CN116042838A - 与肝细胞癌抗嘧啶类药物副作用相关的miRNA标志物及其靶基因和应用 - Google Patents
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Abstract
本发明公开了一种与肝细胞癌抗嘧啶类药物副作用相关的miRNA标志物及其靶基因和应用。所述标志物为miR‑223‑3p。通过下调miR‑223‑3p的表达量，可以减轻5‑氟尿嘧啶带来的副作用。与正常肝细胞相比，其靶基因FBXW7低表达时，肝细胞形态、数目等发生变化，为肝癌早期的快速诊断提供依据。同时为后期的抗嘧啶类药物副作用的靶点治疗以及从分子水平诊断原发性肝癌和开发减低癌症药物副作用药物提供了新的思路。
Description
技术领域
本发明属于基因工程和肿瘤学领域，具体涉及一种与肝细胞癌抗嘧啶类药物副作用相关的miRNA生物标志物及其靶基因和应用。
背景技术
原发性肝癌（Hepatocellular cancer，HCC）是最常见的肝细胞癌，约占全球肝癌总负担的80%。由于肝癌早期的藏匿性，导致大部分患者错过了最佳的治疗时间，因此开发一种高敏感性的肝癌诊断方法，达到早发现、早诊断、早治疗的目的，延长肝癌患者的生命是亟待解决的问题。
目前，癌症的治疗药物主要是传统的细胞毒性药物。虽然，随着肿瘤研究的不断深入，使得抗癌药物更加的高选择，高效，低毒，但是仍然具有明显的副作用。5-氟尿嘧啶（5-fluorouracil，5-FU）作为最常用的细胞毒性药物之一有着包括胃肠道，血细胞生成系统，骨髓毒性和通过其在消化道中的磷酸化给药引起的心脏毒性的副作用。其中，嘧啶类药物诱发的炎症反应尤为明显。因此，研发更有效的抑制肿瘤和减小药物副作用的新抗癌药物对保证抗癌药物的预后和癌症患者的生活质量有着重要意义。
石莼作为叶绿藻门的大型藻类，因其有着众多成分和活性物质而越来越受到重视。目前为止，石莼多糖（
Ulva Lactucapolysaccharide，ULP）已被公认为具有多种潜在的生物活性，如抗氧化、抗高脂血症和抗糖尿病。近年来，有研究表明，石莼多糖能够通过多种机制发挥抗肿瘤和起到免疫调节的作用。
MicroRNA（miRNA）是高度保守的长约22nt的非编码RNA，广泛存在于各种生物中。这些微小RNA能够与mRNA结合阻断相应基因的表达，能够调控胚胎发育、细胞分化、器官生成等重要过程，与包括肝癌在内的多种人类疾病有关。近年来研究发现，miRNA在肿瘤早期阶段表现出异常，可以用于肿瘤高危人群的预警或者肿瘤早期检测。将异常的miRNA作为生物标志物，为肝癌的发展进行检测和后续的靶点治疗打下良好的基础。
发明内容
为了解决现有技术的不足，本发明提供了一种与肝细胞癌抗嘧啶类药物副作用相关的miRNA标志物及其靶基因和应用。
为实现上述目的，本发明采用如下技术方案：
一种与肝细胞癌抗嘧啶类药物化疗副作用相关的miRNA标志物，所述miRNA标志物为miR-223-3p，在NCBI中的Gene ID为723814。
miR-223-3p在降低抗肝癌药物毒副作用中的应用，所述抗肝癌药物为5-氟尿嘧啶。
进一步的，通过降低miR-223-3p的表达量来降低抗肝癌药物毒副作用。
miR-223-3p在筛选可降低5-氟尿嘧啶毒副作用的药物中的应用。
一种用于筛选可降低5-氟尿嘧啶毒副作用的药物的试剂盒，包括检测miR-223-3p表达量的引物，所述的引物为核苷酸序列如序列表中SEQ ID NO.1所示的DNA片段。
miR-223-3p的靶基因在制备肝癌诊断或肝癌辅助诊断的产品中的应用，所述靶基因为
FBXW7，在NCBI中的Gene ID为55294。
附图说明
图1：ULP对H22荷瘤小鼠脾脏指数的影响。
图2：ULP对H22荷瘤小鼠胸腺指数的影响。
图3：miR-223-3p在5Fu和5Fu+ULP组的表达量。
图4：差异miRNA所预测靶基因KEGG富集分析。
图5：miR-223-3p与其靶基因
FBXW7结合位点。
图6：
FBXW7介导的肝脏切片情况。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到.
实施例1 H22肝癌荷瘤小鼠模型的构建
用RPMI 1640培养基稀释肝癌H22细胞为106cells/mL，取0.2 mL腹腔注射到小鼠的左前腋窝中以构建H22肝癌荷瘤小鼠模型。然后将建模成功的小鼠随机分组。Normal组不使用任何药物；5Fu组小鼠腹腔注射单剂量20 mg/kg/d的5-氟尿嘧啶；5Fu+ULP组小鼠腹腔注射单剂量20 mg/kg/d的5-氟尿嘧啶，并口服300mg/kg/d石莼多糖；ULP组小鼠口服300mg/kg/d石莼多糖。经过连续21天给药后，使用颈椎脱位法处死小鼠，测定胸腺指数、脾脏指数，并收集小鼠的肿瘤组织。
作为免疫抑制剂，5-FU可降低免疫器官（脾脏和胸腺）指数。本申请中，如图1和图2所示，5Fu+ULP组的免疫器官指数高于5-FU组，但低于Normal组；其中，5Fu+ULP组的胸腺指数显著高于5Fu组。以上结果表明，ULP可以降低5-FU药物的副作用，本申请ULP降低5-FU药物副作用模型成立，可用于5-FU药物副作用生物标志物进一步探索。
实施例2差异表达miRNA的筛选
连续21天给药后，使用颈椎脱位法处死小鼠，在5Fu组和5Fu+ULP组中各取3只小鼠的肿瘤组织，剪取称重约50-100 mg组织，提取样本总RNA和miRNA。以总RNA为起始样品，使用small RNA Sample Pre Kit试剂盒（Illumina，美国）构建文库，首先在small RNA 5'端和3'端连接接头，反转录合成cDNA，PCR扩增后利用PAGE胶电泳筛选目的片段，切胶回收得到的片段即为small RNA文库。构建small RNA文库后，使用Qubit2.0进行初步定量，再使用Agilent 2100对文库的插入大小进行检测，后使用qPCR对文库的有效浓度进行定量，最后检查合格后再进行高通量测序。
对测序数据质控以确保信息分析的可靠。删除以下数据：低质量的reads（sQ≤20的碱基数占整个片段的30%以上的reads）；无法确定的碱基信息且比例大于10%的reads；polyA/T/G/C；没有3’接头序列的reads；插入片段和有5’接头污染的reads。修剪3’接头序列。
利用bowtie对sRNA进行筛选，在参考序列上将其长度进行定位并分析小RNA的分布情况。在miRBase数据库指定的范围序列中，将mapped比对到参考序列上的reads，以获取sRNA在各样品匹配上的详细情况。小鼠ncRNA序列来注释所测得的sRNA。接下来去除其中的重复序列tRNA、rRNA、snRNA、snoRNA。最后将mRNA的外显子和内含子和进行比对以找出由mRNA降解片段得来的sRNA。每个样品的Q30均大于96.0%，说明测序结果可靠，可进行后续分析。
使用R语言中的DESeq2筛选5Fu组和5Fu+ULP组中显著差异的miRNA，以log2FoldChange＜0的为下调miRNA，反之为上调miRNA。筛选过程中要符合Pvalue<0.05。分析测序结果后，共筛选出104个差异表达的miRNA。5Fu+ULP组中有40个miRNA相对于5Fu组是显著上调的，有64个miRNA为显著下调。这些差异表达的miRNA可能受石莼多糖影响，在缓解5-氟尿嘧啶药物副作用有重要的影响。
实施例3差异表达miRNA的RT-qPCR验证
对实施例2中差异表达的miRNA进行RT-qPCR验证。
采用Poly（A）加尾反应和cDNA合成反应同步进行的方法，使用生工miRNA第一链cDNA合成试剂盒（货号：#B532451）将提取到的miRNA逆转录为cDNA作为RT-qPCR的模板。使用生工的miRNA荧光定量PCR试剂盒（货号：#B532461）进行RT-qPCR。PCR反应体系包括：10μL2×miRNA qPCR master mix、0.5μL Forward Prime，0.5μL Universal PCR Primer R，1-2μL cDNA模板、1μL ROX Reference Dye(L)/(H)，加入RNase-free water定容至20µL；除Forward Prime和cDNA模板外，反应体系中的其他试剂均由试剂盒配套提供。PCR反应条件为：95℃ 30秒、95℃ 5秒、60℃ 30秒，以上阶段进行40个循环。最终得到10个符合测序结果的miRNA；其中，miR-223-3p在5Fu+ULP组中表达极显著（
p<0.0001）低于5Fu组（图3），miR-223-3p在NCBI中的Gene ID为723814。miR-223-3p的Forward Primer序列为5’-CGTGTCAGTTTGTCAAATACCCCA-3’，Universal PCR Primer R由生工试剂盒配套提供。
实施例4差异表达miRNA的靶基因预测
利用miRWalk、TargetScan、miRDB预测miR-223-3p的靶基因，利用DAVID网站对筛选到的靶基因进行功能富集分析，以获得KEGG通路富集分析和GO基因富集。如图4所示，miR-223-3p的靶基因富集到FoxO信号通路、Hippo信号通路和mTOR信号通路。其中，与癌症相关的靶基因为
FBXW7，其在NCBI中的Gene ID为55294，miR-223-3p与
FBXW7结合位点如图5所示。
通过The Human Protein atlas数据库挖掘与
FBXW7有关的肝癌组织和正常肝组织中的组织切片图。如图6所示，
FBXW7低表达时肝癌组织细胞肿胀，气球样变，细胞球形，胞质几乎透明；而正常肝组织肝细胞核大而圆，双核细胞较多。
Claims (6)
1.一种与肝细胞癌抗嘧啶类药物副作用相关的miRNA标志物，其特征在于：所述miRNA标志物为miR-223-3p，其在NCBI中的Gene ID为723814。
2.权利要求1所述的miRNA标志物在降低抗肝癌药物毒副作用中的应用，其特征在于：所述抗肝癌药物为5-氟尿嘧啶。
3.根据权利要求2所述的应用，其特征在于：通过降低miR-223-3p的表达量来降低抗肝癌药物毒副作用。
4.miR-223-3p在筛选可降低5-氟尿嘧啶毒副作用的药物中的应用。
5.一种用于筛选可降低5-氟尿嘧啶毒副作用的药物的试剂盒，其特征在于：包括检测miR-223-3p表达量的引物，所述的引物为核苷酸序列如序列表中SEQ ID NO.1所示的DNA片段。
6.miR-223-3p的靶基因在制备肝癌诊断或肝癌辅助诊断的产品中的应用，其特征在于：所述靶基因为FBXW7，在NCBI中的Gene ID为55294。
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| CN110117652A (zh) * | 2018-02-05 | 2019-08-13 | 箐健科技(天津)有限公司 | 肝癌早期诊断方法 |
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| US20130064810A1 (en) * | 2010-02-26 | 2013-03-14 | Hans-Guido Wendel | Methods and Compositions for the Detection and Treatment of Cancer involving miRNAs and miRNA Inhibitors and Targets |
| CN110117652A (zh) * | 2018-02-05 | 2019-08-13 | 箐健科技(天津)有限公司 | 肝癌早期诊断方法 |
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