CN109055564A - 用于慢性淋巴细胞白血病诊断及预后评估的CircRNA标志物 - Google Patents
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Abstract
本发明涉及一种用于慢性淋巴细胞白血病诊断及预后评估的CircRNA标志物，所述CircRNA标志物为hsa_circRPL15_001，其核苷酸序列如SEQ ID NO.1所示。通过定量PCR对本发明的CircRNA标志物进行检测，可实现对慢性淋巴细胞白血病的早期诊断，前期芯片筛选结合临床大样本验证发现，该CircRNA标志物在CLL患者中的表达水平显著高于正常人，且其表达水平与临床分期及生存期高度相关，临床检测仅需采集微量外周血,创伤性小，更易被受检者接受，更可成为CLL患者的早诊、病情进展的判断以及预后评估的有效手段，并且特异性强，敏感性高，结果稳定，具有广泛的临床应用前景。
Description
技术领域
本发明涉及一种用于慢性淋巴细胞白血病诊断及预后评估的CircRNA标志物，属于生物技术领域。
背景技术
慢性淋巴细胞白血病(chronic lymphocytic leukemia,CLL)是主要发生在中老年人群的一种淋巴细胞克隆增殖性的肿瘤性疾病，以淋巴细胞在外周血、骨髓、脾脏和淋巴结聚集为特征。CLL是一种具有特定免疫表型特征的成熟B淋巴细胞克隆增殖性肿瘤，95％以上的CLL为B细胞的克隆性增殖，仅不到5％的病例为T细胞表型。CLL起病隐袭,在发病个体、疾病进展、治疗反应、临床预后等方面存在很大的异质性。CLL患者的中位生存期约为10年，但不同患者的预后呈高度异质性。性别、年龄、体能状态、伴随疾病、外周血淋巴细胞计数及倍增时间，以及LDH、β2微球蛋白(β2-MG)、胸苷激酶1(TK1)等临床和实验室检查指标是传统的预后因素。目前临床上主要通过以下3项标准可以诊断为CLL：①外周血单克隆B淋巴细胞计数≥5×109/L；②外周血涂片特征性的表现为小的、形态成熟的淋巴细胞显著增多，其细胞质少、核致密、核仁不明显、染色质部分聚集，并易见涂抹细胞；③外周血淋巴细胞中不典型淋巴细胞及幼稚淋巴细胞。
根据外周血淋巴细胞计数明显升高、典型的淋巴细胞形态及免疫表型特征，大多数CLL患者可以诊断，但尚需与其他疾病，特别是其他B细胞慢性淋巴增殖性疾病相鉴别。根据CLL免疫表型积分系统(CD5+、CD23+、FMC7-、sIgdim、CD22/CD79bdim/-各积1分)，CLL积分为4～5分，其他B-CLPD为0～2分。积分≤3分的患者需要结合淋巴结、脾脏、骨髓组织细胞学及遗传学、分子生物学检查等进行鉴别诊断(特别是套细胞淋巴瘤)(具体参照《B细胞慢性淋巴增殖性疾病诊断与鉴别诊断中国专家共识(2018年版)》)。目前CLL的诊断通常需要结合临床表现、细胞形态、流式分析、免疫组化等联合分析，诊断较复杂且易误诊，鉴别困难。
环状RNA(circular RNAs，circRNAs)是一类广泛存在的非编码RNA。在早期研究中，circRNAs被认为是形成于外显子转录本的错误剪接，作为随机产物并不具备生物学功能。CircRNAs是缺少5'端帽和3'端poly(A)尾的特殊闭环结构，具有核酸酶抗性；与哺乳动物细胞中线性mRNA、微小RNA和长链非编码RNA相比，具有更高的稳定性和序列保守性。随着生物信息学及高通量测序技术的迅速发展，大量circRNAs被发现并引起研究者的关注，成为近年来的研究热点。越来越多的证据表明，circRNAs并非偶然产生，其丰度、结构稳定性和时空表达特异性，在多种疾病的发生发展中发挥重要作用。
近些年研究发现，circRNAs在胃癌、肝细胞癌、胰腺导管腺癌、乳腺癌等肿瘤的发生过程中起着非常关键的作用，Yin等人发现hsa-circ-0001785可以作为乳腺癌诊断的分子靶标，Ma D等发现hsa_circ_0004277可以作为急性髓细胞白血病的诊断标志物，并且发现化疗可以显著恢复该circRNA的表达量，提示其与预后存在相关性，说明circRNAs可作为一类新颖的、极具潜力的肿瘤诊断、治疗以及与预后相关的新型靶标。目前，circRNAs在血液病中的表达及功能研究甚少，尚未发现CLL相关的血浆circRNA分子标志物或治疗靶点。
发明内容
本发明的目的在于解决现有技术的不足，提供一种血浆中的hsa_circRPL15_001(circBase ID：hsa_circ_0064574，Position：chr3:23959340-23960054)，其对于慢性淋巴细胞白血病有很高的灵敏度和特异性，可作为CLL患者诊断、临床分期及预后的分子标志物。
技术方案
一种用于慢性淋巴细胞白血病诊断的CircRNA标志物，所述CircRNA标志物为hsa_circRPL15_001，其核苷酸序列如SEQ ID NO.1所示。
针对上述CircRNA标志物的特异性引物，包括SEQ ID NO.2所示的上游引物和SEQID NO.3所示的下游引物。
上游引物序列：5'-TCGAAGCCTTCAGTAAGCCA-3'(SEQ ID NO.2)
下游引物序列：5'-CCCTCAGAAGAAAGCGCATG-3'(SEQ ID NO.3)。
上述特异性引物在制备或筛选慢性淋巴细胞白血病诊断药物中的应用。
一种慢性淋巴细胞白血病的诊断试剂盒，包括上述特异性引物。
有益效果：本发明提供了一种用于慢性淋巴细胞白血病诊断的CircRNA标志物，通过定量PCR对CircRNA标志物进行检测，可实现对慢性淋巴细胞白血病的早期诊断及预后评估，前期芯片筛选结合临床大样本验证发现，该CircRNA标志物在CLL患者中的表达水平显著高于正常人，且其表达水平与临床分期及病人生存期高度相关。临床检测仅需采集微量外周血(小于1ml),创伤性小，更易被受检者接受，更可成为CLL患者的早诊、病情进展的判断以及预后评估的有效手段，通过ROC曲线分析，AUC值为0.85，P<0.0001，反映了该指标用于CLL诊断的特异性强，敏感性高。结果稳定，具有广泛的临床应用前景。
附图说明
图1为hsa_circRPL15_001对CLL患者的血浆与健康人群的血浆的ROC曲线；
图2为hsa_circRPL15_001的表达量与CLL患者Rai分期的相关性；
图3为生存曲线分析hsa_circRPL15_001表达量与患者生存率的相关性。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明。
实施例1
1、RNA提取：
从江苏省人民医院血液科采集50例CLL患者和30例健康人群血浆样本，分别使用TIANamp Virus RNA Kit(TIANGEN,Cat.#DP315-R)按照说明书提取血浆总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GoScript Reverse 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 480SYBR GREENⅠMaster混合液(购于美国Roche公司)中，再加入1.6ul特异性引物的上、下游引物(上、下游引物浓度都为10pmol/ul，上游引物的序列如SEQ ID NO.2所示，下游引物的序列如SEQ ID NO.3所示，上游引物和下游引物均由上海生功生物工程股份有限公司合成)，4.4ul无RNA酶水，组成20ul反应体系在罗Roche lightCycler 480Ⅱ仪器上进行PCR扩增反应；反应程序：94℃，10分钟；94℃20s，55℃30s，72℃30s，共进行45次循环的退火反应；得到PCR扩增产物；
反应完成后是95℃1min，59℃30s，95℃30s进入熔解曲线分析，发现相同样品各管的溶解曲线都只有一个尖锐的峰，且不是Dimer，说明扩增产物是稳定正确的，数据可以采用。
3、对PCR扩增产物进行测序，结果比对后为目的片段，该目的片段包含RPL15基因环化位点，为hsa_circRPL15_001特异性片段。CLL患者与健康人群的血浆中hsa_circRPL15_001表达比较见表1：
表1：CLL患者与健康人群的血浆中hsa_circRPL15_001表达比较
| 组别 | hsa_circRPL15_001表达 |
| CLL患者 | 1.6137±0.2793 |
| 健康人群 | 0.4023±0.2847 |
由表1的结果可以看出：与健康人群相比，被检测的环状RNA分子标记物在CLL患者中表达水平明显上调，具有显著差异。
图1为hsa_circRPL15_001对CLL患者与健康人群的ROC曲线，可以看出，AUC为0.85(95％CI＝0.74–0.95，P<0.0001)，这说明，hsa_circRPL15_001可以作为CLL患者诊断的标志物，其对CLL患者具有较高的诊断价值。
4、通过荧光定量PCR对50例CLL患者和30例健康人群血浆样本进行分析发现，hsa_circRPL15_001的表达量与CLL患者Rai分期相关，见图2，进一步说明hsa_circRPL15_001可以作为CLL患者诊断的标志物。
5、对50例患者预后生存曲线进行分析，图3为生存曲线分析hsa_circRPL15_001表达量与患者生存率的相关性。可以看出，hsa_circRPL15_001在Rai III/IV的CLL患者中的表达量显著高于Rai 0/I/II的患者(P<0.05)，并且hsa_circRPL15_001高表达的病人生存期明显缩短，提示该环状RNA指标与CLL临床分期及生存期高度相关。说明hsa_circRPL15_001可以作为CLL患者诊断及预后评估的标志物。
序列表
<110> 金晖
<120> 用于慢性淋巴细胞白血病诊断及预后评估的CircRNA标志物
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 306
<212> DNA
<213> 环状RNa(circular RNas)
<400> 1
gtaagccaag atgggtgcat acaagtacat ccaggagcta tggagaaaga agcagtctga 60
tgtcatgcgc tttcttctga gggtccgctg ctggcagtac cgccagctct ctgctctcca 120
cagggctccc cgccccaccc ggcctgataa agcgcgccga ctgggctaca aggccaagca 180
aggttacgtt atatatagga ttcgtgttcg ccgtggtggc cgaaaacgcc cagttcctaa 240
gggtgcaact tacggcaagc ctgtccatca tggtgttaac cagctaaagt ttgctcgaag 300
ccttca 306
<210> 2
<211> 20
<212> DNA
<213> 环状RNa(circular RNas)
<400> 2
tcgaagcctt cagtaagcca 20
<210> 3
<211> 20
<212> DNA
<213> 环状RNa(circular RNas)
<400> 3
ccctcagaag aaagcgcatg 20
Claims (4)
1.一种用于慢性淋巴细胞白血病诊断及预后评估的CircRNA标志物，其特征在于，所述CircRNA标志物为hsa_circRPL15_001，其核苷酸序列如SEQ ID NO.1所示。
2.针对权利要求1所述CircRNA标志物的特异性引物，其特征在于，所述特异性引物包括SEQ ID NO.2所示的上游引物和SEQ ID NO.3所示的下游引物。
3.权利要求2所述特异性引物在制备或筛选慢性淋巴细胞白血病诊断药物中的应用。
4.一种慢性淋巴细胞白血病的诊断试剂盒，其特征在于，包括权利要求2所述特异性引物。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113252900A (zh) * | 2021-06-19 | 2021-08-13 | 山东第一医科大学附属省立医院(山东省立医院) | 基于ApoA的慢性淋巴细胞白血病预后风险评估系统及其应用 |
| CN113782087A (zh) * | 2021-11-09 | 2021-12-10 | 山东第一医科大学附属省立医院(山东省立医院) | 一种慢性淋巴细胞白血病sscr风险模型及其建立方法和应用 |
| CN114306606A (zh) * | 2021-11-29 | 2022-04-12 | 江苏省人民医院（南京医科大学第一附属医院） | cGAS抑制剂在制备治疗T细胞淋巴瘤的药物中的应用 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113252900A (zh) * | 2021-06-19 | 2021-08-13 | 山东第一医科大学附属省立医院(山东省立医院) | 基于ApoA的慢性淋巴细胞白血病预后风险评估系统及其应用 |
| CN113252900B (zh) * | 2021-06-19 | 2022-04-08 | 山东第一医科大学附属省立医院(山东省立医院) | 基于ApoA的慢性淋巴细胞白血病预后风险评估系统及其应用 |
| CN113782087A (zh) * | 2021-11-09 | 2021-12-10 | 山东第一医科大学附属省立医院(山东省立医院) | 一种慢性淋巴细胞白血病sscr风险模型及其建立方法和应用 |
| CN114306606A (zh) * | 2021-11-29 | 2022-04-12 | 江苏省人民医院（南京医科大学第一附属医院） | cGAS抑制剂在制备治疗T细胞淋巴瘤的药物中的应用 |
| CN114306606B (zh) * | 2021-11-29 | 2023-05-26 | 江苏省人民医院（南京医科大学第一附属医院） | cGAS抑制剂在制备治疗T细胞淋巴瘤的药物中的应用 |
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