CN113699234A - 长链非编码RNA Linc01605在作为胃癌诊断性试剂盒及靶向药物开发中的应用 - Google Patents
长链非编码RNA Linc01605在作为胃癌诊断性试剂盒及靶向药物开发中的应用 Download PDFInfo
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Abstract
本发明公开了长链非编码RNA Linc01605在作为胃癌诊断性试剂盒及靶向药物开发中的应用。本研究通过实时荧光定量PCR检测了胃癌组织以及其对应的癌旁组织中长链非编码RNA Linc01605的mRNA表达水平,在胃癌组织中Linc01605的表达明显高于癌旁组织,具有统计学差异。我们研究还发现,敲低Linc01605可以在体外抑制胃癌细胞AGS的增殖与迁移。根据Linc01605的表达差异以及敲低之后细胞表型实验结果,Linc01605将可能作为胃癌的一个潜在生物标志物以及作为胃癌诊断性试剂盒的潜在靶点。
Description
技术领域
本发明属于生物医药领域,具体涉及Linc01605作为胃癌诊断的生物标志物、在诊断性试剂盒以及靶向药物开发中的应用。
背景技术
癌症是21世纪全球普遍存在的健康问题,也是导致死亡的主要原因。胃癌是癌症相关死亡的第三大原因,也是全球第五大最常见的癌症。由于缺乏敏感和特异的生物标志物,大多数新诊断的GC患者已达到晚期。因此,进一步研究GC的发病分子机制,并确定与GC相关的生物诊断标志物成为了目前的研究热点。
胃癌是起源于胃黏膜上皮的恶性肿瘤,它的发生是一个从正常的胃黏膜到慢性胃炎、萎缩性胃炎、肠上皮化生、不典型增生、癌的连续发展过程。虽然对胃癌的研究已取得一定的进展,但目前仍不能完全明确其确切的分子机制。胃癌的发病原因主要和环境因素、一般性及特异性的遗传变异累积相关。诱发胃癌的环境因素主要是幽门螺杆菌的感染,感染后引起胃黏膜急、慢性炎症反应,细胞增生与凋亡平衡失调从而促进胃癌进展;一些不健康的生活方式,如饮食问题、吸烟饮酒也会导致胃癌;胃癌相关基因变异,会引起氧化性损伤,亚硝酸盐和亚硝基化合物增加,人端粒酶RNA的表达及端粒酶活性增加,环加氧酶表达增加,也会促进胃癌的发生和发展。
长链非编码RNA(lncRNA)是指在基因组中广泛转录,长度大于200nt并且无法翻译成功能性蛋白质的RNA分子。lncRNA最初被认为是RNA聚合酶II(pol II酶)转录的副产物,是一种“噪音分子”,不具有生物学功能。但是随着对于lncRNA的深入研究,发现lncRNA虽然与mRNA相似由基因座中起始经pol II酶介导转录,但是lncRNA可能与染色质相互作用,再通过低效剪接经RNA结合蛋白(RBP)加工修饰,形成与mRNA类似在5’端7甲基鸟苷封端帽状结构,3’端被多聚腺苷酸化的成熟体。然后lncRNA会经一种独特的转运方式,一部分lncRNA根据核保留机制停留于细胞核中,剩下一部分的lncRNA与mRNA共享相同的加工转运方式,被运输到细胞质中,由于这种独特的运输模式,造就了lncRNA亚细胞定位不同,从而在一定程度上决定了lncRNA的功能。近年来的研究表明,lncRNA的功能主要是,第一,作为信号分子调控下游靶基因转录;第二,作为诱饵分子阻断一些分子途径,比如与蛋白结合阻断信号通路以及行使内源性竞争性RNA(ceRNA)功能;第三,作为向导分子,指导特定蛋白质到达其靶标位置并发挥其生物学功能;第四,作为支架分子,募集并组装大分子复合物,比如lncRNA可以募集染色质重塑复合物核染色质修饰复合物,行使表观遗传调控作用。总之,lncRNAs参与了染色体修饰和基因组修饰、转录激活、转录干扰、核内运输以及维持mRNA稳定性等过程。lncRNAs影响到了具有重要生理意义的几种细胞功能,其表达的改变是癌症细胞所固有的。这些功能性lncRNA的特定表达模式有可能被用作癌症生物标志物,并且针对其治疗靶向性的策略成为目前研究热点与难点。
Linc01605是一种产生于基因间的lncRNA。有研究报道,较高的Linc01605水平在肠癌、肝细胞癌以及膀胱癌中预后较差,Linc01605通过促进癌症细胞的增殖、迁移,从而达到促进癌症进展过程。通过TCGA数据库以及GEO数据集分析,与胃正常组织相比Linc01605在胃癌组织中的表达显著升高,与临床预后密切相关。但到目前为止,Linc01605在胃癌中的作用及其机理还未曾报道。
因此本发明将检测长链非编码RNA Linc01605在胃癌组织中的表达情况,旨在用于制备诊断性试剂盒,并在此基础上研究Linc01605表达下调对胃癌细胞增殖与迁移情况的影响,这对于提高胃癌的诊断率及改善胃癌的生存状况提供有利的理论依据。
发明内容
发明目的:为了克服上述现有技术的缺点,本发明的一个目的在于提供一种长链非编码RNA Linc01605作为胃癌诊断的生物标志物的应用,满足癌症诊断指标的使用需求。本发明的另一个目的是提供一种长链非编码RNA Linc01605在制备用于癌症诊断性试剂盒中的应用,适于大规模推广应用。
技术方案:为了实现上述发明目的,本发明采用的技术方案如下:
(1)长链非编码RNA Linc01605(NCBI数据库,Gene ID:100507420,NR_121620.2)在作为癌症诊断的生物标志物的作用。所述的癌症为胃癌。
(2)长链非编码RNA Linc01605制备用于检测Linc01605表达异常的胃癌的诊断性试剂盒中的应用。
(3)长链非编码RNA Linc01605的抑制剂,为Linc01605-siRNA,其序列为:5’-CCAGTGAGAGAACATACAA-3’。
(4)长链非编码RNA Linc01605的抑制剂在制备用于治疗Linc01605表达异常的癌症的药物中的应用。所述的癌症为胃癌。
有益效果:与现有的技术相比,本发明经实时荧光定量PCR检测证实,长链非编码RNA Linc01605的表达量在胃癌中表达明显高于癌旁正常组织;应用特异性siRNA序列高效抑制Linc01605基因在人胃癌细胞株的表达,经MTT、细胞划痕及实时荧光定量PCR等实验方法,验证降低Linc01605的表达可以抑制胃癌细胞增殖与迁移能力。可见,长链非编码RNALinc01605在作为胃癌生物标志物及制备用于治疗胃癌药物、用于癌症诊断性试剂盒中具有广泛应用。
附图说明
图1:实时荧光定量PCR(RT-PCR)分析长链非编码RNA Linc01605在胃癌组织(n=12)及癌旁组织(n=12)中mRNA的表达情况;18s rRNA作为内参对照基因。
图2:AGS细胞通过转染Linc01605-siRNA靶向敲低长链非编码RNA Linc01605后Linc01605在mRNA上的水平变化。
图3:AGS细胞转染Linc01605-siRNA后对细胞增殖的影响。A:胃癌细胞AGS转染Linc01605-siRNA后,在0-96h中,通过MTT比色法测定细胞增殖情况。B:胃癌细胞AGS转染Linc01605-siRNA后,RT-PCR测定相关促增殖基因mRNA水平变化。
图4:AGS细胞转染Linc01605-siRNA后对胃癌细胞迁移的影响。A:胃癌细胞AGS转染Linc01605-siRNA后,24h、72h通过细胞伤口愈合实验分析敲低Linc01605对细胞迁移的影响。B:胃癌AGS细胞转染Linc01605-siRNA 24、72h后,AGS细胞的划痕愈合率示意图。
具体实施方式
本发明所述的干扰长链非编码RNA Linc01605的表达不仅限于siRNA的序列,还包括敲除长链非编码RNA Linc01605的编码基因,干扰Linc01605的转录等生物过程,虽然干扰的具体机制较多且尚未完全清楚,但并不妨碍“敲低”的效果实现。
在一些具体的实施方式中,所述药物可以加入一种或多种药学上可接受的佐剂,包括但不限于颗粒剂、缓冲剂、表面活性剂等公认的药物佐剂。
在一些具体的实施方式中,所述药物可以制成包括但不限于显微注射剂、适于转染的剂型,这些剂型可以按照药学领域常规的方法制备。
以下通过实施例进一步描述本发明,其中包括使用材料及具体来源。但应当理解的是,这些只是示例性的,而非限制本发明。与如下组织、细胞、试剂、仪器的类型及型号、或性质或功能相似或相同的材料均可以用于本发明的实施。
下述示例中的方法如无特殊说明均为普通方法。
主要材料:
注:除非另有所指,本发明中用到的试剂可以是任何合适的市售试剂;细胞系均可以通过市售获得。
一.长链非编码RNA Linc01605在组织中表达分析检测
1.癌症临床样品的采集
胃癌及癌旁正常组织均采集来源自河南大学附属淮河医院。整个采集及后续实验过程符合医学伦理道德要求并严格遵循病例资料的保密原则。组织样品经手术取出后,切成小块放入冻存管,置于液氮中长期保存备用。
2.组织RNA的提取
准备冰盒,将装有组织的离心管、刀片、镊子放于冰上备用。用刀片将组织切下约100ng放入新的管中。在冰上使用组织破碎仪破碎组织,破碎完后加入1mL Trizol溶液,在加入100μL的BCP溶液,用涡旋仪涡旋充分混匀15秒后室温静置8分钟。提前预冷离心机,离心机设置4℃,12000g,离心15min。将上清液取出转移至无RNA酶的1.5mL离心管中,加入等体积的异丙醇溶液,颠倒10次混匀后,静置10min。离心机4℃,12000g离心15分钟后,弃去上清液,用含75%(V/V)乙醇的DEPC水溶液清洗RNA。离心机常温,7500g离心5min后,弃上清液,置于烘箱中37℃晾干1h。加入30μL RNAase free水,置于55℃金属浴加热10分钟充分溶解RNA,溶解后用Nanodrop 2000仪器测定RNA含量。一般认为A260/A280指标在1.8-2.1之间可以判定总RNA质量较好。
3.RNA反转为cDNA
采用Nanodrop 2000仪器测定RNA的浓度为cRNA,根据以下公式计算反转体系。
根据以上计算结果配制反转体系,将RNA反转为cDNA,如表1所示。5×Buffer、M-MLV均为cDNA合成试剂盒中试剂。
表1反转试剂与过程
4.实时荧光定量PCR检测长链非编码RNA Linc01605在mRNA水平上的表达
将反转出的cDNA按照体积比1∶10的比例用水稀释至100μL。以cDNA为模板,使用针对Linc01605的引物及2×SYBR Green PCR Master Mix,按照表2配制检测体系,在ABI7500实时荧光定量PCR仪上进行扩增。
表2实时荧光定量PCR检测体系
PCR条件为:50℃20秒;95℃10分钟;95℃10秒;60℃1分钟,重复40个循环。测得样本Linc01605扩增的CT值,以内参基因18s rRNA的CT值和对应的样品浓度进行标准化校正。所得CT值使用ΔΔCT法进行计算,比较不同样本间Linc01605表达的差异。所用Linc01605正向引物如SEQ ID NO:1所述;Linc01605反向引物如SEQ ID NO:2所述。内参对照18s rRNA正向引物如SEQ ID NO:7所述;内参对照18s rRNA反向引物如SEQ ID NO:8所述。
结果:如图1所示,Linc01605在胃癌组织的表达高于癌旁组织。(p=0.0025)
二、敲低长链非编码RNA Linc01605后在AGS细胞中mRNA水平上的变化
1.细胞培养
人胃癌细胞系AGS细胞在1640完全培养基中进行培养。完全培养基中含有90%(V/V)1640不完全培养基、10%(V/V)胎牛血清(Gibco,USA)、青霉素(100U/mL)和链霉素(100U/mL)。所有细胞置于37℃细胞培养箱、含5%(V/V)CO2和95%(V/V)空气条件下培养。
2.siRNA设计
针对Linc01605基因序列,委托苏州吉玛基因股份有限公司构建siRNA,Linc01605-siRNA序列为5’-CCAGTGAGAGAACATACAA-3’。
3.细胞转染
首先,将2×105个状态较好的AGS细胞种于6孔的培养板中,细胞种板约24h后至长到70%左右密度后开始转染,转染Linc01605-siRNA(50nM)、NC-siRNA(50nM)通用阴性对照,转染8-10h后将培养基换成完全培养基。所用转染试剂为Lipofectamine 2000,转染方法参照说明书进行。
4.AGS细胞中Linc01605 mRNA水平的检测
转染48h后,用Trizol消化AGS细胞。按照具体实施方法一中的方法,提取细胞中的RNA,反转录后通过实时荧光定量PCR的方法,检测转染siRNA后AGS细胞内长链非编码RNALinc01605的mRNA水平的变化。
结果:如图2所示,实时荧光定量PCR分析表明,转染si-Linc01605后可显著抑制AGS细胞内Linc01605的mRNA表达水平,P<0.05。该结果验证通过外源性方法可以有效降低胃癌细胞内异常升高的长链非编码RNA Linc01605的mRNA水平。
三、敲低长链非编码RNA Linc01605可以抑制胃癌细胞的增殖
1.细胞增殖MTT检测
采用MTT检测细胞的增殖情况。将2×105个AGS细胞种于6孔培养板中,按照具体实施二中的方法,转染Linc01605-siRNA及NC-siRNA通用阴性对照。于转染后8h换完全培养基,待转染后48h收集细胞,将两个转染组细胞重新种至96孔板中,密度1×104/孔,每个转染组设置3个重复孔。细胞贴壁后向每孔加入10μL的MTT溶液(5mg/mL),将96孔板置于培养箱孵育4h后,再加入二甲基亚砜溶液萃取10min后,用酶标仪测定在450nm处的吸光度。分别在种入96孔板0h、24h、48h、72h、96h进行检测。根据测定的OD值绘制细胞增殖曲线。
2.转染siRNA后细胞增殖相关基因在mRNA水平上的表达变化
转染48h后,收集细胞。按照具体实施一中的方法,提取细胞总RNA,反转录后利用实时荧光定量PCR的方法,检测转染siRNA后AGS细胞内与增殖相关基因mRNA水平的变化。所用Cyclin E1正向引物如SEQ ID NO:3所述;Cyclin E1反向引物如SEQ ID NO:4所述;所用Cyclin E2正向引物如SEQ ID NO:5所述;Cyclin E2反向引物如SEQ ID NO:6所述;内参对照18s,同上。
结果:如图3,与阴性对照组相比,敲低长链非编码RNA Linc01605后,AGS细胞的增殖速度逐步降低,在48h时Linc01605-si组与NC组细胞的生长速度已经有统计学的差异(P<0.05)。在72h时siRNA组细胞生长速度明显低于NC组(P<0.01)。表明敲低内源性的长链非编码RNA Linc01605可以有效抑制胃癌细胞AGS的增殖。Cyclin E1、Cyclin E2是典型的促增殖基因,在转染了Linc01605-si后,通过提取RNA、反转录、实时荧光定量PCR检测。发现与NC组相比较,转染si组Cyclin E1、Cyclin E2的mRNA水平下降较为明显(P<0.05)。表明敲低长链非编码RNA Linc01605后可以抑制胃癌细胞AGS种促增殖基因的表达。
四、敲低长链非编码RNA Linc01605可以抑制胃癌细胞的迁移能力
1.细胞伤口愈合实验
将AGS细胞种于24孔培养板中,每孔1×105个。转染前细胞生长至70%左右密度,按照具体实施二中的方法,分别转染Linc01605-siRNA和NC-siRNA通用阴性对照。转染8h后更换新鲜完全培养基,待转染48h用200μL无菌移液枪枪头在单层细胞上呈“十”字划痕,PBS清洗三次去除飘落的细胞,加入完全培养基继续培养细胞。在4倍显微镜下先选一处划痕光滑的位置标记,并拍照记为0h对照,然后将培养皿置于培养箱继续培养,并在24h和72h采集相同位置细胞图像。用Image Pro Plus 6.0软件测量划痕的面积和宽度。
宽度1为0h时的相对划痕宽度,宽度2为24或72h的时的相对划痕宽度,划痕宽度是划痕面积与长度的比值。
结果:如图4所示,转染Linc01605-siRNA后转染组与阴性对照组相比,24h时,胃癌细胞的划痕愈合率即出现明显降低(P<0.05);待转染后72h后,NC组细胞的划痕伤口已经完全愈合,而敲低组细胞的划痕愈合率仅50%左右(P<0.01),表明转染Linc01605-siRNA能够显著抑制胃癌细胞的迁移能力。
序列表
序列表
<110> 北京化工大学
<120> 长链非编码RNA Linc01605在作为胃癌诊断性试剂盒及靶向药物开发中的应用
<141> 2021-07-26
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 长链非编码RNA Linc01605的特异性检测引物序列F
<400> 1
gaccctagcc atgtgtggtc 20
<210> 2
<211> 20
<212> DNA
<213> 长链非编码RNA Linc01605的特异性检测引物序列R
<400> 2
tgtgtcgctg tggtcttctc 20
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<211> 19
<212> DNA
<213> 长链非编码RNA Linc01605的抑制剂为Linc01605-siRNA
<400> 3
ccagtgagag aacatacaa 19
Claims (8)
1.长链非编码RNA Linc01605作为胃癌诊断的生物标志物或诊断性试剂盒的应用。
2.长链非编码RNA Linc01605在制备用于治疗Linc01605表达异常的胃癌的药物中的应用。
3.根据权利要求1或2所述的应用,其特征在于,靶点均以长链非编码RNA Linc01605设计而成。
4.根据权利1所述的应用,长链非编码RNA Linc01605的特异性检测引物序列为F:GACCCTAGCCATGTGTGGTC;R:TGTGTCGCTGTGGTCTTCTC。
5.根据权利要求2所述的应用,具体步骤如下:
(1).以长链非编码RNA Linc01605为目的基因,构建载体;
(2).利用步骤(1)中的载体制备离体所用药物制剂。
6.根据权利要求5所述的应用,长链非编码RNA Linc01605的抑制剂为Linc01605-siRNA,其序列为5’-CCAGTGAGAGAACATACAA-3’。
7.根据权利要求5所述应用,载体除RNAi干扰外还包括病毒载体或其他非病毒基因沉默载体。
8.根据权利要求7所述应用,所述病毒载体为腺病毒载体、腺相关病毒载体、逆转录病毒载体或疱疹病毒载体;所述非病毒基因沉默载体为CRISPR/Cas9系统基因敲除载体或在RNAi沉默基础上改造的载体。
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CN104877998A (zh) * | 2015-05-13 | 2015-09-02 | 中国人民解放军总医院 | 长链非编码rna以及检测细胞及组织中该长链非编码rna表达水平的引物对及试剂盒 |
WO2020051293A1 (en) * | 2018-09-07 | 2020-03-12 | The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc. | Recurrence gene signature across multiple cancer types |
CN111518913A (zh) * | 2020-06-04 | 2020-08-11 | 桂林医学院附属医院 | 用于胃癌诊治的非编码rna-linc01819 |
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WO2020051293A1 (en) * | 2018-09-07 | 2020-03-12 | The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc. | Recurrence gene signature across multiple cancer types |
CN111518913A (zh) * | 2020-06-04 | 2020-08-11 | 桂林医学院附属医院 | 用于胃癌诊治的非编码rna-linc01819 |
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