CN114752673B - 一种检测isyna1表达水平的试剂在制备卵巢癌干性鉴别试剂中的应用 - Google Patents

一种检测isyna1表达水平的试剂在制备卵巢癌干性鉴别试剂中的应用 Download PDF

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CN114752673B
CN114752673B CN202210469757.XA CN202210469757A CN114752673B CN 114752673 B CN114752673 B CN 114752673B CN 202210469757 A CN202210469757 A CN 202210469757A CN 114752673 B CN114752673 B CN 114752673B
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杨玲玲
郎廷元
周琦
杨牧垚
崔晨曦
邹冬玲
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Abstract

本发明公开了一种检测ISYNA1表达水平的试剂在制备卵巢癌干性鉴别试剂中的应用，所述试剂指能够检测ISYNA1mRNA水平的试剂和能够检测ISYNA1蛋白水平的试剂，本发明提供了一种新的卵巢癌干性评估方法，可应用于卵巢癌恶性程度和患者预后情况的评估。

Description

一种检测ISYNA1表达水平的试剂在制备卵巢癌干性鉴别试剂 中的应用
技术领域
本发明涉及医学分子诊断领域，具体涉及一种检测ISYNA1表达水平的试剂在制备卵巢癌干性鉴别试剂中的应用。
背景技术
卵巢癌(Ovarian cancer)是严重危害女性健康的生殖系统恶性肿瘤，全球每年新发29.54 万例，死亡18.48万例，是最致命的女性恶性肿瘤之一。由于卵巢癌发病部位隐匿，早期症状不典型，且缺乏有效的筛查和诊断方法，因此，75％的卵巢癌患者就诊时即为晚期，尽管接受规范的治疗方案，5年生存率依然不到40％。耐药是导致卵巢癌死亡率高的主要原因，而肿瘤干细胞是驱动卵巢癌耐药及预后不良的主要因素。目前，卵巢癌的诊断主要依靠临床和病理，尚不能判断卵巢癌的干性程度。因此，亟待需要标志物判断卵巢癌的干性程度，从而为卵巢癌患者治疗、预后提供更多信息。
ISYNA1为3-磷酸肌醇合酶1，为细胞内经葡萄糖途径调节肌醇代谢中的限速酶，它的主要作用为控制肌醇合成。但是目前并无关于ISYNA1表达水平与卵巢癌患者的生存、干性标志物CD44、CD133的表达呈负相关的报道。因此，研究ISYNA1表达水平与卵巢癌相关性对卵巢癌的诊断和治疗具有重要意义。
发明内容
有鉴于此，本发明的目的在于提供一种新的卵巢癌干性判断试剂，研究发现ISYNA1的表达水平与卵巢癌患者的生存成反比、与卵巢癌干性标志物CD44、CD133的表达呈负相关、其在卵巢癌细胞中的外源过表达抑制卵巢癌的成微球能力，利用此现象，将检测ISYNA1表达水平的试剂用于制备卵巢癌干性的鉴别试剂；为卵巢癌干性判断提供了新的试剂。
为达到上述目的，本发明提供如下技术方案：
一种检测ISYNA1表达水平的试剂在制备卵巢癌干性鉴别试剂中的应用，所述试剂指能够检测ISYNA1 mRNA水平的试剂和能够检测ISYNA1蛋白水平的试剂。
本发明优选的，所述检测ISYNA1 mRNA水平的试剂应用的方法包括探针法、RT-PCR法和测序法；更优选的，所述试剂为RT-PCR试剂；更优选的，RT-PCR的检测引物如SEQ IDNO.1 和SEQ ID NO.2所示。
本发明优选的，所述检测ISYNA1蛋白水平的试剂应用的方法包括免疫组化法、Western blot法和ELISA法。
本发明优选的，所述卵巢癌干性指卵巢癌组织的分离细胞或卵巢癌细胞系细胞的体外成微球能力。
本发明优选的，所述卵巢癌干性程度高应代表卵巢癌恶性程度高和患者预后情况差。
本发明优选的，所述干性程度高应代表卵巢癌组织、卵巢癌组织分离细胞或卵巢癌细胞系细胞中卵巢癌干性标志物表达水平高。
本发明优选的，所述卵巢癌干性标志物指CD44和CD133。
本发明的有益效果在于：本发明首次公开了检测ISYNA1表达水平的试剂可应用于卵巢癌干性鉴别试剂的制备，为判断卵巢癌干性提供了新方法。检测ISYNA1表达水平的方法可使用探针法、RT-PCR法、免疫组化法和Western blot法等。本发明试剂可实现卵巢癌组织样本、细胞系细胞、卵巢癌组织分离的原代细胞的干性的判断。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图进行说明：
图1为ISYNA1在卵巢癌病人组织中的表达与CD44和CD133成反比；
图2为ISYNA1低表达预示卵巢癌病人不良预后；
图3为ISYNA1在悬浮培养的卵巢癌干细胞中的表达低于其在贴壁培养的分化卵巢癌细胞中的表达；
图4为ISYNA1外源过表达抑制卵巢癌细胞的干性；
图5为ROC曲线分析ISYNA1表达水平用于判断体外培养卵巢癌细胞干性的准确性；
图6为ROC曲线分析ISYNA1表达水平用于判断患者卵巢癌组织干性的准确性；
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好的理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
本发明中Caov3卵巢癌干细胞和分化Caov3卵巢癌细胞参见：Jingshu Liu，Jiangfeng Qiu,Zhiqi Zhang,Lei Zhou,Yunzhe Li,Dongyan Ding,Yang Zhang,DonglingZou,Dong Wang,Qi Zhou,Tingyuan Lang，SOX4 maintains the stemness of cancercells via transcriptionally enhancing HDAC1 revealed by comparativeproteomics study.Cell Biosci.2021Jan 22。
实施例1、ISYNA1在卵巢癌病人组织中的表达与CD44和CD133成反比
利用TCGA数据下载卵巢癌病人基因表达测序数据以及生存信息，提取ISYNA1、CD44、 CD133的表达数据，利用Pearson相关性方法分析ISYNA1与CD44和CD133的mRNA表达相关性，Ρ＜0.05为具备统计意义。结果显示ISYNA1的表达与CD44、CD133呈负相关，具体如图1所示。该结果说明ISYNA1与卵巢癌的干性具有一定相关性。
实施例2、ISYNA1低表达预示卵巢癌病人不良预后
下载TCGA(The Cancer Genome Atlas)数据库卵巢癌基因表达数据和生存数据，将数据进行清洗和标准化等预处理后，根据ISYNA1表达水平将患者样本为高表达和低表达两组，利用Kaplan-meier分析比较两组患者的生存情况。结果显示，相对于ISYNA1高表达组，ISYNA1 低表达组的卵巢癌患者预后更差，生存时间更短说明ISYNA1低表达能够预示了卵巢癌患者的不良预后，如图2所示。
实施例3、ISYNA1在悬浮培养的卵巢癌干细胞中表达低于其在贴壁培养的分化卵巢癌细胞中表达
将Caov3卵巢癌干细胞培养于超低吸附培养皿，培养方式为悬浮培养，培养基为含10ng/ml FGF和20ng/ml EGF的DMEM/F-12培养基，培养14天。分化Caov3卵巢癌细胞培养于10mm培养皿，培养皿为含10％胎牛血清的DMEM培养基，培养方式为贴壁培养，培养至汇合度为80％-90％。细胞或微球培养结束后，分别收集mRNA，用qRT-PCR法检测 ISYNA1的mRNA表达水平。ISYNA1的检测引物为F：5’-gcagttccgctctaaggagg-3’(SEQ ID NO.1)，R：5’-gcagtggtcaggctcttcg-3’(SEQ ID NO.2)，内参GAPDH的检测引物序列为F: 5’-ctgggctacactgagcacc-3’(SEQ ID NO.3),R:5’-aagtggtcgttgagggcaatg-3’(SEQ IDNO.4)。结果显示Caov3卵巢癌干细胞的ISYNA1表达水平低于Caov3贴壁卵巢癌细胞ISYNA1mRNA 表达水平，经student’s t检验结果为具体结果见图3。
实施例4、ISYNA1外源过表达抑制卵巢癌细胞的干性
构建ISYNA1过表达Caov3卵巢癌细胞株以及相应的对照细胞株。ISYNA1过表达慢病毒载体通过将ISYNA1编码区克隆于pcSLenti-CMV-MCS-3xFLAG-PGK-Puro-WPRE的 MCS得到pcSLenti-CMV-ISYNA1-3xFLAG-PGK-Puro-WPRE载体。对照细胞株通过感染由 pcSLenti-CMV-MCS-3xFLAG-PGK-Puro-WPRE空载体包装的病毒获得。将ISYNA1过表达 Caov3卵巢癌干细胞和对照Caov3卵巢癌干细胞培养于超低吸附培养皿，培养方式为悬浮培养，培养基为含10ng/ml FGF和20ng/ml EGF的DMEM/F-12培养基。培养14天，在普通显微镜下观察微球形成情况，统计微球形成数目。结果显示ISYNA1过表达组的微球形成数目显著低于对照组，经student’s t检验结果为说明ISYNA1外源过表达抑制卵巢癌细胞的干性，具体如图4所示。
实施例5、ROC曲线分析ISYNA1表达水平用于判断体外培养卵巢癌细胞干性的准确性
为了评估ISYNA1作为标志物判断卵巢癌干性程度的效果，选择卵巢癌10个细胞系(Caov3、SKOV3、A2780、Ovcar4、ES2、Ovcar8、Caov4、Nihovcar3、Heya8、IGROV1)，分别将以上细胞进行常规培养和干细胞培养。干细胞培养于超低吸附培养皿，培养方式为悬浮培养，培养基为含10ng/ml FGF和20ng/ml EGF的DMEM/F-12培养基，培养14天。将卵巢癌细胞培养于10mm培养皿中，培养至汇合度为80％-90％。培养完成后，分别提取微球干细胞和常规培养细胞的mRNA，采用qRT-PCR测定ISYNA1 mRNA的表达水平。悬浮培养的微球样本为真阳性，贴壁培养的分化细胞为真阴性。根据检测结果绘制ROC曲线，如图5所示，曲线下面积的AUC值为0.772，说明以ISYNA1 mRNA水平判断体外卵巢癌细胞干性具有较高的准确度。ISYNA1mRNA表达水平作为检测标准的最佳阈值可以选择8.269、8.251 和8.228三个，选择该三个阈值时的检测灵敏度和特异性分别为：当ISYNA1 mRNA表达量＜8.269时，ISYNA1作为标志物判断卵巢癌干性的灵敏度为86.67％，特异度为66.67％，当 ISYNA1 mRNA表达量＜8.251时，ISYNA1作为标志物判断卵巢癌干性的灵敏度为80％，特异度为70％，当ISYNA1 mRNA表达量＜8.228时，ISYNA1作为标志物判断卵巢癌干性的灵敏度为76.67％，特异度为73.33％。
实施例6、ROC曲线分析ISYNA1表达水平用于判断患者卵巢癌组织干性的准确性
为了评估ISYNA1在TCGA数据库卵巢癌样本中判断卵巢癌干性程度的效果，下载TCGA 卵巢癌数据，整理出同时高表达干性标志物CD44和CD133的样本，将这些样本对应的ISYNA1表达水平也进行整理，绘制ROC曲线，结果如图6所示，曲线下面积的AUC值为0.6382，当ISYNA1的表达水平＜-0.07808时，ISYNA1用于判断卵巢癌干性程度的灵敏度为60％，特异度为61.43％，对于卵巢癌样本的干性程度判断具有一定的参考意义。
因此，ISYNA1可以作为卵巢癌干性程度的标志物，通过检测ISYNA1 mRNA水平和检测ISYNA1蛋白水平判断卵巢癌的干性程度，ISYNA1 mRNA水平和ISYNA1蛋白水平低表达，卵巢癌患者的不良预后，表明卵巢癌干性程度高。
最后说明的是，以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
序列表
<110> 重庆大学附属肿瘤医院
<120> 一种检测ISYNA1表达水平的试剂在制备卵巢癌干性鉴别试剂中的应用
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<170> SIPOSequenceListing 1.0
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<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ctgggctaca ctgagcacc 19
<210> 4
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
aagtggtcgt tgagggcaat g 21

Claims (8)

1.一种检测ISYNA1表达水平的试剂在制备卵巢癌干性鉴别试剂中的应用，其特征在于：所述试剂指能够检测ISYNA1 mRNA水平的试剂和能够检测ISYNA1蛋白水平的试剂。
2.根据权利要求1所述的应用，其特征在于：所述检测ISYNA1 mRNA水平的试剂应用的方法包括探针法、RT-PCR法和测序法。
3.根据权利要求2所述的应用，其特征在于：所述试剂应用的方法为RT-PCR法，所述RT-PCR法使用的检测引物如SEQ ID NO.1和SEQ ID NO.2所示。
4.根据权利要求1所述的应用，其特征在于：所述检测ISYNA1蛋白水平的试剂应用的方法包括免疫组化法、Western blot法和ELISA法。
5.根据权利要求1所述的应用，其特征在于：所述卵巢癌干性指卵巢癌组织的分离细胞或卵巢癌细胞系细胞的体外成微球能力。
6.根据权利要求1所述的应用，其特征在于：所述卵巢癌干性程度高应代表卵巢癌恶性程度高和患者预后情况差。
7.根据权利要求1所述的应用，其特征在于：所述干性程度高应代表卵巢癌组织、卵巢癌组织分离细胞或卵巢癌细胞系细胞中卵巢癌干性标志物表达水平高。
8.根据权利要求7所述的应用，其特征在于：所述卵巢癌干性标志物指CD44和CD133。
CN202210469757.XA 2022-04-30 2022-04-30 一种检测isyna1表达水平的试剂在制备卵巢癌干性鉴别试剂中的应用 Active CN114752673B (zh)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111781358A (zh) * 2020-07-13 2020-10-16 张瑜 Xab2蛋白作为卵巢癌预后和/或预测铂类耐药标志物
WO2021036620A1 (zh) * 2019-08-23 2021-03-04 伯克利南京医学研究有限责任公司 一组卵巢癌预后相关基因的应用
KR20210036676A (ko) * 2019-09-26 2021-04-05 서울대학교산학협력단 난소암의 예후 예측용 바이오마커 및 이의 용도

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JP6143920B1 (ja) * 2016-06-20 2017-06-07 国立研究開発法人国立がん研究センター 卵巣がんの予後診断マーカーとしてのｍｍｐ１遺伝子転写産物と検査法

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Publication number Priority date Publication date Assignee Title
WO2021036620A1 (zh) * 2019-08-23 2021-03-04 伯克利南京医学研究有限责任公司 一组卵巢癌预后相关基因的应用
KR20210036676A (ko) * 2019-09-26 2021-04-05 서울대학교산학협력단 난소암의 예후 예측용 바이오마커 및 이의 용도
CN111781358A (zh) * 2020-07-13 2020-10-16 张瑜 Xab2蛋白作为卵巢癌预后和/或预测铂类耐药标志物

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