凋亡等生理过程。1993年，Lee等在线虫中发现第一个miRNA(lin-4)，此后大量miRNAs在动植及人类中均被发现。
MiRNA作为基因表达的内源性抑制物，主要通过与mRNA的3’非编码区(UTR)结合导致翻译抑制或mRNA降解。约30％的人类编码基因受到miRNA的调控，超过50％的miRNA基因位于肿瘤相关基因组区域(称为脆性位点)，提示miRNA在人类肿瘤发生发展过程中发挥重要的调控作用。研究已证实，miRNAs在不同的肿瘤微环境下可作为癌基因或抑癌基因参与肿瘤的发生发展。一般情况下，在肿瘤中发挥癌基因作用的miRNA，如miR-17～92簇，可通过负向调控细胞凋亡相关基因的表达，引起肿瘤细胞永生化；相反，在肿瘤中发挥抑癌作用的miRNA，通常为低表达，可通过负向调节细胞增殖及周期相关基因的表达，抑制细胞增殖，加速细胞凋亡。每个miRNA能调控约100个mRNAs，导致mRNA降解或翻译抑制，而同一个mRNA也会受到多个miRNAs调控，最终形成了一个全方面、多层次、立体的调控网络。近年研究证实，作为重要的调控型分子，miRNA在肿瘤发生发展中起调控的枢纽作用。因此，通过筛选肿瘤中差异表达的miRNAs，探讨其下游分子参与肿瘤发生发展、侵袭与转移的分子机理，确定miRNAs在肿瘤恶性行为中的作用，将有利于增进我们对其癌变分子机理的理解，并为寻找特异性靶分子及高效预后分子标志物提供了可能，为肿瘤筛查与生物治疗开辟一条新的途径，具有重要的经济效益和社会价值。
发明内容
本发明通过qPCR检测miR-3942-5p结果显示，miR-3942-5p在结直肠癌、肝细胞癌、胃癌、宫颈癌及子宫内膜癌组织中较癌旁高表达，在癌症组血清中较健康及良性组均显著增高，且在转移组血清中的水平显著高于未转移组，提示miR-3942-5p可作为无创型泛癌诊断及预后肿瘤标志物；功能实验显示，miR-3942-5p可促进结肠癌细胞转移及侵袭。此外，本发明将为肿瘤筛查及预后提供新思路，并对阐明肿瘤恶性行为的分子机制具有重要意义。并且可以根据肿瘤标志物miR-3942-5p来开发无创型泛癌诊断及预后药物。
附图说明
图1为miR-3942-5p在结直肠癌组织中高表达(NT：癌旁正常组织；CRC：结直肠癌；***P＜0.001)；
图2为miR-3942-5p在结直肠癌、肝细胞癌、胃癌、宫颈癌及子宫内膜癌组织中高表达(NT：癌旁正常组织；CRC：结直肠癌；HCC：肝细胞癌；GC：胃癌；CC：宫颈癌；EC：子宫内膜癌；**P＜0.01；***P＜0.001)；
图3为miR-3942-5p在结直肠癌、肝细胞癌、胃癌、宫颈癌及子宫内膜癌血清中高表达(HC：健康对照；BE：良性病变；CRC：结直肠癌；HCC：肝细胞癌；GC：胃癌；CC：宫颈癌；EC：子宫内膜癌；*P＜0.05；**P＜0.01)；
图4为miR-3942-5p在转移组结直肠癌血清中显著高表达(CRC-NM：非转移结直肠癌；CRC-M：转移结直肠癌；*P＜0.05)；
图5为miR-3942-5p影响结肠癌细胞的体外转移及侵袭能力(NC：阴性对照；In：抑制物；Mi：模拟物；*P＜0.05；*P＜0.05；**P＜0.01)；
图6为三个miRNA靶基因预测软件共同预测到292个靶基因；
图7为13个靶基因的3'-UTR存在miR-3942-5p“种子序列区”的潜在结合位点；
图8为SMAD7可能是miRNA3942-5p的直接靶基因。
具体实施方式
1、miR-3942-5p作为泛癌诊断标志物的鉴定
(1)前期qPCR结果提示miR-3942-5p可作为泛癌的血清标志物。本发明拟分别收集12种常见的恶性肿瘤，包括结直肠癌、肝细胞癌、胃癌、宫颈癌、子宫内膜癌、肺癌、食管癌、乳腺癌、膀胱癌、鼻咽癌癌、前列腺癌及卵巢癌的血清标本数量各120例，与癌症组患者年龄及性别匹配的各良性组患者血清(预计收集120例)及健康对照血清标本(预计收集120例)分别作为良性组及健康组(高血脂、糖尿病、复合癌症及合并其他自身免疫性疾病患者的标本均不纳入该研究)。
(2)提取总RNA，逆转录获得cDNA，设计特异性PCR引物，通过qPCR检测miR-3942-5p在癌症组、良性组和健康组间的表达水平差异，探究miR-3942-5p的预警功能；
(3)分析上述实验结果，计算miR-3942-5p在每一种癌症诊断中的特异性及灵敏度，探究其作为泛癌血清诊断标志物的潜能；
(4)分析肿瘤Ⅰ+Ⅱ分期血清中miR-3942-5p的表达水平，探究miR-3942-5p在肿瘤早期诊断中的作用。
2、miR-3942-5p作为泛癌转移及预后标志物的鉴定
(1)预实验结果提示miR-3942-5p可作为潜在的肿瘤转移及预后标志物。本发明拟收集不同临床等级的结直肠癌患者病例标本，提取总RNA，逆转录获得cDNA，设计特异性PCR引物，qPCR检测miR-3942-5p的表达水平；
(2)利用统计软件分析miR-3942-5p与临床患者病理分级、TNM分期及预后情况的相关性；
(3)通过对比不同病理分级及TNM分期间miR-3942-5p的表达差异，探究miR-3942-5p作为预后标志物的潜能。
3、miR-3942-5p体内及体外生物学功能的鉴定
(1)前期工作中，我们通过瞬时转染结合细胞功能学实验，初步证实miR-3942-5p可在体外促进结肠癌细胞的侵袭转移能力。本发明将通过慢病毒感染的方法构建miR-3942-5p的稳定细胞系SW480和LS174-T(过表达和沉默)；
(2)通过MTT实验分别比较SW480和LS174-T细胞miR-3942-5p过表达和沉默后增殖能力的改变；
(3)通过Transwell小室实验分别比较SW480和LS174-T细胞miR-3942-5p过表达和沉默后运动迁移能力的改变；
(4)通过Matrigel基质胶实验分别比较SW480和LS174-T细胞miR-3942-5p过表达和沉默后侵袭能力的改变；
(5)通过流式检测技术分别比较SW480和LS174-T细胞miR-3942-5p过表达和沉默后细胞周期分布的改变；
(6)通过Annexin-V/PI实验分别比较SW480和LS174-T细胞miR-3942-5p过表达和沉默后细胞凋亡水平的改变；
(7)通过顺铂及5-氟尿嘧啶耐药实验分别比较SW480和LS174-T细胞miR-3942-5p过表达和沉默后细胞耐药能力的改变；
(8)通过构建裸鼠皮下成瘤模型，分别比较LS174-T细胞miR-3942-5p过表达和沉默后肿瘤细胞体内成瘤能力的改变；
(9)通过构建尾静脉注射肺转移模型，分别比较LS174-T细胞miR-3942-5p过表达和沉默后肿瘤细胞体内转移能力的改变。
4、miR-3942-5p靶基因的挖掘及鉴定
前期工作中，我们利用miRWalk 2.0(http://zmf.umm.uni-heidelberg.de/apps/ zmf/mirwalk2/)、TargetScan(http://www.targetscan.org/vert_71/)及DIANA-microT(http://diana.imis.athena-innovation.gr/DianaTools/index.php？r＝mr microt/ index)三个在线软件共同预测到292个miR-3942-5p的靶基因，通路富集结果显示，靶基因主要集中在TGF-β/SMAD通路、VEGF信号通路、细胞粘附分子及Rho GTPase激活通路等。本发明拟结合生物信息学分析及分子生物学实验验证，进一步挖掘miR-3942-5p的靶基因。
(1)分别构建靶基因3’-UTR-WT(Wild Type)及结合位点序列突变的3’-UTR-MT(Mutant Type)荧光素酶报告载体，与miR-3942-5p共转染细胞，检测荧光素酶活性；
(2)提取稳定过表达及沉默表达细胞(SW480和LS174-T)总RNA，逆转录获得cDNA，设计特异性PCR引物，qPCR检测靶基因在不同处理后的表达水平变化；
(3)提取稳定过表达及沉默表达细胞(SW480和LS174-T)总蛋白SDS-PAGE电泳蛋白，稳压转膜至PVDF膜，4℃过夜孵育一抗，37℃1小时孵育二抗，ECL显色发光记录结果。
5、miR-3942-5p上游表达调控机制的鉴定
(1)利用在线软件PROMO(http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/ promoinit.cgi？dirDB＝TF_8.3)、JASPAR(http://jaspar.binf.ku.dk/)和TRANSFAC(http://gene-regulation.com/pub/databases.html)对miR-3942-5p的基因启动子区进行生物信息学分析，预测可能发挥调控作用的转录因子；
(2)根据预测到的转录因子及其识别序列，构建Luciferase报告基因系统；
(3)克隆含识别序列在内的启动子调控区，克隆至pGL3载体荧光素酶启动子的上游，构建质粒(含识别序列在内的调控区域+Promoter+Luciferase)；
(4)转染HEK193细胞，检测细胞荧光素酶的表达活性，以验证miR-3942-5p是否受转录因子的调控；
(5)检测各种肿瘤组织及对应癌旁标本中，miR-3942-5p的基因启动子区域甲基化水平，探究miR-3942-5p是否受到甲基化调控。
6、miR-3942-5p的作用结果
本发明前期在TCGA数据库下载27种肿瘤的miRNAseq数据。首先，根据癌旁组织样本数小于3例不予纳入的标准排除了10种肿瘤；然后，对剩余的17种肿瘤数据(结直肠癌，肾上腺癌，胆管癌，膀胱尿路上皮癌，脑胶质瘤，乳腺癌，宫颈癌，食管癌，头颈部鳞状细胞癌，肾癌，肝细胞癌，肺癌，胰腺癌，前列腺癌，胃腺癌，甲状腺癌，子宫癌)按照FDR＜0.05，FC＞2的标准分别进行差异miRNAs筛选[注FDR(False Discovery Rate)：错误发现率；FC(FoldChange)：差异倍数]；最后，选择在结直肠癌中特异性高表达且未见功能报道的4个miRNAs(miR-3912,miR-3942,miR-6733,miR-6755)进行深入研究。
由于低表达的miRNAs不适合做临床血清标志物，因此不做重点研究。MiRNA一般分为3p和5p，通过引物设计，排除GC含量不适宜者，最后选取miR-3912-3p,miR-3912-5p,miR-3942-5p,miR-6733-3p,miR-6755-5p，采用qPCR检测8例结肠癌组织及相应的癌旁组织中这些miRNAs的表达水平，发现miR-3942-5p与数据挖掘结果一致(图1)。然而采用不同的FC标准，所筛选出来的特异性表达的miRNA也会有所不同，经计算，如果按照FDR＜0.05，FC＞1.5的标准，TCGA数据挖掘结果显示miR-3942在四种肿瘤中均有特异性高表达(结直肠癌，乳腺癌，肝细胞癌，胃腺癌)，qPCR实验结果显示miR-3942-5p在肝细胞癌、胃癌、宫颈癌及子宫内膜癌组织中表达显著增高(图2)，两者结果基本吻合。
我们继续收集结直肠癌、肝细胞癌、胃癌、宫颈癌及子宫内膜癌血清标本各8例，检测发现，miR-3942-5p在癌症患者血清较健康及良性组表达水平均显著增高(图3)；且在转移组结直肠癌血清中的表达水平显著高于未转移组(P＜0.05)(图4)，上述结果提示miR-3942-5p具备作为无创型泛癌诊断及预后标志物的潜能。
为了初步探究miR-3942-5p的生物学功能，我们利用人工合成的miRNA模拟物(Mimics)及抑制物(Inhibitor)，通过瞬时转染结肠癌细胞系SW480和LS174-T，通用Transwell小室迁移实验及Matrigel基质胶实验发现，miR-3942-5p显著改变结肠癌细胞的体外转移及侵袭能力(图5)。
为了寻找miR-3942-5p发挥功能所涉及的下游靶基因，我们利用miRWalk 2.0(http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/)、TargetScan(http://www.targetscan.org/vert_71/)及DIANA-microT(http://diana.imis.athena-innovation.gr/DianaTools/index.php？r＝mrmicrot/index)三个在线软件共同预测到292个miR-3942-5p的靶基因(图6)。通路富集结果显示，靶基因主要集中在TGF-β/SMAD通路、VEGF信号通路、细胞粘附分子及Rho GTPase激活通路等(表1)。
表1 miR-3942-5p靶基因Pathway富集分析
生物信息学分析发现13个靶基因(SMAD7、NCAM2、NLGN1、RAN、ABI1、MKL2、CTNNA2、TMEM2、MPRIP、PRKCA、PIK3R1、RASAL2、RASA1)的3'-UTR存在miR-3942-5p“种子序列区”的潜在结合位点(图7)。我们进一步选择TGF-β/SMAD pathway的关键负反馈调节因子——SMAD7，对靶基因预测结果进行初步验证。Western blot实验证实，miR-3942-5p的表达水平与SMAD7呈负相关，而与TGF-β的表达水平正相关，提示SMAD7可能是miR-3942-5p的直接靶基因(图8)。
Claims (2)
1.miR-3942-5p在制备无创型泛癌诊断及预后的肿瘤标志物中的应用。
2.miR-3942-5p作为肿瘤标志物在制备无创型泛癌诊断及预后药物中的应用。
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