CN114958856B - 长链非编码rna cyp1b1-as1作为乳腺癌生物标志物和治疗靶点的应用 - Google Patents
长链非编码rna cyp1b1-as1作为乳腺癌生物标志物和治疗靶点的应用 Download PDFInfo
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- CN114958856B CN114958856B CN202210733639.5A CN202210733639A CN114958856B CN 114958856 B CN114958856 B CN 114958856B CN 202210733639 A CN202210733639 A CN 202210733639A CN 114958856 B CN114958856 B CN 114958856B
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- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
本发明公开了长链非编码RNA CYP1B1‑AS1,其核苷酸序列如SEQ ID NO.1所示。本发明还公开了CYP1B1‑AS1慢病毒过表达载体、慢病毒稳定表达细胞株及其在制备乳腺癌诊断试剂盒、预防或治疗乳腺癌药物或预后产品中的应用。CYP1B1‑AS1在乳腺癌中显著下调表达,可作为乳腺癌的诊断标志物。CYP1B1‑AS1的表达水平与患者的预后相关,可作为乳腺癌的预后标志物。上调CYP1B1‑AS1可抑制乳腺癌细胞的恶性进展,具有作为基因治疗靶点的应用价值。上调CYP1B1‑AS1的表达可提高乳腺癌细胞对化疗药物的敏感性。本发明能够用于乳腺癌相关的诊断、治疗、提高靶向药物敏感性等产品的制备中,具有一定的临床转化前景。
Description
技术领域
本发明属于肿瘤分子生物学技术领域,涉及长链非编码RNA CYP1B1-AS1作为乳腺癌诊断、预后生物标志物及治疗靶点的应用。
背景技术
乳腺癌作为全球发病率第一的恶性肿瘤,是女性癌症死亡的主要原因。我国乳腺癌的发病率不断升高,由于缺乏及时有效的诊断及治疗,常常导致患者最终治疗失败。因此,深入探索乳腺癌的发展机制,寻找新的诊疗靶标,挖掘其应用价值,开发其临床转化潜能刻不容缓。
近年来的研究成果表明,长度超过200nt的长链非编码RNA(long noncoding RNA,lncRNA)可以与DNA、RNA或蛋白质相互作用,参与染色体修饰、转录、核内运输、翻译及翻译后修饰等重要调控,作用于多种细胞信号传导途径,参与肿瘤进展的全过程。LncRNA表达失调是人类癌症转录组的特征性变化之一,具备成为肿瘤生物标志物及治疗靶分子的潜能。
本发明依托单位为江苏省肿瘤医院,申请人承担了多项肿瘤相关的国家级和省级自然科学基金项目,累积了丰富的肿瘤相关分子标志物的研究经验。近年来,基因工程技术在肿瘤研究中迅速发展,基因诊断和分子靶向治疗在多种恶性肿瘤的临床中显示出巨大的优势。因此,深入了解乳腺癌的发病机制,明确相关分子标志物在疾病发展中的作用,可为乳腺癌个体化精准诊疗奠定基础。
发明内容
发明目的:本发明所要解决的技术问题是提供了有利于乳腺癌诊疗的长链非编码RNA CYP1B1-AS1,揭示了lncRNA CYP1B1-AS1在解决乳腺癌临床现状中的应用潜能。
本发明还要解决的技术问题是提供了CYP1B1-AS1慢病毒过表达载体、慢病毒稳定表达细胞株在制备乳腺癌诊断试剂盒、预防或治疗乳腺癌药物或预后产品中的应用。
本发明还要解决的技术问题是提供了一种荧光定量PCR检测试剂盒。
技术方案:为了解决上述技术问题,本发明提供了长链非编码RNA CYP1B1-AS1,其核苷酸序列如SEQ ID NO.1所示。
CYP1B1-AS1基因全称为:Cytochrome P450 family 1 subfamily B member 1-antisense RNA 1,位于2号染色体正义链,长度为1776nt并带有polyA尾。
Seq ID NO.1
>NR_027252.1Homo sapiens CYP1B1 antisense RNA 1(CYP1B1-AS1),longnon-coding RNA
本发明还包括CYP1B1-AS1在乳腺癌细胞中发挥的生物学功能,揭示其作为分子治疗靶点的应用价值。
本发明内容还包括CYP1B1-AS1慢病毒过表达载体,所述CYP1B1-AS1慢病毒过表达载体含有所述的长链非编码RNA CYP1B1-AS1。
本发明内容还包括慢病毒稳定表达细胞株,所述慢病毒稳定表达细胞株是将所述的CYP1B1-AS1慢病毒过表达载体包装成病毒颗粒感染宿主细胞获得。
其中,所述宿主细胞为MCF7或MDA-MB-231细胞。
本发明通过慢病毒过表达载体上调乳腺癌细胞株MCF7和MDA-MB-231中CYP1B1-AS1的表达,并通过细胞增殖实验、克隆形成实验、流式细胞周期检测等,证实上调CYP1B1-AS1抑制乳腺癌细胞的恶性进展。
本发明内容还包括所述的长链非编码RNA CYP1B1-AS1在作为乳腺癌诊断标志物中的应用。
本发明内容还包括所述的长链非编码RNA CYP1B1-AS1、所述的CYP1B1-AS1慢病毒过表达载体、所述的慢病毒稳定表达细胞株在制备乳腺癌诊断试剂盒、预防或治疗乳腺癌药物或预后产品中的应用。
本发明内容还包括一种荧光定量PCR检测试剂盒,所述荧光定量PCR检测试剂盒包括特异性引物对,其引物对序列如SEQ ID NO.2和SEQ ID NO.3。
其中,所述检测试剂盒还包括逆转录试剂和实时荧光定量PCR试剂,实时荧光定量PCR检测乳腺癌组织中CYP1B1-AS1表达量的制剂。
其中,所述检测试剂盒还包括内参GAPDH引物对,所述内参GAPDH引物对序列如SEQID NO.4和SEQ ID NO.5所示。
用于实时荧光定量PCR的CYP1B1-AS1检测引物为:
CYP1B1-AS1:
SEQ ID NO.2:Forward Primer(5’to3’)
SEQ ID NO.3:Forward Primer(5’to3’)
内参GAPDH:
SEQ ID NO.4:Forward Primer(5’to3’)
SEQ ID NO.5:Forward Primer(5’to3’)
本发明内容还包括所述的长链非编码RNA CYP1B1-AS1、所述的CYP1B1-AS1慢病毒过表达载体、所述的慢病毒稳定表达细胞株在制备提高乳腺癌细胞对化疗的敏感性产品中的应用。
本发明的CYP1B1-AS1慢病毒过表达载体为LV6-Homo CYP1B1-AS1重组质粒,是上海吉玛制药技术有限公司构建得到。
用于构建CYP1B1-AS1慢病毒过表达载体的引物加上酶切位点NotI和NsiI的序列如下:
SEQ ID NO.6:Forward Primer(5’to3’)
SEQ ID NO.7:Forward Primer(5’to3’)
本发明还包括将慢病毒重组载体LV6-Homo CYP1B1-AS1进行包装后感染细胞获得稳定表达的细胞株。
本发明利用慢病毒过表达载体上调CYP1B1-AS1,有利于提高乳腺癌细胞对常用化疗药物顺铂的敏感性。
其中,所述应用通过加入不同浓度的顺铂处理乳腺癌细胞,然后通过加入CCK-8测定CYP1B1-AS1对癌细胞化疗药物敏感性的影响。
其中,所述顺铂的浓度为25μg/mL,12.5μg/mL,6.25μg/mL,3.13μg/mL,1.56μg/mL,0.78μg/mL。
有益效果:相对于现有技术,本发明具备以下优点:
1、本发明首次发现CYP1B1-AS1在乳腺癌中显著下调表达,可作为乳腺癌的诊断标志物。
2、本发明首次揭示了CYP1B1-AS1的表达水平与患者的预后相关,可作为乳腺癌的预后标志物。
3、本发明首次明确上调CYP1B1-AS1可抑制乳腺癌细胞的恶性进展,具有作为基因治疗靶点的应用价值。
4、本发明首次证实上调CYP1B1-AS1的表达可提高乳腺癌细胞对化疗药物的敏感性。
综上,本发明能够用于乳腺癌相关的诊断、治疗、提高靶向药物敏感性等产品的制备中,具有一定的临床转化前景。
附图说明
图1乳腺癌组织lncRNA芯片检测结果的热图分析。注:T,肿瘤组织,N,癌旁正常组织。
图2TCGA数据分析CYP1B1-AS1在乳腺癌中的表达。
图3NCBI数据库展示CYP1B1-AS1的结构特征。
图4LNCipedia数据库分析CYP1B1-AS1编码能力和保守性。
图5结合临床乳腺癌组织样本评价CYP1B1-AS1检测的应用价值。
图6分析CYP1B1-AS1表达水平与乳腺癌患者预后的关系。
图7CYP1B1-AS1的生物学功能研究。A、qPCR检测在乳腺癌细胞中上调表达CYP1B1-AS1的效果。B、CCK-8法检测上调CYP1B1-AS1对乳腺癌细胞增殖能力的影响。C、克隆形成实验检测上调CYP1B1-AS1对乳腺癌单细胞克隆形成能力的影响。D、流式细胞分析上调CYP1B1-AS1对乳腺癌细胞周期的影响。注:上调CYP1B1-AS1表达的乳腺癌细胞及其阴性对照分别标记为MCF7-exp,MCF7-NC和MDA-231-exp,MDA-231-NC。
图8过表达CYP1B1-AS1对乳腺癌细胞药物敏感性的影响。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1 lncRNA芯片检测的在乳腺癌组织中差异表达的lncRNA-CYP1B1-AS1
1.实验材料与方法
经伦理委员会批准,从生物样本库中选取6例乳腺癌组织和癌旁正常组织,使用Arraystar人类lncRNA芯片检测组织中lncRNA和mRNA的表达谱,由上海康成生物公司完成。该芯片能够检测40,173条lncRNA和20,730个蛋白质编码转录本。通过GeneSpring GXv12.1软件分析,并经Quantile标准化,两组样品间具有统计学意义的差异表达lncRNAs或差异表达mRNAs通过Fold change≥2.0,且P<0.05筛选。
2.实验结果
本实施例中以癌旁正常组织为对照,Fold change≥2,P<0.05为筛选指标,发现CYP1B1-AS1在乳腺癌组织中显著下调,且GEPIA2分析癌症基因组图谱(the Cancer GenomeAtlas,TCGA)数据表明:CYP1B1-AS1在乳腺癌中下调表达(图1,2)。CYP1B1-AS1是一条反义lncRNA,有polyA尾,位于2号染色体正义链,长度为1776nt并带有polyA尾。CYP1B1-AS1不具备编码蛋白质的能力(图3,4)。
实施例2CYP1B1-AS1检测在制备乳腺癌诊断、预后产品中的应用
1.实验材料与方法
1.1乳腺癌组织样本收集
经伦理委员会批准,收集30例原发性乳腺癌及癌旁正常组织样本,立即液氮保存直至RNA提取。
1.2RNA提取、逆转录及qPCR反应
使用Trizol试剂(Invitrogen)提取细胞中的总RNA,单次提取RNA的细胞总数不少于106个,用OD260/280检测RNA纯度,当OD260/280在1.9-2.1之间时,可用于后续检测。
使用PrimeScript RT试剂盒(TaKaRa)将用总量为1μg RNA合成cDNA,并保存于-20℃备用。CYP1B1-AS1的引物如下:上游引物:5′-ACTGGTATGACCACCCGAGA-3′;下游引物:5′-ACTGGCATACTGGTCACTGC-3′,Tm=60℃。内参GAPDH的引物如下,上游引物:5′-ATGGGTGTGAACCATGAGAA-3′;下游引物:5′-GTGCTAAGCAGTTGGTGGTG-3′,Tm=60℃。引物交由上海生工公司合成。
使用美国Roche公司的LightCycler PCR仪进行实时荧光定量PCR检测并进行熔解曲线分析,使用Takara公司的TBPremix Ex TaqTM II试剂盒进行PCR反应,使用两步法扩增40个循环并收集荧光信号,采用仪器自带软件分析扩增曲线和熔解曲线,计算平均循环阈值(cycle threshold,Ct),用2-ΔΔCt法计算获得相对基因的表达量。
具体操作及反应体系如下:
1.2.1RNA提取
①组织样品按50-100mg/mL加入Trizol。用电动匀浆器充分匀浆约需1-2min,细胞完全裂解后,转移至离心管中。
②在装有裂解物的离心管中加入0.2mL的氯仿(1mL Trizol加入0.2mL氯仿),振荡器上充分振荡混匀20s,室温放置5min。
③12000rpm 4℃离心10min,然后吸取含总RNA的上层水相至一新的离心管中,每毫升Trizol约可吸取0.5mL上层水相。应避免吸取有机相和中间层的DNA和蛋白质。
④加入和上层水相等体积的异丙醇,颠倒数次混匀,室温沉淀5min。12000rpm 4℃离心15min。
⑤弃上清,按每毫升Trizol加入1mL 75%乙醇,轻轻颠倒混匀,以清洗RNA沉淀两遍。12000rpm 4℃离心2min,弃去液体,室温倒置晾干。
⑥加入适量DEPC处理水使RNA沉淀溶解。存放于-80℃。
1.2.2逆转录:
①去除基因组DNA
反应条件:42℃,2min,4℃,保存。
②逆转录反应:
将上述反应产物加入以下试剂:
反应条件为37℃,15min;85℃,5s;4℃,保存。
1.2.3PCR扩增:
将上述逆转录cDNA吸取2μL加入以下反应体系:
1.3Kaplan-Meier Plotter分析CYP1B1-AS1表达与乳腺癌患者预后的关系。
1.4数据处理与分析
Wilcoxon符号秩检验用于比较乳腺癌组织和癌旁正常组织样本。使用GraphPadPrism 8.0软件做统计分析。P值小于0.05被认为具有统计学意义(*,P<0.05,**P<0.01,***P<0.001)。
2.实验结果
本实施例中使用RT-PCR检测30例乳腺癌组织和癌旁正常组织中的CYP1B1-AS1,发现癌组织中CYP1B1-AS1显著低于癌旁正常组织,且受试者工作特征曲线(receiveroperating characteristic curve,ROC)显示曲线下面积(area under the ROC curve,AUC)为0.909,[95%置信区间:0.839-0.979,P<0.001],提示CYP1B1-AS1检测具有较高的诊断价值(图5)。
另外,Kaplan-Meier Plotter分析表明:CYP1B1-AS1表达高的乳腺癌患者,其无复发生存率(RFS)和总生存率(OS)都更高,显示出其作为乳腺癌临床预后因子的应用价值(图6)。
实施例3CYP1B1-AS1在乳腺癌细胞中的生物学功能
1.实验材料与方法
1.1细胞培养
乳腺癌细胞株MCF7和MDA-MB-231购自中国科学院上海细胞库,根据ATCC方案培养:MCF7细胞使含有10%胎牛血清的RPMI 1640培养,置于37℃含有5%的CO2培养箱中。MDA-MB-231细胞使用含有10%FBS的L-15培养,置于37℃的空气培养箱中。293T细胞购自中国科学院上海细胞库,使用含有10%FBS的DMEM培养。
1.2慢病毒稳定表达细胞株的建立
利用慢病毒过表达载体(LV6,上海吉玛制药技术有限公司)建立CYP1B1-AS1的稳定表达细胞株:扩增目的基因HomoCYP1B1-AS1的引物加上酶切位点NotI和NsiI的序列如下:上游引物:5′-AGGGTTCCAAGCTTAAGCGGCCGCCAACTATGGTTGCTAACACAAGAATCGGCA-3′,下游引物:5′-GATCCATCCCTAGGTAGATGCATTTTTTTTTTTTTTTTTTTTTTTTTTATTAAACTG-3′,Tm=55℃;将目的基因Homo CYP1B1-AS1克隆到慢病毒过表达载体(LV6,上海吉玛制药技术有限公司)得到LV6-Homo CYP1B1-AS1重组质粒(H2470-2),分别利用脂质体Lipofectamine 3000(Invitrogen)将慢病毒过表达载体LV6-Homo CYP1B1-AS1重组质粒和慢病毒过表达载体LV6(阴性对照)转染293T细胞进行病毒包装,72h后收集细胞培养上清中的病毒液,并分别感染MCF7和MDA-MB-231细胞,48h后加入嘌呤霉素10μg/mL进行筛选,得到稳定表达CYP1B1-AS1的MCF7和MDA-MB-231细胞及其阴性对照(分别标记为MCF7-exp,MCF7-NC和MDA-231-exp,MDA-231-NC)。慢病毒表达载体的构建和包装交由上海吉玛制药技术有限公司完成。使用实施例2中的RT-PCR方法检测过表达CYP1B1-AS1的效果,结果参见图7A。
1.3细胞增殖实验
使用CCK-8检测MCF7-exp,MCF7-NC和MDA-231-exp,MDA-231-NC细胞的增殖能力。将处理好的待测细胞铺在96孔培养板中(每孔3×103个),分别在0h、24h,48h,72h和96h,更换含10%的CCK-8的培养液孵育2h,在450nm波长处测量吸光度。CCK-8购自日本同仁化学研究所。
1.4克隆形成实验
将MCF7-exp,MCF7-NC和MDA-231-exp,MDA-231-NC细胞以300、500、800个细胞的梯度密度分别接种含2mL RPMI 1640(MCF7)或L-15(MDA-MB-231)培养液的六孔板中,并轻轻转动,使细胞分散均匀。置细胞培养箱中培养1-2周。当培养皿中出现肉眼可见的克隆时,弃去上清液,PBS浸洗2次。加4%多聚甲醛固定细胞5mL固定15min。去固定液,加适量结晶紫染色液染20min,然后用流水缓慢洗去染色液,干燥后显微镜下观察细胞克隆形成情况。
1.5细胞周期检测
使用碘化丙啶染色法进行细胞周期分析:将MCF7-exp,MCF7-NC和MDA-231-exp,MDA-231-NC细胞用胰蛋白酶消化,PBS清洗,在-20℃用70%乙醇固定。离心后,将细胞在含有50μg/mL碘化丙啶,0.1%柠檬酸钠,0.1%TritonX-100和20μg/mL的低渗溶液中,37℃温育30min。然后使用流式细胞仪以线性模式分析细胞。
1.6数据处理与分析
t检验和方差分析(analysis of variance,ANOVA)用于组间比较。使用GraphPadPrism 8.0软件做统计分析。P值小于0.05被认为具有统计学意义(*,P<0.05,**P<0.01)。
2.实验结果
本实施例中的慢病毒过表达载体可稳定上调乳腺癌细胞MCF7和MDA-MB-231中CYP1B1-AS1的表达。细胞增殖实验、克隆形成实验和流式细胞周期检测结果表明,上调CYP1B1-AS1的表达能有效抑制细胞的增殖(图7)。
实施例5CYP1B1-AS1在制备提高乳腺癌细胞对化疗的敏感性产品中的应用
1.实验材料与方法
1.1药物敏感性实验
将MCF7-exp,MCF7-NC和MDA-231-exp,MDA-231-NC细胞以每孔5×103个接种于96孔板中,孵育24h后,将顺铂(溶解在生理盐水中)以不同浓度(25μg/mL,12.5μg/mL,6.25μg/mL,3.13μg/mL,1.56μg/mL,0.78μg/mL)添加到细胞中。加入等体积的溶剂作为药物浓度为0的对照组。处理48h后,将培养液更换为100μL含有10%CCK-8的反应液(分别用RPMI 1640或L-15培养液中配置10%CCK-8,现用现配),孵育3h后在波长450nm处测量吸光度值,绘制剂量-反应曲线。
1.2数据处理与分析
ANOVA用于组间比较,使用GraphPad Prism8软件进行统计分析。P值小于0.05被认为具有统计学意义(*,P<0.05,**P<0.01)。
2.实验结果
本实施例我们用不同浓度的顺铂处理乳腺癌细胞,加入CCK-8测定细胞活性,结果表明上调CYP1B1-AS1表达有利于提高乳腺癌细胞对化疗药物的敏感性(图8)。
序列表
<110> 江苏省肿瘤医院
<120> 长链非编码RNA CYP1B1-AS1作为乳腺癌生物标志物和治疗靶点的应用
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1776
<212> DNA
<213> CYP1B1-AS1基因(Cytochrome P450 family 1 subfamily B member 1-antisense RNA 1)
<400> 1
caactatggt tgctaacaca agaatcggca ctggtgactt ttgagccatc aatcatcctt 60
tcctaacaca tgaatctttt ggaaaatggt ctcaaatagt caagaccccc tgctgggagg 120
agcttctatc ctgctctgtg gaagaaatgg gcatgccaga tgcctggaag atcagaggct 180
cacattccag ttcaaagtta gagaaacctg agaggggtgt actggtatga ccacccgaga 240
tcccatcgct gagaaagata atggccctgg agacaaagac tgatgaagaa agacagtatc 300
cagctgtgat gcagatgaaa catgcggcat ggttgcactc actatgcagc caaggctgga 360
agccactctg gatccagcca ctctggcaca gcgttcttgc tctaggaaga acaactgaac 420
atggccacac aggcctcaca gctggcatat ggaggctgtt aataagctga gcttgaggct 480
tcaaaggact cttgtagcct gtgctggatc tagcagtgac cagtatgcca gtggccatgg 540
atgcccaggc ccatgttcag tagtgaagca ggtgctagca gcagaagcaa tagaaatgga 600
aactgggaga tctgctgtgg atgaatctca ttatcttctt tctctgaccc tctttggact 660
aaataaactc ccagtattct tgaccaacag aggtagggtt ggaacagaac ctcaaaagtc 720
atcattagac ttcctactaa tagggcattg gaagttcaag cgactaacta ggaaacaggt 780
tagtatttgt acccctacac tgtgcaacag attacatacc atttgctcta tttggcatcc 840
tttccttagg ctagattccc agaagaaaga ttactgattc gaagggcatg aacattttta 900
tgactcttgg cctaaattac taggccaagg ttttttcaaa gggatgcact aattttcact 960
atttccccac accctttcag gtgataaata attgacttga tttgtttaca ctgacgccaa 1020
gaattcttgg gaagaaggat caaagtgaaa agtggtagcc catggtgggg aggccacact 1080
cagtgcagtt gtgaagtcag cagcattttt aagcccattc attgccagta gccctgagat 1140
gagcattccc aagctgcttc ccaggtccct cctccactct ctcagggaca tccattctgt 1200
accagccccc tcagatactc atgcccttgc cctcccttct ccatgctcat taaaaccagc 1260
ttaagagcag atatgacatc agtcacttac cttttcgtgt ggctctaaat caaagtgtca 1320
cctctcccaa gcacatgctt gcatcttagc tctgattgag gaatgaaaag ctttgttctc 1380
tgttaaaaga actttgagct tagatgcccc agactgagaa atccaccaaa agctggacct 1440
ttgacttgag cggcaaagga cccccatagt caggctgtga tttgaggagc atcaagaggc 1500
cagagtttcc aagttgggac ccagaaggtg gggttggcag ggggagggta ggagcagctg 1560
gagactctgc tctaccaggg gaatacggag gtggggacca atcccagggt aaggaatgaa 1620
agtaggagcc ccagaagctg aaaacatact tcaccgatgt cagcatttta cccagagcca 1680
ttccaaggtg accataactc acttaaaagc ctgaaagctg ttttatgcca actaataaag 1740
tgcagtttaa taaaaaaaaa aaaaaaaaaa aaaaaa 1776
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
actggtatga ccacccgaga 20
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
actggcatac tggtcactgc 20
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgggtgtga accatgagaa 20
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gtgctaagca gttggtggtg 20
<210> 6
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
agggttccaa gcttaagcgg ccgccaacta tggttgctaa cacaagaatc ggca 54
<210> 7
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gatccatccc taggtagatg catttttttt tttttttttt ttttttttat taaactg 57
Claims (5)
1.CYP1B1-AS1慢病毒过表达载体在制备治疗乳腺癌的药物中的应用,其特征在于,所述CYP1B1-AS1慢病毒过表达载体含有长链非编码RNA CYP1B1-AS1,所述长链非编码RNACYP1B1-AS1的核苷酸序列如SEQ ID NO.1所示。
2.慢病毒稳定表达细胞株在制备治疗乳腺癌的药物中的应用,所述慢病毒稳定表达细胞株是将CYP1B1-AS1慢病毒过表达载体包装成病毒颗粒感染细胞获得,所述CYP1B1-AS1慢病毒过表达载体含有长链非编码RNA CYP1B1-AS1,所述长链非编码RNA CYP1B1-AS1的核苷酸序列如SEQ ID NO.1所示。
3.长链非编码RNA CYP1B1-AS1在制备提高乳腺癌细胞对化疗药物的敏感性的产品中的应用,其特征在于,所述长链非编码RNA CYP1B1-AS1的核苷酸序列如SEQ ID NO.1所示,所述化疗药物为顺铂。
4.CYP1B1-AS1慢病毒过表达载体在制备提高乳腺癌细胞对化疗药物的敏感性产品中的应用,其特征在于,所述CYP1B1-AS1慢病毒过表达载体含有长链非编码RNA CYP1B1-AS1,所述长链非编码RNA CYP1B1-AS1的核苷酸序列如SEQ ID NO.1所示,所述化疗药物为顺铂。
5.慢病毒稳定表达细胞株在制备提高乳腺癌细胞对化疗药物的敏感性产品中的应用,所述慢病毒稳定表达细胞株是将CYP1B1-AS1慢病毒过表达载体包装成病毒颗粒感染细胞获得,所述CYP1B1-AS1慢病毒过表达载体含有长链非编码RNA CYP1B1-AS1,所述长链非编码RNA CYP1B1-AS1的核苷酸序列如SEQ ID NO.1所示,所述化疗药物为顺铂。
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GenBank Accession:NR_027252.1.Homo sapiens CYP1B1 antisense RNA 1 (CYP1B1-AS1), long non-coding RNA.《GenBank》.2020, * |
Homo sapiens CYP1B1 antisense RNA 1 (CYP1B1-AS1), long non-coding RNA;GenBank Accession:NR_027252.1;《GenBank》 * |
Landscape of tumor suppressor long noncoding RNAs in breast cancer;Boran Pang等;《Journal of Experimental & Clinical Cancer Research》;第38卷;文章编号:79 * |
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