CN108588068B - 急性红白血病kel基因及其转录出的环状rna分子标记物 - Google Patents
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Abstract
本发明公开了一种急性红白血病KEL基因及其转录出的环状RNA(circ‑KEL)分子标记物以及相关的检测试剂。与现有技术相比，本发明的检测试剂具有检测灵敏度高、特异性强、快速且检测方法简单。
Description
技术领域
本发明涉及分子生物学技术领域，具体的涉及一种急性红白血病KEL基因及其转录出的环状RNA(circ-KEL)分子标记物。
背景技术
急性红白血病(acute erythroid leukemia，AEL)是一类少见的血液系统恶性疾病，可同时累及多个细胞系，其主要不成熟成分是原始红细胞。该病可发生于任何年龄组，儿童罕见，在白血病中占2.89％。AEL作为急性髓细胞白血病(acute myeloidleukemia，AML)中的一种少见亚型(M6型AML)，在AML中发病率低于5％。关于该病大宗临床报道较少，临床表现各异，且具有较强的异质性。目前，临床上AEL的治疗方案和其他类型AML基本一致，仍以蒽环类药物联合阿糖胞苷为主。AEL发病机制不明，且至今未发现相关分子标志物作为诊断标准。
白血病在我国人群中发病率高，近年来其发病率呈上升趋势，病因至今尚未阐明。目前公认的观点是白血病是环境-基因相互作用引起的。基因方面涉及代谢、DNA损伤修复基因等的变异。研究发现，致癌物代谢通路中的代谢酶家族在人群中呈多态性，不同个体对特定致癌物的代谢能力有巨大的差异。同样，DNA修复能力也存在显著的个体差异。人类基因组计划研究成果表明，每个人的基因都是一样的，但在序列上有极小的变异如单核苷酸多态(SNPs)。正是这些基因的SNP多态性导致了基因产物的功能差异，从而决定个体对引起白血病发病的特定致癌物的代谢能力及DNA损伤后修复能力的不同。因此，代谢类基因、DNA修复基因等的多态性是决定人群对白血病易感性的遗传因素。
白血病患者的遗传学特征异常复杂多变，包括染色体的易位、缺失、突变和倒位等，这些染色体易位畸变时大部分情况下会产生新的融合基因，这些融合基因可作为诊断不同类型白血病的分子标志，此外，不同类型的白血病化疗和放疗的治疗方案亦不同。因此，融合基因的检测对白血病的诊断、预后、监测微小残留病和指导用药等具有重要的指导价值，已成为白血病诊断标准最重要的指标之一。
现有技术检测白血病易感人群，采用杂交芯片法或玻璃板芯片法，以及利用taqman探针，该方法准确率低、假阴性和假阳性率较高，且不能批量检测；另一种直接测序法，虽准确率高，但其成本高、同样不能实现批量检测，因此现有方法均无法满足大规模基因筛选的要求。
环状RNA(circRNA)是新近确认的一类特殊的非编码RNA，是RNA剪接后所形成的一类极稳定的保守型产物，circRNA可作为miRNA“海绵”来影响对应靶基因的调控与表达，通过与疾病关联的miRNA相互作用，在疾病中发挥着重要的调控作用。有研究人员发现circRNA在动脉粥样硬化、神经系统紊乱、糖尿病和肿瘤等疾病发生过程中发挥着非常重要的作用。目前，已有研究证实circRNA在胃癌和非癌组织的表达量存在显著差异，是临床上具有潜在诊断价值的新型生物标志物。迄今为止，KEL基因在AEL中的作用尚无报道，对AEL发病机制的研究仍处于起步阶段，国内外还没有公开任何关于利用KEL基因及其转录出的circRNA分子作为急性红白血病预测和诊断的分子标志物的相关研究报道。
发明内容
本发明的目的在于提供一种急性红白血病KEL基因及其转录出的环状RNA(circ-KEL)分子标记物，以便于特异性、简单的对白血病进行检测。为了实现本发明的目的，拟采用如下技术方案：
本发明一方面涉及一种急性红白血病KEL基因，其特征在于在急性红白血病患者的血液样本中特异性高表达。
本发明另一方面涉及上述急性红白血病KEL基因所转录出的环状RNA(circ-KEL)分子标记物。
本发明涉及一种检测试剂，其特征在于该检测试剂包括如下引物：
KEL-F：5’-GGATCTCCGGGATCATTACCT-3’和
KEL-R：5’-ATTCTCCGAAGTCGGTTTTTGT-3’；
circ-KEL-F：5’-GCTCGATGTGCT-3’和
circ-KEL-R：5’-ACAGGCGACGAG-3’。
本发明另一方面涉及上述检测试剂的应用，所述的应用优选作为急性红白血病的检测试剂。
与现有技术相比，本发明的检测试剂具有检测灵敏度高、特异性强、快速且检测方法简单。
具体实施方式
下面结合具体实施例对本发明做进一步的详细说明，所述是对本发明的解释而不是限定，本发明中所用的方法及其相关的试剂，可以有其他可选择和替代方案，能够达到相同技术结果即可。
实施例1
针对急性红白血病KEL基因及其转录出的环状RNA(circ-KEL)分子标记物，分别设计两对特异性引物作为检测试剂：KEL：5’-GGATCTCCGGGA TCATTACCT-3’和5’-ATTCTCCGAAGTCGGTTTTTGT-3’；circ-KEL：5’-GCTC GATGTGCT-3’和5’-ACAGGCGACGAG-3’。引物由上海生功生物工程股份有限公司合成。
实施例2
利用实施例1的一对引物进行病例检测，具体检测过程如下：
1、RNA提取：我们从本地三级医院采集15例AEL患者血液样本、15例其他类型AML患者的血液样本和15例健康人群血液样本，分别采用TRIZOL试剂(Invitrogen LifeTechnologies Co,USA)按照说明书提取组织总RNA，通过NanoDrop2000超微量分光光度计(Thermo Fisher Scientific，USA)检测所得RNA的浓度及纯度，然后将总RNA用GoScriptReverse Transcription(RT)System试剂盒(购于美国Promega公司)按照说明书反转录成cDNA，cDNA用无酶水稀释4倍得到cDNA样本。
2、PCR反应：取4ul cDNA样本溶液加入到10ul Roche lightCycler 480SYBRGREENⅠMaster混合液(购于美国Roche公司)中，再加入1.6ul特异性上、下游引物(上、下游引物浓度都为10pmol/ul)，4.4ul无RNA酶水，组成20ul反应体系在罗Roche lightCycler480Ⅱ仪器上进行PCR扩增反应；扩增步骤包括初始激活在94℃，10分钟；94℃20s，55℃30s，72℃30s，共进行45次循环的退火反应；反应完成后是95℃1min，59℃30s，95℃30s进入熔解曲线分析，判断产物的特异性。
3、完毕后PCR产物测序，结果比对后为目的片段，该目的片段包含KEL基因环化位点，为KEL基因特异性片段。采用2-△△CT方法对数据进行整理，具体结果如表1所示。
表1：AEL患者与其他类型AML患者以及健康人群的血液组织中KEL基因及其转录出的环状RNA表达比较
| 实验组 | KEL基因表达 | circ-KEL表达 |
| AEL患者 | 0.305±0.015 | 1.5275±0.2135 |
| 其他类型AML患者 | 0.028±0.010 | 0.5225±0.1548 |
| 健康人群 | 0.023±0.008 | 0.4356±0.1243 |
从表1中可以看出，在三类不同患者中，通过KEL基因以及circ-KEL的检测试剂检测，AEL患者血细胞中的KEL基因以及circ-KEL的表达水平明显高于其他类型AML患者和正常人群，而在其他类型AML患者和正常人群中二者的表达均无显著差异，说明本发明的检测方法特异性高。
以上具体实施方式仅为本创作的较佳实施例，并不用以限制本发明，凡在本发明的精神及原则之内所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。以上所述是本发明的优选实施例，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明所述原理的前提下，还可以作出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (2)
1.一种急性红白血病KEL基因作为急性红白血病标志物的应用，其特征在于所述KEL基因在急性红白血病患者的血液样本中特异性高表达。
2.一种检测试剂在制备诊断急性红白血病试剂盒中的应用，其特征在于该检测试剂包括如下引物：
KEL-F：5’-GGATCTCCGGGATCATTACCT-3’和
KEL-R：5’-ATTCTCCGAAGTCGGTTTTTGT-3’。
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