CN116904591A - 一种甲状腺乳头状癌的LncRNA标志物及其应用 - Google Patents
一种甲状腺乳头状癌的LncRNA标志物及其应用 Download PDFInfo
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Abstract
本发明涉及一种甲状腺乳头状癌的LncRNA标志物及其应用,属于分子生物学技术领域。本发明发现了一种新的LncRNA标志物,即LncPTCaTS,其互补DNA的核苷酸序列如SEQ ID NO.1所示。LncPTCaTS在甲状腺乳头状癌组织中的表达水平明显低于正常组织,其低表达水平与甲状腺乳头状癌患者的生存不良有关。通过实验发现,LncPTCaTS在体外可以抑制甲状腺乳头状癌细胞的迁移和侵袭,在体内也可以抑制甲状腺乳头状癌细胞的转移,表明LncPTCaTS可以成为甲状腺乳头状癌精准治疗的新靶点,也是潜在的预测甲状腺乳头状癌靶向治疗疗效的指标,是一种新的甲状腺乳头状癌分子生物学标志物。
Description
技术领域
本发明涉及一种甲状腺乳头状癌的LncRNA标志物及其应用,属于分子生物学技术领域。
背景技术
甲状腺癌是最常见的内分泌恶性肿瘤,世界范围内发病率第九,女性中发病率第五。在甲状腺癌中甲状腺乳头状癌(Papillary thyroid carcinoma,PTC)占比约80%,是甲状腺癌的主要分类,也是近年来导致甲状腺癌发病率快速增长的主要原因。虽然一般来说,甲状腺乳头状癌预后普遍较好,甚至有些患者可以无需介入处理,但仍有少数表现出高侵袭性,比如具有淋巴结转移的患者,其术后复发率明显升高。因此,如果能够筛选出甲状腺乳头状癌致病及进展相关的标志因素,并以此作为是否需要手术的辅助判断指标和治疗靶点,将会产生巨大的社会经济学效益。
表观遗传学改变,包括非编码RNA的改变,已被确定为基本的致癌机制。长链非编码RNA(Long noncoding RNA,LncRNA)是指长度大于200bp的非编码RNA,近年来,随着高通量测序技术的发展和普及,发现了一大批在癌症发生发展中起重要作用的LncRNA。
LncRNA是在甲状腺癌发病机制中研究最多的表观遗传因素之一。一方面,AB074169、PTCSC3、PTCSC2、Klhl14-AS、COMET和RAIN等多个LncRNA是控制甲状腺乳头状癌细胞增殖的重要元素,例如LncRNA AB074169可以通过结合KHSRP蛋白抑制甲状腺乳头状癌细胞的增殖,降低KHSRP蛋白水平,提高CDKN1a表达,从而下调CDK2水平。另一方面,像Linc00941、COMET、GLTC、SLC26A4-AS1、FAM230B和MFSD4A-AS1等一些LncRNA已被证明在调节甲状腺乳头状癌细胞转移的生物学过程中起关键作用,例如TGF-β诱导的linc00941可通过上调促转移基因CDH6水平,促进细胞骨架重排,减少细胞膜粘连来促进癌细胞转移。
中国专利文献CN 114171200 A(申请号202111597619.1)提出一种PTC的预后标志物及其应用。PTC预后的生物标志物包括与m6A相关的LncRNA:AC025175.1,CCDC13 AS1,AC093249.2,AL356481.1,AC008556.1,AC103957.2,AL049796.1,AC012213.4中的一种或多种,并提出以m6A相关LncRNA构建PTC复发风险评估模型,基于每位患者风险基因的表达情况进行判断,能够更加精准个体化的预测患者的复发风险,更好的指导临床决策;采用基因组学的方法,可通过采取少量癌组织即可检测出术后复发风险,及时对手术方案的选择和后续治疗进行更加精准化的制定,降低患者术后复发的风险。
针对甲状腺乳头状癌,发现更多与其诊断、治疗、预后等相关的分子生物学标志物,以期对甲状腺乳头状癌患者进行更精准的判断,选择更适合的治疗方案,显著提高患者生存率,具有重大的研究意义和实际的临床应用价值。
发明内容
针对现有技术的不足,本发明提供了一种甲状腺乳头状癌的LncRNA标志物及其应用。本发明发现了一种在甲状腺乳头状癌中低表达,能够抑制甲状腺乳头状癌细胞的增殖、侵袭以及上皮-间充质转化和MAPK信号通路的激活的LncRNA,命名为LncPTCaTS,LncPTCaTS可作为甲状腺乳头状癌良好的预后预测指标和潜在的分子治疗靶点。
本发明的技术方案如下:
一种甲状腺乳头状癌的LncRNA标志物,所述LncRNA标志物为LncPTCaTS,其互补DNA的核苷酸序列如SEQ ID NO.1所示。
本发明发现,相对于正常组织,LncPTCaTS在甲状腺乳头状癌组织中的表达水平显著降低。
上述LncRNA标志物的检测试剂在制备甲状腺乳头状癌诊断产品或甲状腺乳头状癌预后监测产品中的应用。
根据本发明优选的,所述LncRNA标志物的检测试剂包括针对上述LncRNA标志物设计的特异性引物对。
进一步优选的,所述特异性引物对的核苷酸序列如SEQ ID NO.2和SEQ ID NO.3所示。
根据本发明优选的,所述甲状腺乳头状癌诊断产品或甲状腺乳头状癌预后监测产品为试纸、芯片或试剂盒。
进一步优选的,所述试剂盒为荧光定量PCR检测试剂盒。
上述LncRNA标志物的拟合剂在制备治疗甲状腺乳头状癌药物中的应用。
根据本发明优选的,所述LncRNA标志物的拟合剂包括核酸分子、脂类、小分子化学药物、抗体药物、多肽或干扰慢病毒,用以提高甲状腺乳头状癌细胞中LncRNA标志物的表达水平。
进一步优选的,所述LncRNA标志物的拟合剂为含有LncRNA标志物的互补DNA的重组质粒。
上述LncRNA标志物在筛选甲状腺乳头状癌治疗药物中的应用。
在筛选时,所述甲状腺乳头状癌治疗药物可以促进甲状腺乳头状癌细胞中LncRNA标志物的表达,提高LncRNA标志物的表达水平。
上述LncRNA标志物在预测甲状腺乳头状癌靶向药物敏感性中的应用。
在预测敏感性时,可以根据靶向药物对于甲状腺乳头状癌细胞中LncRNA标志物表达水平的影响程度判断其敏感性。
一种甲状腺乳头状癌诊断试剂盒或甲状腺乳头状癌预后监测试剂盒,包括根据上述LncRNA标志物设计的特异性引物对。
根据本发明优选的,所述特异性引物对的核苷酸序列如SEQ ID NO.2和SEQ IDNO.3所示。
根据本发明优选的,所述试剂盒为荧光定量PCR检测试剂盒。
一种治疗甲状腺乳头状癌的药物,为含有上述LncRNA标志物的互补DNA的重组质粒。
有益效果:
1、本发明发现了一种新的LncRNA标志物,即LncPTCaTS,LncPTCaTS在甲状腺乳头状癌组织中的表达水平明显低于正常组织,其低表达水平与甲状腺乳头状癌患者的生存不良有关。通过实验发现,LncPTCaTS在体外可以抑制甲状腺乳头状癌细胞的迁移和侵袭,在体内也可以抑制甲状腺乳头状癌细胞的转移,表明LncPTCaTS可以成为甲状腺乳头状癌精准治疗的新靶点,也是潜在的预测甲状腺乳头状癌靶向治疗疗效的指标,是一种新的甲状腺乳头状癌分子生物学标志物。
2、作为一种新的甲状腺乳头状癌分子生物学标志物,LncPTCaTS的应用范围广泛。其中,本发明将LncPTCaTS的检测试剂应用于制备甲状腺乳头状癌诊断产品或甲状腺乳头状癌预后监测产品,主要是将LncPTCaTS作为检测目标物,制备相应的荧光定量PCR检测试剂盒,用于检测LncPTCaTS的表达情况,操作简便,稳定性好,灵敏度高。另外,本发明提供的含有LncPTCaTS互补DNA的过表达质粒能够促进甲状腺乳头状癌细胞中LncPTCaTS的表达,进而抑制甲状腺乳头状癌细胞的增殖和侵袭转移能力,在制备治疗甲状腺乳头状癌药物中具有重要的指导意义。
附图说明
图1为LncPTCaTS在癌旁正常组织(N)、癌灶组织(T)以及颈部转移淋巴结组织(LN)中的表达水平。
图2为LncPTCaTS的分子结构、基因定位及编码潜力评分示意图。
图3为在TCGA数据库中LncPTCaTS在正常组织(Normal)、癌灶组织(PTC)中的表达水平。
图4为LncPTCaTS与甲状腺乳头状癌患者预后的关系图;图中,纵坐标表示无进展存活率,横坐标表示无进展生存时间。
图5为LncPTCaTS在癌旁正常组织(N)、癌灶组织(T)中的表达水平。
图6为LncPTCaTS过表达和基因敲除甲状腺乳头状癌细胞(TPC-1/BCPAP)中LncPTCaTS表达水平。
图7为LncPTCaTS基因敲除甲状腺乳头状癌细胞(TPC-1/BCPAP)的细胞增殖曲线。
图8为LncPTCaTS过表达甲状腺乳头状癌细胞(TPC-1/BCPAP)的细胞增殖曲线。
图9为LncPTCaTS过表达和基因敲除甲状腺乳头状癌细胞(TPC-1/BCPAP)的细胞克隆图片。
图10为LncPTCaTS过表达和基因敲除甲状腺乳头状癌细胞(TPC-1)在体内生长时肿瘤体积变化曲线。
图11为LncPTCaTS过表达和基因敲除甲状腺乳头状癌细胞(TPC-1/BCPAP)迁移至小室下面的细胞图片。
图12为LncPTCaTS过表达和基因敲除甲状腺乳头状癌细胞(TPC-1/BCPAP)迁移至小室下面的细胞数量柱状图。
图13为LncPTCaTS过表达和基因敲除甲状腺乳头状癌细胞(TPC-1/BCPAP)中E-cadherin、N-cadherin、Vimentin、β-catenin的Western blot检测图。
图14为LncPTCaTS过表达和基因敲除甲状腺乳头状癌细胞(TPC-1)经血行转移后在肺部的荧光强度水平。
图15为LncPTCaTS过表达和基因敲除甲状腺乳头状癌细胞(TPC-1/BCPAP)中p-ERK、ERK的Western blot检测图。
具体实施方式
下面结合具体实施例,对本发明进一步阐述,但本发明的保护范围不限于此。实施例中使用的药品及试剂,若无特殊说明,均为普通市售产品,实施例中未详加说明的内容,均按本领域现有技术。
材料来源:
TPC-1细胞(人甲状腺乳头状癌细胞)、BCPAP细胞(人甲状腺乳头状癌细胞)和HEK293T细胞(人胚肾细胞)由市售购买获得。
实施例中甲状腺乳头状癌细胞所用培养基,DMEM或1640培养基,Gibco公司有售;
胰酶、Opti-MEM,Gibco公司有售;RIPA裂解液,碧云天公司有售;PMSF蛋白酶抑制剂,金泰宏达公司有售;Trizol,Invitrogen公司有售;SYBR Premix Ex Taq,Takara公司有售;siRNA转染试剂(INTERFERin)、质粒转染试剂(JetPRIME)、JetPRIME Buffer,Polyplus公司有售;Transwell用小室,Corning公司有售。
实施例1:甲状腺乳头状癌LncRNA标志物的筛选
通过对来源于10位甲状腺乳头状癌患者的癌旁正常组织、癌灶组织以及颈部转移淋巴结组织中的总RNA进行测序,针对得到的RNAseq数据进行分析,筛选得到一个LncRNA,它的表达量在癌旁正常组织、癌灶组织以及颈部转移淋巴结组织中依次递减(|logFC|≥2,FDR<0.05,图1),将该LncRNA命名为LncPTCaTS(lncRNAin Papillary Thyroid Carcinomaas Tumor Suppressor)。所述LncPTCaTS的分子结构及基因定位示意图如图2所示。LncPTCaTS是位于16号染色体、1159548-1160176碱基位置的反义链,长度476nt,PhyloCSF编码评分均为负值,提示其为非编码RNA,其互补DNA(cDNA)序列如SEQ ID NO.1所示。
随后在TCGA数据库中检索分析发现,LncPTCaTS在甲状腺乳头状癌的癌灶组织中表达量显著低于正常组织(p<0.01,图3);对TCGA数据库中的429例甲状腺乳头状癌患者进行随访,Kaplan-Meier生存曲线(图4)结果显示,甲状腺乳头状癌的癌灶组织中LncPTCaTS低表达的患者具有更短的无进展生存时间,差异均具有统计学意义(P<0.05)。进一步分析发现,在有淋巴结转移的患者中,癌灶组织中LncPTCaTS的表达量低于无淋巴结转移的患者(p<0.01),而临床分期较晚的患者(ACGG StageⅢ/Ⅳ)癌灶组织内LncPTCaTS的表达量也低于早期患者(ACGG StageⅠ/Ⅰ)(p<0.01)。此外,我们还发现LncPTCaTS还与肿瘤大小的分期(T)相关,较大肿瘤(TⅢ+Ⅳ)的LncPTCaTS表达量低于较小肿瘤(TⅠ+Ⅰ)。
实施例2:甲状腺乳头状癌LncRNA标志物的验证
收集自2021年1月至12月,于山东省烟台毓璜顶医院甲状腺外科进行手术治疗的60位甲状腺乳头状癌患者及7位良性肿瘤患者组织标本。所有患者均为汉族,术前没有接受过局灶或系统性抗肿瘤治疗。术中从每位患者的标本中留取1份癌灶组织和1份癌旁正常组织。该研究经烟台毓璜顶医院伦理委员会审批。
通过RT-qPCR检测癌灶组织和癌旁正常组织中LncPTCaTS的表达水平,
其中,所用引物序列如下:
上游引物:5’-CACTCCCAGCCTCTGTCCCT-3’,SEQ ID NO.2;
下游引物:5’-GGCGTCACAACTGCTCCTT-3’,SEQ ID NO.3。
所述RT-qPCR反应体系为:2×SYBR Premix Ex Taq(TliRNaseH Plus)5μL,上下游引物(10μM)1μL,cDNA0.2μL,超纯水3.8μL,共10μL。用2-ΔΔCt法计算LncPTCaTS的表达水平。
结果显示,甲状腺乳头状癌的癌灶组织中LncPTCaTS表达明显降低,差异有统计学意义(p<0.05),如图5所示。将患者按是否出现颈部淋巴结转移区分,结果显示伴有淋巴结转移的患者癌灶组织中LncPTCaTS的表达量低于无淋巴结转移患者,与实施例1中高通量筛选及分析结果一致。还进一步对少数良性肿瘤病灶内LncPTCaTS的表达量进行了检验,发现其高于恶性组织。以上结果提示,LncPTCaTS在甲状腺乳头状癌的发生发展中可能发挥着重要作用,初步将LncPTCaTS确定为甲状腺乳头状癌的LncRNA标志物。
实施例3:LncPTCaTS过表达和基因敲除甲状腺乳头状癌细胞的构建
LncPTCaTS的互补DNA(cDNA)序列如SEQ ID NO.1所示,由金唯智公司合成,并将其克隆到pCDH-CMV-MCS-EF1α-Puro载体上,重组质粒命名为lncPTS,即为LncPTCaTS的过表达质粒。将lncPTS和对照用空白载体(NC)分别瞬时转染HEK293T细胞,转染时需同时转染包装质粒psPAX2和pMD2.G,转染48h后,收集上清液,得到含有目的质粒的重组慢病毒。
合成LncPTCaTS的两个短发夹RNA(shRNA,shPTS-1或shPTS-2)和一个阴性对照shRNA(shNC),合成的shRNA序列如表1所示,并将其克隆到pLKO.1质粒中构建重组质粒,即为LncPTCaTS的敲除质粒。将含有shPTS-1、shPTS-2或对照用shNC的重组质粒分别瞬时转染HEK293T细胞,转染时需同时转染包装质粒psPAX2和pMD2.G,转染48h后,收集上清液,得到含有目的质粒的重组慢病毒。
表1.shRNA序列
用含有8μg/mL聚凝胺的上述重组慢病毒上清液分别感染甲状腺乳头状癌细胞株TPC-1细胞及BCPAP细胞。用2μg/mL的嘌呤霉素筛选稳定的LncPTCaTS过表达细胞;用10μg/mL的杀稻瘟素筛选出稳定的LncPTCaTS基因敲除细胞。筛选出的重组慢病毒转染细胞,用RT-qPCR检测LncPTCaTS的表达水平。
RT-qPCR结果如图6所示,可以看出,shRNA(shPTS-1和shPTS-2)显著降低了TPC-1细胞和BCPAP细胞的LncPTCaTS的表达水平;过表达质粒lncPTS显著提高了TPC-1细胞和BCPAP细胞的LncPTCaTS的表达水平。LncPTCaTS过表达和基因敲除甲状腺乳头状癌细胞(TPC-1/BCPAP)构建成功。
实施例4:LncPTCaTS对甲状腺乳头状癌细胞增殖的影响(细胞计数法)
采用细胞计数实验检测LncPTCaTS对甲状腺乳头状癌细胞增殖能力的影响,具体步骤如下:
(1)将稳定的LncPTCaTS过表达或基因敲除细胞(TPC-1/BCPAP)消化离心后,用1640培养基重悬细胞,吹打混匀,计数后稀释细胞,取2500个细胞加入12孔板中,每孔含培养基1mL,每组细胞铺板3个孔;
(2)分别于铺板后第24小时、第48小时及第72小时分别消化一个培养孔内的细胞,稀释至1mL后显微镜下进行细胞计数操作。
细胞计数结果如图7和图8所示,结果显示,LncPTCaTS过表达(lncPTS)可抑制甲状腺乳头状癌细胞的增殖能力,而LncPTCaTS基因敲除(shPTS-1和shPTS-2)的甲状腺乳头状癌细胞的增殖能力显著增强。
实施例5:LncPTCaTS对甲状腺乳头状癌细胞增殖的影响(细胞克隆法)
采用细胞克隆形成实验检测LncPTCaTS对甲状腺乳头状癌细胞增殖能力的影响,具体步骤如下:
(1)将稳定的LncPTCaTS过表达或基因敲除细胞(TPC-1/BCPAP)消化离心后,用1640培养基重悬细胞,吹打混匀,计数后进行梯度稀释,混匀细胞后再次计数细胞,取2000个细胞加入6孔板中充分吹匀,每孔含培养基2mL,每组细胞铺板3个孔;
(2)定期更换培养基,并在显微镜下观察细胞团生长情况,待生长较快组长至每个细胞团约50个细胞的时候终止培养;
(3)弃掉孔内培养基,每孔加入2mL PBS缓冲液冲洗2次,加入2mL甲醇溶液,室温固定30min;
(4)弃掉甲醇,每孔加入2mL PBS缓冲液冲洗2次;
(5)弃掉PBS缓冲液,加入2mL 0.5%结晶紫溶液,常温染色30min;
(6)染色完成后,回收结晶紫,用PBS缓冲液冲洗,放于常温晾干,显微镜下拍照,储存图片。
显微镜照片如图9所示,结果显示,LncPTCaTS过表达(lncPTS)可抑制甲状腺乳头状癌细胞的克隆形成能力,而LncPTCaTS基因敲除(shPTS-1和shPTS-2)的甲状腺乳头状癌细胞克隆形成能力显著增强,与实施例4的实验结论一致。
实施例6:LncPTCaTS在体内对甲状腺乳头状癌细胞增殖的影响
为了研究LncPTCaTS在体内对肿瘤细胞增殖的作用,将稳定表达萤火虫荧光素酶的LncPTCaTS过表达或基因敲除TPC-1细胞接种到5周龄BALB/c雌性裸鼠(中国北京生命河实验室)的肩胛区皮下(每组5只)。观测肿瘤生长过程,每两天测量肿瘤直径。待生长较快组的肿瘤长至直径约1cm时处死小鼠,将肿瘤完整剥下进行仔细测量,并将结果进行统计分析后做图。
结果如图10所示,结果显示,LncPTCaTS基因敲除(shPTS-1和shPTS-2)可明显增强甲状腺乳头状癌细胞的增殖能力(P<0.001),肿瘤生长速度较快;稳定的LncPTCaTS过表达(lncPTS)显著抑制了其增殖能力(P<0.001),肿瘤生长速度较慢。
实施例7:LncPTCaTS对甲状腺乳头状癌细胞侵袭的影响
采用Transwell实验检测LncPTCaTS对甲状腺乳头状癌细胞侵袭能力的影响,具体步骤如下:
(1)取出小室放于24孔板内,加入1mL纯1640培养基(无双抗、无血清)浸泡30min;培养箱内放置30min,进行小室预处理;
(2)将稳定的LncPTCaTS过表达或基因敲除甲状腺乳头状癌细胞(TPC-1/BCPAP)消化离心后,加入营养含量低的1640培养基(1%FBS)重悬细胞,吹打混匀,计数后稀释细胞至300个/μL;
(3)取100μL稀释后的细胞悬液(3万个细胞)于新的EP管内,吹打数次,使之混匀;
(4)将700μL高营养含量的1640培养基(10%FBS)加入24孔板内,然后放入步骤(1)中预处理的小室,将步骤(3)中的细胞悬液转入小室的上室内,操作完成后放进培养箱内继续培养,24h结束培养;
(5)弃掉上室和下室的培养基,每孔加入1mL PBS缓冲液冲洗2次,加入1mL甲醇溶液,室温固定30min;
(6)弃掉甲醇,每孔加入1mL PBS缓冲液冲洗2次;
(7)弃掉PBS缓冲液,加入1mL 0.5%结晶紫溶液,常温染色30min;
(8)染色完成后,回收结晶紫,用PBS缓冲液冲洗,用棉签轻轻擦掉上室的细胞,留下小室下面的细胞,放于常温晾干,显微镜下拍照,储存图片。
小室下面的细胞图片如图11所示,统计各组细胞数量绘制柱状图如图12所示,实验结果显示,LncPTCaTS过表达细胞(lncPTS)迁移至小室下面的细胞数量显著低于对照组,表明LncPTCaTS过表达可抑制甲状腺乳头状癌细胞的侵袭能力,而LncPTCaTS基因敲除细胞(shPTS-1和shPTS-2)迁移至小室下面的细胞数量显著高于对照组,表明LncPTCaTS基因敲除可使甲状腺乳头状癌细胞侵袭能力显著增强。以上结果提示,LncPTCaTS与甲状腺乳头状癌细胞的侵袭能力有关。
实施例8:
采用Western blot实验从蛋白质水平分析LncPTCaTS对甲状腺乳头状癌细胞侵袭能力的影响,具体方法为:
裂解稳定的LncPTCaTS过表达或基因敲除甲状腺乳头状癌细胞(TPC-1/BCPAP),提取总蛋白,用BCA试剂盒测定各组细胞蛋白浓度,取等量蛋白进行SDS-PAGE凝胶电泳,将分离的蛋白电转至PVDF膜上。用含5%脱脂奶粉的TBST溶液室温封闭2小时,加入按比例稀释的E-cadherin、N-cadherin、Vimentin、β-catenin及GAPDH一抗,4℃孵育过夜。第二天加入对应的辣根过氧化物酶联二抗,室温孵育2小时。洗膜后用超敏ECL化学发光试剂盒显色并曝光。
采用Image J对图像结果进行量化处理并作图,结果如图13所示,结果表明,LncPTCaTS基因敲除(shPTS-1和shPTS-2)后会明显增加神经钙黏附蛋白(N-cadherin)、β-连环蛋白(β-catenin)、波形蛋白(Vimentin)的表达量,而降低上皮细胞钙粘蛋白(E-Cadherin)的表达,LncPTCaTS过表达(lncPTS)的结果则相反。
上皮-间充质转化(EMT),是指上皮细胞通过特定程序转化为具有间质表型细胞的生物学过程。间充质标志蛋白如神经钙黏附蛋白(N-cadherin)、β-连环蛋白(β-catenin)、波形蛋白(Vimentin)的表达量反应了肿瘤细胞由上皮细胞源性向间充质特点转化的程度,表达量增加说明上皮细胞失去了细胞极性,失去与基底膜的连接等上皮表型,获得了较高的迁移与侵袭、抗凋亡和降解细胞外基质的能力等间质表型。而上皮标志蛋白上皮细胞钙粘蛋白(E-Cadherin)则相反,代表了上皮细胞特性的程度,其表达增加说明细胞极性增加,与基底膜的链接更紧密,也意味着较低的迁移与侵袭。以上结果表明,LncPTCaTS过表达可以降低甲状腺乳头状癌细胞的迁移与侵袭能力。
实施例9:LncPTCaTS对甲状腺乳头状癌细胞转移能力的影响
为了研究LncPTCaTS在体内甲状腺乳头状癌细胞血行转移过程中的作用,将稳定表达萤火虫荧光素酶的LncPTCaTS过表达或基因敲除TPC-1细胞接种到5周龄BALB/c雌性裸鼠(中国北京生命河实验室)的尾静脉中(每组5只)。LncPTCaTS过表达组小鼠饲养8周,LncPTCaTS基因敲除组小鼠饲养6周,然后进行小动物活体成像,将麻醉后的小鼠腹腔注射Luci底物10分钟后进行成像,观察细胞肺部转移的荧光强度。将得到的图像进行归一化处理,并将结果进行统计分析后做图。
结果如图14所示,结果显示LncPTCaTS基因敲除(shPTS-1和shPTS-2)可明显增强甲状腺乳头状癌细胞的血行转移(P<0.001),肺部的荧光强度更强,稳定的LncPTCaTS过表达(lncPTS)则显著抑制了远处甲状腺乳头状癌细胞的血行转移(P<0.001),肺部的荧光强度较弱。
实施例10:
本发明还对LncPTCaTS作用的信号通路进行了研究,通过对LncPTCaTS过表达的BCPAP细胞进行基因组RNA测序(RNA-seq),在众多上调/下调的基因中进行KEGG通路富集分析,发现MAPK通路被明显富集。
磷酸化细胞外调节蛋白激酶(Phosphorylation extracellular regulatedprotein kinases,p-ERK)是MAPK信号通路激活的标志物之一,利用p-ERK一抗,通过Western blot实验,检测LncPTCaTS过表达或基因敲除甲状腺乳头状癌细胞(TPC-1/BCPAP)中p-ERK的变化,结果如图15所示。
结果显示,LncPTCaTS过表达(lncPTS)可以抑制p-ERK蛋白的表达,LncPTCaTS基因敲除(shPTS-1和shPTS-2)的作用相反,同时都对非磷酸化ERK蛋白没有明显作用,提示LncPTCaTS过表达能够抑制MAPK信号通路的激活,从而抑制肿瘤的发生。
本发明发现了一种新的长链非编码RNA(LncRNA),即LncPTCaTS,LncPTCaTS在甲状腺乳头状癌组织中的表达明显低于正常组织,其低表达水平与甲状腺乳头状癌患者的生存不良有关,LncPTCaTS在体外可以抑制细胞的增殖以及迁移和侵袭,在体内可以抑制甲状腺癌的生长和转移,是潜在的预测甲状腺乳头状癌预后的指标,是一种新的甲状腺乳头状癌分子标志物。本发明提供的过表达质粒能够促进LncPTCaTS的表达,进而抑制甲状腺乳头状癌细胞的增殖和侵袭转移能力,在制备治疗甲状腺乳头状癌药物中具有重要意义。
Claims (10)
1.一种甲状腺乳头状癌的LncRNA标志物,其特征在于,所述LncRNA标志物为LncPTCaTS,其互补DNA的核苷酸序列如SEQ ID NO.1所示。
2.权利要求1所述的LncRNA标志物的检测试剂在制备甲状腺乳头状癌诊断产品或甲状腺乳头状癌预后监测产品中的应用。
3.如权利要求2所述的应用,其特征在于,所述LncRNA标志物的检测试剂包括针对权利要求1所述的LncRNA标志物设计的特异性引物对;
进一步优选的,所述特异性引物对的核苷酸序列如SEQ ID NO.2和SEQ ID NO.3所示;
优选的,所述甲状腺乳头状癌诊断产品或甲状腺乳头状癌预后监测产品为试纸、芯片或试剂盒;
进一步优选的,所述试剂盒为荧光定量PCR检测试剂盒。
4.权利要求1所述的LncRNA标志物的拟合剂在制备治疗甲状腺乳头状癌药物中的应用。
5.如权利要求4所述的应用,其特征在于,所述LncRNA标志物的拟合剂包括核酸分子、脂类、小分子化学药物、抗体药物、多肽或干扰慢病毒,用以提高甲状腺乳头状癌细胞中LncRNA标志物的表达水平;
进一步优选的,所述LncRNA标志物的拟合剂为含有LncRNA标志物的互补DNA的重组质粒。
6.权利要求1所述的LncRNA标志物在筛选甲状腺乳头状癌治疗药物中的应用。
7.权利要求1所述的LncRNA标志物在预测甲状腺乳头状癌靶向药物敏感性中的应用。
8.一种甲状腺乳头状癌诊断试剂盒或甲状腺乳头状癌预后监测试剂盒,包括根据权利要求1所述的LncRNA标志物设计的特异性引物对。
9.如权利要求8所述的甲状腺乳头状癌诊断试剂盒或甲状腺乳头状癌预后监测试剂盒,其特征在于,所述特异性引物对的核苷酸序列如SEQ ID NO.2和SEQ ID NO.3所示;
优选的,所述试剂盒为荧光定量PCR检测试剂盒。
10.一种治疗甲状腺乳头状癌的药物,其特征在于,为含有权利要求1所述的LncRNA标志物的互补DNA的重组质粒。
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