CN108486247B - piRNA-54265在结直肠癌的诊治和预后评价方面的应用 - Google Patents
piRNA-54265在结直肠癌的诊治和预后评价方面的应用 Download PDFInfo
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Abstract
本发明公开了piRNA‑54265在结直肠癌的诊治和预后评价方面的应用。基于本发明的首席研究发现,piRNA‑54265可作为结直肠癌诊断标志物、特异性指示结直肠癌患者的化疗敏感性及预后判断,有助于临床治疗方案更精准的个体化选择及改善预后;还可作为结直肠癌治疗靶点、用于制备结直肠癌治疗药物,尤其是piRNA‑54265表达抑制剂或敲除试剂可作为结直肠癌治疗药物、可作为提高结直肠癌化疗效果的辅助药物,能够抑制结直肠癌细胞的增殖、侵袭、转移和/或定植。因此,piRNA‑54265是一个十分有潜力、有前景的结直肠癌特异性分子标志物,能方便地经血标本进行检测,具有很好的应用前景。
Description
技术领域
本发明属于生物医药技术领域。更具体地,涉及piRNA-54265在结直肠癌的诊治和预后评价方面的应用。
背景技术
结直肠癌是世界范围内常见的胃肠道肿瘤,在我国各种恶性肿瘤中居首位,是全球范围内致人死亡的主要病因之一,发生率及死亡率较高,并且临床治疗存在很大的困难。结直肠癌的诊断对于治疗来说具有重要的意义。
结直肠癌的化疗方案也受肿瘤耐药问题的困扰。据美国癌症协会估计,死于不同程度的耐药的肿瘤患者占90%以上,肿瘤的耐药问题已经成为肿瘤化疗成功与否的关键因素。患者的化疗敏感性对于化疗效果具有重要的影响作用。
另外,结直肠癌治疗的预后判断与个体化精准治疗方案的选择也是至关重要的,不同个体的差异性会显著影响治疗效果。
发明内容
本发明要解决的技术问题是克服现有结直肠癌诊断及治疗技术的缺陷和不足,提供一种新的结直肠癌诊断标志物piRNA-54265,并能够特异性指示结直肠癌患者的化疗敏感性及预后判断,有助于临床治疗方案更精准的个体化选择及改善预后;还可作为结直肠癌治疗靶点、用于制备结直肠癌治疗药物。
本发明上述目的通过以下技术方案实现:
本发明首次研究发现公开piRNA-54265在结直肠癌与癌旁组织中的表达具有显著统计学差异,piRNA-54265在癌组织中高表达。piRNA-54265在癌组织中表达量的高低与结直肠癌患者的生存预后相关,piR-54365高表达组结直肠癌患者预后较差。结直肠癌患者血清中能够检测到piRNA-54265的表达,其在血清中的表达水平与在癌组织中的表达水平呈正相关性,并与患者生存预后相关;而在正常人中血清piR-54265表达水平极低,与结直肠癌患者血清piR-54265水平相比,差异显著并区分良好。敲降piRNA-54265后,结直肠癌细胞的增殖及侵袭能力等均明显减弱,能够抑制裸鼠皮下移植瘤的生长及癌细胞的远处转移及定植。扰动piRNA-54265的表达水平,结直肠癌细胞均表现出一定程度的化疗敏感性降低或增强;piRNA-54265高表达患者,化疗近期疗效较差。
因此,以下应用均应在本发明的保护范围之内:
piRNA-54265在作为结直肠癌诊断标志物方面的应用。
piRNA-54265在作为结直肠癌预后评价标志物方面的应用。
piRNA-54265在作为结直肠癌患者化疗敏感性评价标志物方面的应用。
其中,优选地,所述piRNA-54265是指血清中piRNA-54265;能方便地经血标本进行检测。
piRNA-54265在作为结直肠癌治疗靶点方面的应用。
piRNA-54265在制备结直肠癌治疗试剂或药物方面的应用。
piRNA-54265表达抑制剂或敲除试剂在作为结直肠癌治疗药物方面的应用。
piRNA-54265表达抑制剂或敲除试剂在作为提高结直肠癌化疗效果的辅助药物方面的应用。
其中,优选地,上述药物是能够抑制结直肠癌细胞的增殖、侵袭、转移和/或定植的药物。
另外,piR-54265作为抑制剂(Inhibitor)应用,有效序列为原序列的反向互补序列,因此,以该序列为基础或作为有效成分,任何形式的修饰应用,均应受到保护,即下述SEQ ID NO.1-3所示序列及其应用均应在本发明的保护范围之内。
SEQ ID NO.1:tggag gtgat gaact gtctg agcct gacc
SEQ ID NO.2:UGGAG GUGAU GAACU GUCUG AGCCU GACC
SEQ ID NO.3:5’-GGUCAGGCUCAGACAGUUCAUCACCUCCA-3’
本发明具有以下有益效果:
本发明首次发现公开piRNA-54265在结直肠癌与癌旁组织中的表达具有显著统计学差异,piRNA-54265在癌组织中高表达。因此piRNA-54265可作为结直肠癌的诊断标志物用于结直肠癌的诊断应用。
同时,本发明还发现piRNA-54265在癌组织中表达量的高低与结直肠癌患者的生存预后相关,piR-54265高表达组结直肠癌患者预后较差,因此piRNA-54265可作为结直肠癌预后评价的标志物用于结直肠癌预后评价。
再者,本发明还发现结直肠癌患者血清中能够检测到piRNA-54265的表达,其在血清中的表达水平与在癌组织中的表达水平呈正相关性,并与患者生存预后相关;而在正常人中血清piR-54265表达水平极低,与结直肠癌患者血清piR-54265水平相比,差异显著并区分良好。因此,既可以直接方便地经血标本进行检测,又能用于进行早期诊断或初筛。
另外,本发明还发现敲降piRNA-54265后,结直肠癌细胞的增殖及侵袭能力等均明显减弱,能够抑制裸鼠皮下移植瘤的生长及癌细胞的远处转移及定植;因此piRNA-54265可用于结直肠癌治疗靶点用于结直肠癌的治疗。
最后,本发明还发现扰动piRNA-54265的表达水平,结直肠癌细胞均表现出一定程度的化疗敏感性降低或增强;piRNA-54265高表达患者,化疗近期疗效较差。因此基于piRNA-54265可作为化疗辅助治疗手段。
附图说明
图1为TCGA数据分析及病人组织样本验证piR-54265在结直肠癌中表达水平升高。
图2为癌组织piR-54265表达水平影响结直肠癌患者的生存预后。
图3为血清piR-54265表达水平影响结直肠癌患者的生存预后。
图4为扰动piR-54265水平影响系列细胞表型。
图5为扰动piR-54265水平影响裸鼠皮下成瘤及转移灶生长。
图6为piR-54265结合PIWIL2后促进激活STAT3及其下游信号通路。
图7为扰动piR-54265影响肠癌细胞药物敏感性。
图8为特异性piR-54265抑制剂抑制移植瘤生长。
图9为piR-54265影响结直肠癌患者化疗近期疗效。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
实施例1
1、实验方法
提取并分析TCGA及GEO表达谱数据,获得在结直肠癌中最高表达的前20个piRNA,对前20个piRNAs设计特异性引物,并在110例来自广州的结直肠癌患者的癌组织与癌旁组织样本中进行qPCR表达量检测。对鉴定出的piR-54265在苏州来源的108例结直肠癌患者癌与癌旁组织样本中进行再次qPCR验证。对TCGA中COAD样本基因拷贝数变异及DNA甲基化修饰数据进行生物信息学分析。
2、实验结果
如图1中a图和b图所示,只有piRNA-54265在结直肠癌与癌旁组织中的表达具有显著统计学差异,piRNA-54265在癌组织中高表达,两个临床中心(广州、苏州)样本得到同样结果。
piRNA-54265 (Accession: DQ587153)是由其前体snoRNA57在细胞内经过加工后形成的成熟体,序列长度29nt,基因组所在位置chr20:2637585-2637613;其序列如下:
SEQ ID NO.1:tggag gtgat gaact gtctg agcct gacc
或SEQ ID NO.2:UGGAG GUGAU GAACU GUCUG AGCCU GACC
piRNA-54265的有效序列即原序列的反向互补序列为:
SEQ ID NO.3:5’-GGUCAGGCUCAGACAGUUCAUCACCUCCA-3’。
如图1中c图和d图所示,DNA methylation及SCNA结果提示,piRNA-54265在结直肠癌患者中DNA甲基化程度降低、基因拷贝数扩增,与正常组相比差异具有统计学意义,这从源头上基因组水平解释了piRNA-54265在结直肠癌患者中的表达升高。
实施例2
1、实验方法
使用SPSS(19.0)对piR-54265癌组织中表达量与结直肠癌患者预后情况(如前述110例广州及108例苏州的结直肠癌术后化疗患者)进行生存分析。
2、实验结果
piR-54265在癌组织中表达量的高低与结直肠癌患者的生存预后相关,piR-54265高表达组结直肠癌患者预后较差,其5年总生存及5年无进展生存时间较短。两个临床样本中心结果一致(如图2所示)。
实施例3
1、实验方法
调取如前所用的结直肠癌病例对应的治疗前血清样本,随机选4例血清样本行Northern Blot实验检测,直接明确有无血清piR-54265表达;而后对全部样本进行RNA提取及RT-qPCR piR-54265表达量检测,并对血清piR-54265及癌组织piR-54265水平进行相关性分析。对血清piR-54265水平及对应的患者预后情况进行生存分析。申请调取111例正常人血清样本,行RNA提取及RT-qPCR 检测piR-54265表达量,与结直肠癌患者血清piR-54265水平行unpaired Student’s t-test 统计分析。
2、实验结果
如图3中a图所示,结直肠癌患者血清中能够检测到piRNA-54265的表达。图3中b图和c图,其在患者血清中的表达水平与在癌组织中的表达水平呈正相关性,并与患者生存预后负性相关。图3中d图,结直肠癌患者血清piR-54265表达量高于正常人群水平,差异具有显著统计学差异。上述结果表明,血清piR-54265表达水平在正常人、结直肠癌癌发生及预后的进程中具有一定线性关系,有作为结直肠癌早期诊断及预后判断重要标志物的潜能。
实施例4
1、实验方法
成功构建piR-54265稳定过表达及敲降的结直肠癌细胞株HCT116和LoVo,进行平板克隆实验、transwell细胞迁移或侵袭实验、CCK8细胞活力检测、Annexin-V/PI流式细胞凋亡及流式细胞周期检测。
2、实验结果
如图4所示,在两株结直肠癌细胞系中进行piRNA-54265体外功能验证,结果提示:与对照组相比,过表达piRNA-54265后能显著增强结直肠癌细胞的增殖(图4中a图)、克隆形成(图4中b图)、抗凋亡(图4中c图)及侵袭、迁移能力(图4中d图);反之,敲降piRNA-54265后,结直肠癌细胞的增殖及侵袭能力等均明显减弱。扰动piR-54265表达水平不影响细胞周期(未展示)。
实施例5
1、实验方法
将稳定过表达或敲降piR-54265的结直肠癌细胞株分别接种至裸鼠皮下建立皮下移植瘤模型,建模成功后每周记录皮下移植瘤体积大小。构建结直肠癌细胞株同时稳定表达Luciferase及过表达(OE)或敲降(KD)的piR-54265,Luc细胞经尾静脉注射入裸鼠体内构建转移瘤模型,建模成功后用小动物体外活体成像(IVIS)方法定期检测裸鼠体内Luc光子值,监察转移灶进展。
2、实验结果
如图5所示,动物实验结果同样提示:过表达 piRNA-54265能够促进裸鼠皮下移植瘤的生长及癌细胞的远处转移及定植;敲降后则结果反之。
实施例6
1、实验方法
RIP实验,用PIWI蛋白家族抗体分别对细胞总RNA进行IP,对IP下来的RNA进行洗脱后行RT-qPCR验证。Pull Down实验,用生物素合成的piR-54265经链霉素磁珠孵育后分别在HCT116和lovo细胞新鲜裂解蛋白中进行孵育,拉下的蛋白经洗脱后行Western Blot验证。扰动piR-54265表达水平,Western Blot检测兴趣蛋白表达水平变化。Co-IP实验,在扰动piR-54265水平的情况下,分别用PIWIL2或STAT3抗体对新鲜细胞裂解蛋白进行孵育,IP后的蛋白进行Western Blot验证。
2、实验结果
如图6中a图和b图所示,双向证明piR-54265特异性与PIWIL2蛋白结合。扰动piR-54265表达水平,STAT3、SRC蛋白水平没有变化,但p-STAT3表达水平受到影响,过表达piR-54265时p-STAT3蛋白表达量上调(图6中c图)。
进而经Co-IP实验证明piR-54265、PIWIL2和STAT3三者相互结合,且过表达或敲降piR-54265能影响PIWIL2和STAT3的结合量(图6中d图)。
扰动piR-54265水平,经WB实验验证,能影响STAT3下游信号通路凋亡及转移相关蛋白的表达水平(图6中e图)。
本研究通过一系列分子生物学实验从分子机制上阐明了piRNA-54265的促癌机制(信号通路及分子机制如图6中f图所示),其可通过结合PIWIL2促进对STAT3的招募及磷酸化激活,从而促进了STAT3介导的抗凋亡、促转移通路。
实施例7
1、实验方法:
分别对稳定过表达或敲降piR-54265的结直肠癌细胞株进行肠癌一线化疗药物5-FU和L-OHP的IC50实验。同样用上述的稳转细胞株构建裸鼠皮下成瘤模型,当皮下移植瘤体积达250mm3时开始腹腔给药(5-FU或L-OHP)进行治疗,定期测量皮下移植瘤大小变化。
2、实验结果:如图7中a图所示,细胞实验药物IC50 结果提示扰动piRNA-54265表达水平能影响结直肠癌细胞系的药物敏感性,过表达piR-54265时肠癌细胞对5-FU和L-OHP的耐药性增强;敲降piR-54265后则敏感性提高。动物实验结果与前一致(图7中b图):过表达piR-54265时,化疗药物治疗皮下移植瘤效果较差,敲降piR-54265后化疗敏感性增强,肿瘤体积显著缩小。
实施例8
1、实验方法:
将稳定过表达或敲降piR-54265的结直肠癌细胞株分别接种至裸鼠皮下建立皮下移植瘤模型,待皮下移植瘤体积达50mm3后开始行瘤内多点注射特异性piR-54265抑制剂,定期监测及记录肿瘤体积(具体操作时间点见图8a)。构建结直肠癌细胞株同时稳定表达Luciferase及过表达(OE)或敲降(KD)的piR-54265,Luc细胞经尾静脉注射入裸鼠体内构建转移瘤模型,IVIS监测Luc发光值,待发光值达1*106后开始定期经尾静脉注射特异性piR-54265抑制剂或联合化疗药物5-FU腹腔给药,IVIS监察转移灶进展(具体操作时间点见图8b)。
2、实验结果:
结果如图8中c图-g图所示,使用特异性的piRNA-54265抑制剂或联合化疗药物进行裸鼠肿瘤治疗,能有效抑制皮下移植瘤及转移瘤的生长,实验组转移模型裸鼠总生存期显著延长。故,特异性piR-54265抑制剂有一定抑癌作用,并可辅助提高化疗药物疗效,piR-54265抑制剂有望成为新一代结直肠癌分子靶向药物。
实施例9
1、实验方法
选取广州(215例,训练集)及北京(102例,验证集)两个临床中心接受了术前新辅助化疗的结直肠癌患者治疗前取样的血清标本进行总RNA提取及随后RT-qPCR piRNA-54265表达水平检测。piR-54265 qPCR表达结果与患者新辅化后疗效评估结果进行相关性分析并进行ROC曲线建模,获取最优cutoff值以助预测化疗疗效。
2、实验结果
为更直接地研究piRNA-54265与化疗的关系,排除手术等多种复杂因素的干扰,本研究选取含疗效评价的新辅化结直肠癌患者治疗前血清进行表达量分析,如图9中a图和b图所示,血清piRNA-54265高表达患者,化疗近期疗效较差;两个临床样本中心结果一致。以血清piR-54265表达水平对患者的化疗近期疗效情况构建ROC曲线,结果如图9中c图所示,训练集中,血清piR-54265表达水平能较准确反映结直肠癌患者的化疗疗效或化疗敏感性(AUC=0.819, P<0.0001, Guangzhou),得到Cutoff值为0.01227(敏感度66.7%,特异度90.0%);以该Cutoff值在验证集中进行模拟,敏感度53.3%,特异度93.1%,AUC=0.808, P<0.0001。 故,piRNA-54265将是一个十分有潜力有前景的结直肠癌特异性分子标志物,能方便地经血标本进行检测,特异性指示结直肠癌患者的化疗敏感性及预后判断,基于血清piR-54265表达水平及Cutoff值,将有助于临床治疗方案更精准的个体化选择及改善预后。
序列表
<110> 中山大学,中山大学肿瘤防治中心
<120> piRNA-54265在结直肠癌的诊治和预后评价方面的应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 29
<212> DNA
<213> piRNA-54265序列1(piRNA-54265)
<400> 1
tggaggtgat gaactgtctg agcctgacc 29
<210> 2
<211> 22
<212> DNA
<213> piRNA-54265序列2(piRNA-54265)
<400> 2
ggagggagaa cgcgagccga cc 22
<210> 3
<211> 23
<212> DNA
<213> piRNA-54265序列3(piRNA-54265)
<400> 3
ggcaggccag acagcacacc cca 23
Claims (16)
1.piRNA-54265在制备结直肠癌诊断产品方面的应用,其特征在于,所述piRNA-54265的序列为SEQ ID NO.1或SEQ ID NO.3所示。
2.piRNA-54265在制备结直肠癌预后评价产品方面的应用,其特征在于,所述piRNA-54265的序列为SEQ ID NO.1或SEQ ID NO.3所示。
3.piRNA-54265在制备结直肠癌患者化疗敏感性评价产品方面的应用,其特征在于,所述piRNA-54265的序列为SEQ ID NO.1或SEQ ID NO.3所示。
4.根据权利要求1~3任一所述应用,其特征在于,所述piRNA-54265是指血清中piRNA-54265。
5.piRNA-54265在制备结直肠癌治疗试剂或药物方面的应用,其特征在于,所述piRNA-54265的序列为SEQ ID NO.1或SEQ ID NO.3所示。
6.piRNA-54265表达抑制剂或敲除试剂在制备结直肠癌治疗药物方面的应用,其特征在于,所述piRNA-54265的序列为SEQ ID NO.1或SEQ ID NO.3所示。
7.piRNA-54265表达抑制剂或敲除试剂在制备提高结直肠癌化疗效果的辅助药物方面的应用,其特征在于,所述piRNA-54265的序列为SEQ ID NO.1或SEQ ID NO.3所示。
8.根据权利要求5~7任一所述应用,其特征在于,所述药物是能够抑制结直肠癌细胞的增殖、侵袭、转移和/或定植的药物。
9.piRNA-54265在制备结直肠癌诊断产品方面的应用,其特征在于,所述piRNA-54265的序列为SEQ ID NO.2所示。
10.piRNA-54265在制备结直肠癌预后评价产品方面的应用,其特征在于,所述piRNA-54265的序列为SEQ ID NO.2所示。
11.piRNA-54265在制备结直肠癌患者化疗敏感性评价产品方面的应用,其特征在于,所述piRNA-54265的序列为SEQ ID NO.2所示。
12.根据权利要求9~11任一所述应用,其特征在于,所述piRNA-54265是指血清中piRNA-54265。
13.piRNA-54265在制备直肠癌治疗试剂或药物方面的应用,其特征在于,所述piRNA-54265的序列为SEQ ID NO.2所示。
14.piRNA-54265表达抑制剂或敲除试剂在制备直肠癌治疗药物方面的应用,其特征在于,所述piRNA-54265的序列为SEQ ID NO.2所示。
15.piRNA-54265表达抑制剂或敲除试剂在制备提高直肠癌化疗效果的辅助药物方面的应用,其特征在于,所述piRNA-54265的序列为SEQ ID NO.2所示。
16.根据权利要求13~15任一所述应用,其特征在于,所述药物是能够抑制直肠癌细胞的增殖、侵袭、转移和/或定植的药物。
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